WO2023233295A1 - Thiadiazolyl derivatives as dna polymerase theta inhibitors and uses thereof - Google Patents

Thiadiazolyl derivatives as dna polymerase theta inhibitors and uses thereof Download PDF

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Publication number
WO2023233295A1
WO2023233295A1 PCT/IB2023/055539 IB2023055539W WO2023233295A1 WO 2023233295 A1 WO2023233295 A1 WO 2023233295A1 IB 2023055539 W IB2023055539 W IB 2023055539W WO 2023233295 A1 WO2023233295 A1 WO 2023233295A1
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compound
cancer
pharmaceutically acceptable
acceptable salt
methyl
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PCT/IB2023/055539
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French (fr)
Inventor
Nicholas Adams
Janos Botyanszki
Kevin Duffy
Chenbo WANG
Marcus Michael FISCHER
Reeja MASKEY
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Ideaya Biosciences, Inc.
Glaxosmithkline Intellectual Property (No.4) Limited
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Publication of WO2023233295A1 publication Critical patent/WO2023233295A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • DNA repair deficient cancers often become dependent on backup DNA repair pathways, which present an “Achilles heel” that can be targeted to eliminate cancer cells, and is the basis of synthetic lethality.
  • Synthetic lethality is exemplified by the success of poly (ADP-ribose) polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers (Audeh M. W., et al., Lancet (2010); 376 (9737): 245-51).
  • PARP poly (ADP-ribose) polymerase
  • DNA damage repair processes are critical for genome maintenance and stability, among which, double strand breaks (DSBs) are predominantly repaired by the nonhomologous end joining (NHEJ) pathway in G1 phase of the cell cycle and by homologous recombination (HR) in S-G2 phases.
  • NHEJ nonhomologous end joining
  • HR homologous recombination
  • a less addressed alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ) pathway is commonly considered as a “backup” DSB repair pathway when NHEJ or HR are compromised.
  • MMEJ microhomology-mediated end-joining
  • Numerous genetic studies have highlighted a role for DNA polymerase theta (Pol0, encoded by POLQ) in stimulating MMEJ in higher organisms (Chan S. H., et al., PLoS Genet.
  • Pole is distinct among human DNA polymerases, exhibiting not only a C-terminal DNA polymerase domain but also an N-terminal helicase domain separated by a long and lesser- conserved central domain of unknown function beyond Rad51 binding (Seki eta. Al, 2003, Shima et al 2003; Yousefzadeh and Wood 2013).
  • the N-terminal ATPase/helicase domain belongs to the HELQ class of SF2 helicase super family.
  • HRD homologous recombination deficient
  • the helicase domain of Pol0 causes suppression of HR pathway through disruption of Rad51 nucleoprotein complex formation involved in initiation of the HR-dependent DNA repair reactions following ionizing radiation. This anti-recombinase activity of Pol0 promotes the alt-EJ pathway.
  • the helicase domain of Pol0 contributes to microhomology-mediated strand annealing (Chan SH et al., PLoS Genet. (2010); 6: el001005; and Kawamura K et al., Int. J. Cancer (2004); 109: 9-16).
  • compositions comprising such compounds and methods of treating and/or preventing diseases treatable by inhibition of PolO such as cancer, including homologous recombination (HR) deficient cancers.
  • HR homologous recombination
  • compositions comprising a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.
  • a method for treating and/or preventing a disease characterized by overexpression of PolO in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or an embodiment thereof disclosed herein).
  • the patient is in recognized need of such treatment.
  • the compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition.
  • the disease is a cancer.
  • a method for treating and/or preventing a homologous recombinant (HR) deficient cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or an embodiment thereof disclosed herein).
  • the patient is in recognized need of such treatment.
  • the compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition.
  • a method for inhibiting DNA repair by Pol0 in a cancer cell comprising contacting the cell with an effective amount of a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof.
  • the cancer is HR deficient cancer.
  • a method for treating and/orpreventingng a cancer in a patient wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition.
  • a compound of Formula (I) or Table 1 or an embodiment thereof disclosed herein, or a pharmaceutically acceptable salt thereof for inhibiting DNA repair by Pol0 in a cell.
  • the cell is HR deficient cell.
  • a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a disease in a patient, wherein the disease is characterized by overexpression of PolO.
  • a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a cancer in a patient, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
  • a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a HR deficient cancer in a patient.
  • a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a cancer that is resistant to poly(ADP-ribose) polymerase (PARP) inhibitor therapy in a patient.
  • PARP poly(ADP-ribose) polymerase
  • cancers resistant to P ARP -inhibitors include, but are not limited to, breast cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, head and neck cancer, pancreatic cancer, gastrointestinal cancer, and colorectal cancer.
  • the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer (data from large scale drop out screens in cancer cell lines indicate that some cell lines from the above cancers are dependent on polymerase theta for proliferation https ://depmap .org/portal/) .
  • a HR-deficient cancer is breast cancer.
  • Breast cancer includes, but is not limited to, lobular carcinoma in situ (LCIS), a ductal carcinoma in situ (DCIS), an invasive ductal carcinoma (IDC), inflammatory breast cancer, Paget disease of the nipple, Phyllodes tumor, Angiosarcoma, adenoid cystic carcinoma, low- grade adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, tubular carcinoma, metaplastic carcinoma, micropapillary carcinoma, mixed carcinoma, or another breast cancer, including but not limited to triple negative, HER positive, estrogen receptor positive, progesterone receptor positive, HER and estrogen receptor positive, HER and progesterone receptor positive, estrogen and progesterone receptor positive, and HER and estrogen and progesterone receptor positive.
  • HR-deficient cancer is ovarian cancer.
  • Ovarian cancer includes, but is not limited to, epithelial ovarian carcinomas (EOC), maturing teratomas, dysgerminomas, endodermal sinus tumors, granulosa-theca tumors, Sertoli-Leydig cell tumors, and primary peritoneal arcinoma.
  • combination therapy comprising methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a DNA polymerase theta (Pol0) inhibitor (e.g. a compound of Formula (I) or Formula (II)) and administering to the subject a therapeutically effective amount of a Poly ADP Ribose Polymerase (PARP) inhibitor, thereby treating the cancer in the subject.
  • a DNA polymerase theta (Pol0) inhibitor e.g. a compound of Formula (I) or Formula (II)
  • PARP Poly ADP Ribose Polymerase
  • a method for treating and/or preventing a homologous recombinant (HR) deficient cancer in a patient in need thereof comprising contacting the cancer cells in the patient with an effective amount of a Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II)) and a Poly ADP Ribose Polymerase (PARP) inhibitor.
  • a Pol0 inhibitor e.g. a compound of Formula (I) or Formula (II)
  • PARP Poly ADP Ribose Polymerase
  • An exemplary Pol0 polymerase domain inhibitor other than those defined by Formula (I) or Formula (II) is known as ART4215 and is developed by Artios Pharma and now in Phase l/2a clinical trials. See “A Study of ART4215 for the Treatment of Advanced or Metastatic Solid Tumors,” NCT04991480 at clinicaltrials.gov.
  • Pol0 polymerase domain inhibitors including ART558, are also reported. See Zatreanu D., et al. “Pol0 inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance,” NATURE COMMUNICATIONS, 2021. 12(1):3636.
  • Formula (II) has the structure wherein Z, R 1 , R 3a , R 3b , and R 3c have the meanings provided hereinbelow.
  • kits for treating cancer in a subject in need thereof comprising administering to the subject a combination comprising a DNA polymerase theta (PolO) (e.g. a compound of Formula (I) or Formula (II)) and a Poly ADP Ribose Polymerase (PARP) inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject.
  • a DNA polymerase theta e.g. a compound of Formula (I) or Formula (II)
  • PARP Poly ADP Ribose Polymerase
  • a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy is a combination of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and a Poly ADP Ribose Polymerase (PARP) inhibitor, for use in therapy.
  • PARP Poly ADP Ribose Polymerase
  • FIG. 1A-1B depict excess over Bliss synergy of Compound 4 (also called Compound A) and Niraparib combinations in MDA-MB-436 cell line.
  • FIG. 2A-2B depict excess over Bliss synergy of Compound 5 (also called Compound B) and Niraparib inhibitor combinations in MDA-MB-436 cell line.
  • FIG. 3 depicts excess over Bliss synergy of Compound 6 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
  • FIG. 4 depicts excess over Bliss synergy of Compound 7 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
  • FIG. 5 depicts excess over Bliss synergy of Compound 8 and Niraparib inhibitor combinations in MDA-MB-436.
  • FIG. 6 depicts excess over Bliss synergy of Compound 9 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
  • FIG. 7 depicts excess over Bliss synergy of Compound 10 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
  • FIG. 8A, 8B, 8C, and 8D show the in vitro efficacy of Compound A and Niraparib in combination in MDA-MB-436 breast cancer cells.
  • FIG. 9A, 9B, 9C, and 9D show the in vitro efficacy of Compound B and Niraparib in combination in MDA-MB-436 breast cancer cells.
  • FIG. 10A, 10B, IOC, and 10D show the in vitro efficacy of Compound A and Niraparib in combination in PEO1 ovarian cancer cells.
  • FIG. 11A, 11B, 11C, and 11D show the in vitro efficacy of Compound B and Niraparib in combination in PEO1 ovarian cancer cells.
  • FIG. 12 shows the efficacy of Compound 4 and Compound 11 in MDA-MB-436 mouse model.
  • FIG. 13A, 13B, 13C, and 13D show individual tumor growth curves for Compound 11 and for Compound 11 combinations with Compound 4.
  • FIG. 14 displays an efficacy study in BRCA1 mutant MDA-MB-436 model.
  • the dotted line represents the mean starting tumor volume.
  • One-way Anova test was applied to calculate statistics, *p ⁇ 0.05.
  • FIG. 15 displays an efficacy study in BRCA1 mutant 134T Ovarian PDX model.
  • One-way Anova test was applied to calculate statistics, *p ⁇ 0.05.
  • FIG. 16 displays an efficacy study with Example 1 in BRCA1 mutant 134T Ovarian PDX model.
  • One-way Anova test was applied to calculate statistics, *p ⁇ 0.05.
  • any definition herein may be used in combination with any other definition to describe a composite structural group.
  • the trailing element of any such definition is that which attaches to the parent moiety.
  • the composite group alkoxyalkyl means that an alkoxy group is attached to the parent molecule through an alkyl group.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a saturated straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. Ci-8 means one to eight carbons).
  • Alkyl can include any number of carbons, such as Ci-2, Ci-3, Ci-4, C1-5, C1-6, C1-7, C1-8, C1-9, C1-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • alkylene refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical.
  • the two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group.
  • a straight chain alkylene can be the bivalent radical of -(CH2)n- where n is 1, 2, 3, 4, 5 or 6.
  • Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, hexylene, and the like.
  • alkoxy refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O-.
  • alkoxy groups can have any suitable number of carbon atoms, such as C1-6, and can be straight or branced.
  • Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.
  • heterocycloalkyl refers to a saturated or partially unsatured monocyclic ring having the indicated number of ring vertices (e.g., a 3- to 7-membered ring) and having from one to five heteroatoms selected from N, O, and S as ring vertices.
  • Partially unsaturated heterocycloalkyl groups have one or more double or triple bonds in the ring, but heterocycloalkyl group are not aromatic.
  • Heterocycloalkyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 7, 4 to 7, or 5 to 7 ring members.
  • heterocycloalkyl groups any suitable number of heteroatoms can be included in the heterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4.
  • Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like.
  • a heterocycloalkyl group can be attached to the remainder of the
  • halo or halogen, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl refers to alkyl, as defined above, where some or all of the hydrogen atoms are replaced with halogen atoms.
  • alkyl group haloalkyl groups can have any suitable number of carbon atoms, such as Ci-6.
  • Ci-6 the term “Ci-4 haloalkyl” is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like.
  • haloalkoxy refers to an alkoxy group where some or all of the hydrogen atoms are substituted with halogen atoms.
  • haloalkoxy groups can have any suitable number of carbon atoms, such as Ci-6, and can be straight or branced, and are substituted with 1, 2, 3, or more halogens.
  • the compounds are per-substituted, for example, perfluorinated.
  • Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2,2,2, -trifluoroethoxy, perfluoroethoxy, etc.
  • heteroatom is meant to include oxygen (O), nitrogen (N), sulfur (S).
  • pharmaceutically acceptable salts is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, 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. Examples of 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- occuring amines and the like, such as arginine, betaine, caffeine, choline, N,N’- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 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.
  • pharmaceutically acceptable acid addition salts 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, benzene sulfonic, p-tolylsulfonic, citric, tartaric, methane sulfonic, 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.
  • the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
  • the present invention provides compounds which are in a prodrug form.
  • Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the parent compounds. Additionally, prodrugs can be converted to the parent compounds by chemical or biochemical methods in an ex vivo environment.
  • Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are intended to be within the scope of the present invention.
  • Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention.
  • a 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%.
  • the compounds of the present invention 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 isotope 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. Additionally, isotopic variants of the compounds of the invention can have altered pharmacokinetic and pharmacodynamic characteristics which can contribute to enhanced safety, tolerability or efficacy during treatment. All isotopic variations of the compounds of the present invention, 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 non-human animal (e.g., a mammal). In one embodiment, the patient is human.
  • administration refers to contact of, for example, an Pol0 modulator, a pharmaceutical composition comprising same, or a diagnostic agent to the subject, 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 a Pol0 modulator 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 subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • 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.
  • in need of treatment refers to a judgment made by a physician or other caregiver that a subject 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. For example, the patient has been diagonosed as having a disease linked to overexpression of Pol0 or a homologous recombination (HR)-deficient cancer.
  • HR homologous recombination
  • therapeutically effective amount refers to the administration of an agent to a subject, 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 subject.
  • 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 subject’s condition, and the like.
  • measurement of the serum level of an PolO modulator (or, e.g., a metabolite thereof) at a particular time post-administration may be indicative of whether a therapeutically effective amount has been used.
  • inhibitors and “activators” refer to inhibitory or activating molecules, respectively, for example, for the activation of, e.g., a ligand, receptor, cofactor, gene, cell, tissue, or organ.
  • Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, or cell.
  • Activators are molecules that increase, activate, facilitate, enhance activation, sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, or cell.
  • An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity.
  • modulate refers to the ability of a molecule (e.g., an activator or an inhibitor) to increase or decrease the function or activity of Pol0, either directly or indirectly.
  • a modulator may act alone, or it may use a cofactor, e.g., a protein, metal ion, or small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules.
  • the "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor; to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity; to the modulation of activities of other molecules; and the like.
  • the term “proliferative activity” encompasses an activity that promotes, that is necessary for, or that is specifically associated with, for example, normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
  • “Pharmaceutically acceptable carrier or excipient” means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier or an excipient that is acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable carrier/excipient” as used in the specification and claims includes both one and more than one such excipient.
  • a wavy line, ""w ", that intersects a single, double or triple bond in any chemical structure depicted herein, represent the point attachment of the single, double, or triple bond to the remainder of the molecule.
  • a bond extending to the center of a ring e.g., a phenyl ring
  • a bond extending to the center of a ring is meant to indicate attachment at any of the available ring vertices.
  • multiple substituents shown as being attached to a ring will occupy ring vertices that provide stable compounds and are otherwise sterically compatible.
  • Disease as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder, " “syndrome, “ and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • “Inhibiting”, "reducing,” or any variation of these terms in relation of Pol0 includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of about, at most about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of Pol0 activity compared to its normal activity.
  • homologous recombination refers to the cellular process of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA.
  • HR deficient 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 of one or more HR-assocated genes or presence of one or more mutations in one or more HR-assocated genes.
  • HR-associated genes include BRCA1, BRCA2, RAD54, RAD51B, CtlP (Choline Transporter-Like 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 (Werner 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, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANJ (BRIP1), FANCL, FANCM, FANCN (RALB2), FANCP (SLX4), FANCS (BRCA1), RAD51C, and XPF.
  • FANCA Fanconi anemia
  • Polynomen overexpression refers to the increased expression or activity of Pol0 in a diseased cell e.g., cancerous cell, relative to expression or activity of Pol0 in a normal cell (e.g., non-diseased cell of the same kind).
  • the amount of Pol0 can be at least 2-fold, at least 3 -fold, at least 4- fold, at least 5- fold, at least 10-fold, or more relative to the Pol0 expression in a normal cell.
  • Pol0 cancers include, but are not limited to, breast, ovarian, cervical, lung, colorectal, gastric, bladder and prostate cancers.
  • PARP Poly ADP Ribose Polymerase
  • PARP inhibitors include, but are not limited to, niraparib, rucaparib, olaparib, talazoparib, and veliparib.
  • R 1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
  • R 3a , R 3b , and R 3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
  • X is -CH 2 O-P(O)(OR a )(OR b ), -CH 2 -O-C(O)-Ci-6 alkylene-CO 2 H,
  • R a and R b are each independently H or Ci -6 alkyl; and each heterocycloalkyl has from 4 to 6 ring members and from 1 to 3 heteroatoms as ring vertices independently selected from N, O, and S; or a pharmaceutically acceptable salt thereof.
  • X in Formula (I) or a subembodiment thereof is -CH2O- P(O)(OR a )(OR b ), -CH 2 -O-C(O)-CI-6 alkylene-CChH, or -CH 2 -O-C(O)-CI-6 alkylene- P(O)(OR a )(OR b ).
  • X in Formula (I) or a subembodiment thereof is -CH 2 O- P(O)(OR a )(OR b ) or -CH 2 -O-C(O)-CI- 6 alkylene-CO 2 H.
  • X in Formula (I) or a subembodiment thereof is -CH 2 -O-C(O)-CI-6 alkylene-piperidinyl .
  • X in Formula (I) or a subembodiment thereof is -CH 2 O- P(O)(OR a )(OR b ).
  • X in Formula (I) or a subembodiment thereof is -CH 2 -O-C(O)-CI- 6 alkylene-CO 2 H. In some embodiments, X in Formula (I) or a subembodiment thereof is
  • X in Formula (I) or a subembodiment thereof is
  • X in Formula (I) or a subembodiment thereof is
  • X in Formula (I) or a subembodiment thereof is
  • X in Formula (I) or a subembodiment thereof is
  • R 1 in Formula (I) or a subembodiment thereof is Ci-4 alkyl. In some embodiments, R 1 in Formula (I) or a subembodiment thereof is methyl.
  • R 3a in Formula (I) or a subembodiment thereof is Ci-4 alkoxy, or Ci-4 haloalkoxy. In some embodiments, R 3a in Formula (I) or a subembodiment thereof is methoxy.
  • R 3b in Formula (I) or a subembodiment thereof is Ci-4 alkyl or halo. In some embodiments, R 3b in Formula (I) or a subembodiment thereof is methyl or chloro. In some embodiments, R 3b in Formula (I) or a subembodiment thereof is methyl. In some embodiments, R 3b in Formula (I) or a subembodiment thereof is chloro. In some embodiments, R 3c in Formula (I) or a subembodiment thereof is H or halo. In some embodiments, R 3c in Formula (I) or a subembodiment thereof is H. In some embodiments, R 3c in Formula (I) or a subembodiment thereof is fluoro. In some embodiments, Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is In some embodiments, Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is
  • Z in Formula (I) or a subembodiment thereof is Representative compounds of Formula (I) are listed in Table 1 below.
  • the compound or pharmaceutically acceptable salt thereof is a compound from Table 1.
  • the compounds of Formula (I) are depicted as a (Z) isomer with respect to the double bond between the thiadiazole moiety and the nitrogen in the amide group:
  • the Formula (I) also encompasses the compounds of Formula (la), an (E) isomer:
  • compositions suitable for administration to a subject may be in the form of compositions suitable for administration to a subject.
  • compositions are pharmaceutical compositions comprising a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable or physiologically acceptable excipients.
  • the compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof is present in a therapeutically effective amount.
  • the pharmaceutical compositions may be used in all the methods disclosed herein; thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic methods and uses described herein.
  • compositions can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. Furthermore, the pharmaceutical compositions may be used 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.
  • compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs.
  • Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets, capsules, and the like.
  • excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • compositions typically comprise a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient.
  • suitable pharmaceutically acceptable excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p- hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents, and/or adjuvants.
  • antioxidants e.g., ascorbic acid and sodium bisulfate
  • preservatives e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p- hydroxybenzoate
  • emulsifying agents suspending agents, dispersing
  • a suitable vehicle may be physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.
  • the buffer components can be water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.
  • Acceptable buffering agents include, for example, a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), and N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).
  • HEPES N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)
  • MES 2-(N-Morpholino)ethanesulfonic acid
  • MES 2-(N- Morpholino)ethanesulfonic acid sodium salt
  • MOPS 3-(N-Morpholino)propanes
  • All the compounds and pharmaceutical compositions provided herein can be used in all the methods provided herein.
  • the compounds and pharmaceutical compositions provided herein can be used in all the methods for treatment and/or prevention of all diseases or disorders provided herein.
  • the compounds and pharmaceutical compositions provided herein are for use as a medicament.
  • Compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and compositions containing the same may be administered in any appropriate manner.
  • Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracistemal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal and inhalation.
  • Depot injections which are generally administered subcutaneously or intramuscularly, may also be utilized to administer the compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof over a defined period of time.
  • Particular embodiments of the present invention contemplate oral administration.
  • the compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof provided herein may be administered to a subject in an amount that is dependent upon, for example, the goal of administration (e.g., the degree of resolution desired); the age, weight, sex, and health and physical condition of the subject to which the formulation is being administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof.
  • the dosing regimen may also take into consideration the existence, nature, and extent of any adverse effects associated with the agent(s) being administered. Effective dosage amounts and dosage regimens can readily be determined from, for example, safety and dose-escalation trials, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.
  • dosing parameters dictate that the dosage amount be less than an amount that could be irreversibly toxic to the subject (the maximum tolerated dose (MTD)) and not less than an amount required to produce a measurable effect on the subject.
  • MTD maximum tolerated dose
  • Such amounts are determined by, for example, the pharmacokinetic and pharmacodynamic parameters associated with ADME, taking into consideration the route of administration and other factors.
  • An effective dose is the dose or amount of an agent that produces a therapeutic response or desired effect in some fraction of the subjects taking it.
  • the “median effective dose” or EDso of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered.
  • the ED50 is commonly used as a measure of reasonable expectance of an agent’s effect, it is not necessarily the dose that a clinician might deem appropriate taking into consideration all relevant factors.
  • the effective amount is more than the calculated ED50, in other situations the effective amount is less than the calculated ED50, and in still other situations the effective amount is the same as the calculated EDso.
  • the combination of agents described in this section may display a synergistic effect.
  • the term “synergistic effect” as used herein refers to action of two agents such as, for example, a DNA polymerase theta (Pol0) inhibitor (e.g. a compound of Formula (I) or Formula (II))and a Poly ADP Ribose Polymerase (PARP) inhibitor producing an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves.
  • a synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet.
  • Pole inhibitor e.g. a compound of Formula (I) or Formula (II)
  • PARP inhibitor used together
  • a combination therapy comprising a therapeutically effective amount of a PolO inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibito.
  • a “therapeutically effective amount” of a combination of agents i.e., a Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor is an amount sufficient to provide an observable improvement over the baseline clinically observable signs and symptoms of the disorders treated with the combination. Observable improvements include those that can be visually ascertained by a clinician and biological tests, biopsies, and assays.
  • a Pole inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a PARP inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a PolO inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a PARP inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a combination comprising a PolO inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject.
  • a combination of a PolO inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a PARP inhibitor for the manufacture of a medicament
  • use of a combination of a Pol0 inhibitor e.g. a compound of Formula (I) or Formula (II)
  • a PARP inhibitor for the treatment of cancer is provided.
  • the cancer is characterized as a homologous recombinant (HR) deficient cancer.
  • the Pol0 inhibitor e.g. a compound of Formula (I) or Formula (II)
  • the Pol0 inhibitor is an inhibitor of the ATPase domain of Pol0.
  • the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
  • Pol0 Inhibitors for combination therapy with a PARP inhibitor for combination therapy with a PARP inhibitor
  • Pol0 inhibitors suitable for the combination therapy treatment with PARP inhibitors described in this section are compounds of Formula (II) wherein:
  • R 1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
  • R 3a , R 3b , and R 3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
  • Z is: or a pharmaceutically acceptable salt thereof.
  • R 1 in Formula II and subembodiments thereof is Ci-4 alkyl. In some embodiments, R 1 in Formula (II) and subembodiments thereof is methyl.
  • R 3a in Formula (II) and subembodiments thereof is Ci-4 alkoxy, or Ci-4 haloalkoxy. In some embodiments, R 3a in Formula (II) and subembodiments thereof is methoxy. In some embodiments, R 3b in Formula (II) and subembodiments thereof is Ci-4 alkyl or halo. In some embodiments, R 3b in Formula (II) and subembodiments thereof is methyl or chloro.
  • R 3b in Formula (II) and subembodiments thereof is methyl. In some embodiments, R 3b in Formula (II) and subembodiments thereof is chloro.
  • R 3c in Formula (II) and subembodiments thereof is H or halo. In some embodiments, R 3c in Formula (II) and subembodiments thereof is H. In some embodiments, R 3c in Formula (II) and subembodiments thereof is fluoro.
  • Z in Formula (II) and subembodiments thereof is In some embodiments, Z in Formula (II) and subembodiments thereof is
  • the PolO inhibitor of Formula (II) is Compound 4:
  • the PolO inhibitor of Formula (II) is Compound 5 :
  • the Pol0 inhibitor for combination therapy is ART558 having the structure:
  • the Pol0 inhibitor for combination therapy is ART4215.
  • the combination therapy described herein provides PARP inhibitors for use with a Pol0 inhibitor ⁇ .g. a compound of Formula (I) or Formula (II)).
  • a Pol0 inhibitor ⁇ e.g. a compound of Formula (I) or Formula (II)
  • a number of agents with PARP inhibitory activity and methods of making the same are known in the art. Each of these embraced by this disclosure.
  • the PARP inhibitor is
  • Niraparib also called Compound 11 herein
  • Rucaparib also called Compound 12 herein
  • Olaparib also called Compound 13 herein
  • Veliparib also called Compound 15 herein
  • a pharmaceutically acceptable salt or hydrate thereof a pharmaceutically acceptable salt or hydrate thereof.
  • niraparib The preparation and activity of niraparib are described in US 8,071,579; US 8,071623; US 8,143,241; US 8,426,185; US 8,859,562; and US 11,091,459, the entire contents of which are hereby incorporated by reference in their entirety.
  • the PARP inhibitor is niraparib tosylate monohydrate.
  • the PARP inhibitor is or pharmaceutically acceptable salt or hydrate thereof.
  • Embodiment 1 A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Pol0 inhibitor, or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
  • Embodiment 2 The method of embodiment 1, wherein the Pol0 inhibitor is an inhbitior of ATPase domain of Pol0.
  • Embodiment 3 The method of embodiment 1 or embodiment 2, wherein the Pol0 inhibitor is a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof.
  • Embodiment 4 The method of embodiment 3, wherein the Pol0 inhibitor is the compound of Example 1, having the structure: 5 or a pharmaceutically acceptable salt thereof.
  • Embodiment 5 The method of embodiment 1 or embodiment 2, wherein the Pol0 inhibitor is a compound of Formula (II): wherein:
  • R 1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
  • R 3a , R 3b , and R 3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy; or a pharmaceutically acceptable salt thereof.
  • Embodiment 6 The method of embodiment 5, wherein R 1 is Ci-4 alkyl.
  • Embodiment 7 The method of embodiment 5, wherein R 1 is methyl.
  • Embodiment 8 The method of any one of embodiments 5 to 7, wherein R 3a is Ci-4 alkoxy, or Ci-4 haloalkoxy/
  • Embodiment 9 The method of any one of embodiments 5 to 7, wherein R 3a is methoxy.
  • Embodiment 10 The method of any one of embodiments 5 to 9, wherein R 3b is Ci-4 alkyl or halo.
  • Embodiment 11 The method of any one of embodiments 5 to 9, wherein R 3b is methyl or chloro.
  • Embodiment 12 The method of any one of embodiments 5 to 9, wherein R 3b is methyl.
  • Embodiment 13 The method of any one of embodiments 5 to 9, wherein R 3b is chloro.
  • Embodiment 14 The method of any one of embodiments 5 to 13, wherein R 3c is H or halo.
  • Embodiment 15 The method of any one of embodiments 5 to 13, wherein R 3c is H.
  • Embodiment 16 The method of any one of embodiments 5 to 13, wherein R 3c is fluoro.
  • Embodiment 17 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 18 The method of any one of embodiments 5 to 16 wherein Z is
  • Embodiment 19 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 20 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 21 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 22 The method of any one of embodiments 5 to 16, wherein Z is Embodiment 23. The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 24 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 25 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 26 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 27 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 28 The method of any one of embodiments 5 to 16, wherein Z is
  • Embodiment 29 The method of embodiment 5, wherein the Pol0 inhibitor of Formula
  • Embodiment 30 The method of embodiment 5, wherein the Pol0 inhibitor of Formula
  • Embodiment 31 The method of embodiment 5, wherein the Pol0 inhibitor of Formula
  • Embodiment 32 The method of any one of embodiments 1-31, wherein the PARP inhibitor is
  • Compound 14 Compound 15 or a pharmaceutically acceptable salt or hydrate thereof.
  • Embodiment 33 The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 11 :
  • Embodiment 34 The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 12:
  • Embodiment 35 The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 13:
  • Embodiment 36 The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 14:
  • Embodiment 37 The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 15:
  • Embodiment 38 The method of any one of embodiments 1-31, wherein the PARP inhibitor is: pharmaceutically acceptable salt thereof.
  • Embodiment 39 A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 4 Compound 4 or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
  • Embodiment 40 The method of embodiment 39, wherein the PARP inhibitor is
  • Embodiment 41 The method of embodiment 39, wherein the PARP inhibitor is
  • Embodiment 42 The method of embodiment 39, wherein the PARP inhibitor is
  • Embodiment 43 The method of embodiment 39, wherein the PARP inhibitor is
  • Embodiment 44 The method of embodiment 39, wherein the PARP inhibitor is
  • Embodiment 45 The method of embodiment 39, wherein the PARP inhibitor is: or pharmaceutically acceptable salts thereof.
  • Embodiment 46 The method of any one of embodiments 1-45, wherein the cancer is a homologous recombinant (HR) deficient cancer.
  • HR homologous recombinant
  • Embodiment 47 The method of any one of embodiments 1-46, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
  • Embodiment 48 The method of any of embodiments 1-47, wherein the cancer is a solid tumor.
  • Embodiment 49 The method of any one of embodiments 1-47, wherein the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.
  • Embodiment 50 The method of any one of embodiments 1-49, wherein the PolO inhibitor and the PARP inhibitor are in separate dosage forms.
  • Embodiment 51 The method of any one of embodiments 1-49, wherein the PolO inhibitor and the PARP inhibitor are in the same dosage form.
  • Embodiment 52 A combination comprising a PolO inhibitor of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
  • Embodiment 53 The combination of embodiment 52, wherein the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof.
  • Embodiment 54 The combination of embodiment 52 or 53, wherein the Pol0 inhibitor is Compound 4 or Compound 5, and the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, or compound 15 or a pharmaceutically acceptable salt thereof.
  • Embodiment 55 A Pol0 inhibitor of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, for use in treating cancer, wherein the Pol0 inhibitor is to be administered simultaneously or sequentially with a PARP inhibitor.
  • Embodiment 56 The Pol0 inhibitor of Formula (I) or Formula (II) for use of embodiment 55, wherein the PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof.
  • Embodiment 57 The use of embodiment 56, wherein the Pol0 inhibitor of Formula (II) is Compound 4 or Compound 5.
  • Embodiment 58 The use of embodiment 56, wherein the Pol0 inhibitor of Formula (I) is compound of Example 1.
  • Embodiment 59 Use of a Pol0 inhibitor of Formula (I) or Formula (II) in the manufacture of a medicament for treating cancer, wherein the Pol0 inhibitor is to be administered simultaneously or sequentially with a PARP inhibitor.
  • Embodiment 60 The use of embodiment 59, wherein PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, AZD5305, or AZD9574 or a pharmaceutically acceptable salt thereof.
  • Embodiment 61 The use of embodiment 60, wherein the Pol0 inhibitor is Compound 4, or a pharmaceutically acceptable salt thereof.
  • Embodiment 61 The use of embodiment 60, wherein the Pol0 inhibitor is compound of Example 1, or a pharmaceutically acceptable salt thereof.
  • Pd(dppf)Ch (l,l’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II);
  • Pd(dppf)Ch- DCM (l,l’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II), complex with dichloromethane;
  • Mel methyliodide;
  • TBS-C1 tert-Butyldimethylsilyl chloride ;
  • TBAF Tetrabutylammonium fluoride;
  • DIBAL-H Diisobutylaluminum hydride;
  • LDA Lithium diisopropylamide ..
  • Step-2 methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3 -carboxylate
  • Step-3 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid
  • Step-4 Synthesis of (R)-O-((l,4-dioxan-2-yl)methyl) S-methyl carbonodithioate
  • Step-5 Synthesis of (R)-O-((l,4-dioxan-2-yl)methyl) hydrazinecarbothioate
  • a stirred solution of (R)-O-((l,4-dioxan-2-yl)methyl) S-methyl carbonodithioate (360.0 mg, 1.73 mmol) in Methanol (4 mL) were sequentially added hydrazine hydrate (96.0 mg, 1.90 mmol) at 25 °C.
  • the resulting solution was stirred at 25 °C for 0.5 h.
  • Step-6 Synthesis of (R)-5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-amine
  • Step-7 Synthesis of (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide
  • Step 8 Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate
  • the reaction vessel was charged with solid (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4- thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (40 g, 84 mmol).
  • NMP N-Methyl -2 -pyrrolidone
  • the aqueous layer was removed and the organic layer was washed once more with 10 vol de-ionized water. The mixture was allowed to stir for an additional 10 min followed by 10 min of standing. The organic layer was washed with 10 vol 15% brine for 10 mins and allowed to separate for 10 min. The aqueous layer was removed and the organic layer was collected (no drying agent) and concentrated under vacuum. The crude mixture was purified by silica gel column chromatography (gradient 0-5% MeOH in DCM, 33 min, 330 g column).
  • Step 9 Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate
  • Step-1 2-chloro-3-fluoro-5-methoxypyridine
  • Step-2 2-chloro-3-fluoro-4-iodo-5 -methoxypyridine
  • reaction mixture was quenched by the addition of saturated sodium thiosulfate aqueous solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum.
  • Step-3 methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate
  • Step-4 methyl 3 '-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3 -carboxylate
  • Step-5 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid
  • Step-6 methyl ( 1 r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane- 1 -carboxylate
  • Step-7 ((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol
  • Step-8 O-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate
  • Step-9 O-((( 1 r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) hydrazinecarbothioate
  • Step-10 5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4-thiadiazol-2- amine
  • Step-11 N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4- thiadiazol-2-yl)-3'-fhioro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide
  • To a solution of 3 '-fhioro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3 -carboxylic acid (160.0 mg, 0.57 mmol, Current Example, Step 5) in dry Acetonitrile (4 mL) were added 5-(((lr,4r)- 4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)- 1 ,3,4-thiadiazol-2-amine (199.0 mg, 0.58 mmol, Current Example, Step 10), 1 -methyl- 1
  • Step-12 3'-fhioro-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'- methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide
  • Step 13 Synthesis of di-tert-butyl (((Z)-2-((3'-fhroro-5'-methoxy-2',6-dimethyl-[4,4'- bipyridine]-3-carbonyl)imino)-5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol- 3(2H)-yl)methyl) phosphate
  • reaction vessel was sealed and the reaction mixture was heated to 40 °C under nitrogen. After 24 h, the reaction mixture was quenched with ice-water (30 mL) and the aqueous mixture was extracted with EtOAc (3 x 20 mL). The combined organics were washed with water (20 mL) and brine (20 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure.
  • Step 14 Synthesis of ((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3- carbonyl)imino)-5 -((( 1 r,4r)-4-hydroxycyclohexyl)methoxy)- 1 ,3 ,4-thiadiazol-3 (2H)-yl)methyl dihydrogen phosphate
  • Step 1 Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate
  • reaction mixture was heated to 50 °C in a sealed vial with stirring under nitrogen. After 3 h, an additional portion of tert-butyl (chloromethyl) succinate (262 mg, 1.18 mmol) was added. After a total of 16 h the reaction temperature was increased to 60 °C and after a further 6 h, additional portions of K2CO3 (217 mg, 1.57 mmol) and tertbutyl (chloromethyl) succinate (262 mg, 1.18 mmol) were added and the reaction mixture was cooled back to 50 °C. After an additional 18 h, the reaction mixture was quenched with water (30 m ) and brine (15 mb) and the aqueous mixture was extracted with DCM (3 x 20 mb).
  • Step 2 Synthesis of (R,Z)-4-((5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6- methyl-[4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methoxy)-4- oxobutanoic acid
  • Compound 5 was prepared as described in Example 2, Steps 1 to 12.
  • Step-1 2-bromo-4-iodo-5 -methoxypyridine
  • Step-2 methyl 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate
  • Step-3 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid
  • Step 4 (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6- methyl-(4,4'-bipyridine)-3-carboxamide
  • Step-1 2-chloro-3-fluoro-5-methoxypyridine
  • Step-2 2-chloro-3-fluoro-4-iodo-5 -methoxypyridine
  • reaction mixture was quenched by the addition of saturated sodium thiosulfate aqueous solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum.
  • Step-4 2'-chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid
  • Step 5 N-(5-(((R)-l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide
  • Step-1 2'-bromo-N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)- 1,3,4- thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide:
  • Step-2 2'-bromo-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide:
  • Step-1 Synthesis of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-l -carboxylate.
  • Step-2 Synthesis of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol.
  • Step-3 Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl) S-methyl carbonodithioate .
  • Step-4 Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazine carbothioate.
  • Step-5 Synthesis of rac-5-(((lS,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)- l,3,4-thiadiazol-2-amine and rac-5-(((lR,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl) methoxy)- 1 ,3,4-thiadiazol-2-amine .
  • Step-6 Synthesis of (lS,2R)-2-(((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol and ( 1 R,2S)-2-(((5 -amino- 1 ,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan- 1 -ol
  • Step-10 Synthesis of 2'-chloro-N-(5-(((lS,2R)-2-hydroxycyclopentyl)methoxy)- 1,3,4- thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (Compound 9)
  • the reaction mixture was stirred at room temperature for 12 h.
  • the reaction mixture was quenched with water (100 mL) and extracted with Ethyl acetate (50 mL x 2).
  • the organic phases were combined and washed with brine solution.
  • the organic layer was dried over anhydrous Na2SC>4 and fdtered.
  • the fdtrate was concentrated under reduced pressure to get the crude product as an off-white solid.
  • the crude product was mixed with another batch of 180 mg material.
  • the combined crude was pre-absorbed on silica using 20 mL DCM and 5g of silica (60-120 mesh), loaded on the pre-packed biotage 45g column and eluted at 10% of methanol in dichloromethane for 60 min with flow rate 30 mL/min.
  • Step- 1 5 -((tetrahydrofuran-3-yl)methoxy)- 1 ,3 ,4-thiadiazol-2 -amine
  • Step-2 Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)- l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide
  • Step-3 Separatino of (R)-2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3- yl)methoxy)-l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide
  • Pol Theta ATPase activity The ability of the compounds of Formula (II) to inhibit ATPase activity of Pol theta (1-899) was determined using the assay described below.
  • Pol Theta ATPase activity was determined by measuring the rate of ATP turn over in a NADH oxidation-coupled enzymatic assay. 10-point dilution series of compounds were used in a 384 well format for the inhibition assays.
  • Pol theta (1-899) (10 nM) in assay buffer (20 mM Tris HC1 (pH 7.80), 80 mM KC1, 10 mM MgCh, 1 mM DTT, 0.01% BSA, 0.01% Tween, 5% glycerol) was transferred to the test wells (20 pL), except the low control wells (20 pL of assay buffer was added to the low control wells). The plate was then incubated at room temperature for 15 min.
  • the high control (DMSO with enzyme) with low absorbance intensity represents no inhibition of ATPase reaction while the low control (DMSO with buffer) with high absorbance intensity represents full inhibition of ATPase activity.
  • Slope of the reaction progress curves were used to calculate the rate of ATP hydrolysis. The rates were used to determine the percent inhibition using a four-parameter inhibition model to generate ICso, Hill slope and max inhibition.
  • the IC50 of the compounds 4-10 are disclosed in Table 2 below:
  • IC50 10 uM > (+) > 1 uM ; 1 uM > (++) > 500 nM;
  • DLD-1 BRCA2-/- cells (Horizon Discovery) were plated in 200 pl growth medium at 500 cells/well in 96-well flat-bottomed plates. After an overnight incubation at 37°C, 5% CO2, compounds were added to cells across a concentration range. Cells were incubated for a further 7 days at 37°C, 5% CO2, to allow for 4-5 population doublings. Cells were then fixed with 4% paraformaldehyde and stained with Hoechst to allow cells to be imaged on the Incell 2200 reader. The cell count data were normalised to control wells containing DMSO (high control) and 100 pM doxorubicin (low control) before analysis using a 4-parameter logistic curve for calculation of IC50 and pICso.
  • pro-drugs are converted by endogenous alkaline phosphatases or esterases present in the assay to give the free parent compounds of equivalent potency to dosing parent compound alone (see Table 3).
  • the solubility of Compound A and Compound B in FASSIF are 17 ug/mL and 37 ug/mL, respectively.
  • the FASSIF solubility for the prodrugs is greater than 1 mg/mL.
  • Combination Therapy Assessing Combination Synergy Index with Compounds of Formula (II) and a PARP inhibitor
  • a 15 -day colony formation assay was performed in BRCA1 mutant MDA-MB-436 cell line. Combinations included double titrations of seven different PolQ inhibitor compounds (Compound 4, 5, 6, 7,8, 9 and 10) with the PARP inhibitor, Niraparib.
  • Optimal cell seeding was determined by assessing the growth of colonies over a range of seeding densities in a 6-well format to identify conditions that permitted growth for 15 days. Cells were then plated at the optimal seeding density (1000 cells per well) and treated with a double titration of a 9-point three-fold dilution series of the PolQ inhibitor compounds and a 3-point three-fold dilution series of Niraparib. This double titration was compared to 9-point 3 -fold dilution series of the PolQ inhibitor compounds single agent or a 3-point three-fold dilution series of Niraparib single agent alone or to 0.1% DMSO.
  • Concentrations tested for the PolQ inhibitors alone or in combination ranged from 4.6 nM to 30,000 nM and Niraparib alone or in combination ranged from 0.56 nM to 5 nM. Plates were incubated for 15 days at 37 °C in 5% CO2. Media containing compounds were replenished at 8 days of the treatment. After 15 days of treatment, cells were fixed with 95% ethanol solution and stained with 0.25% (w/v) crystal violet staining solution (Sinopharm Chemical Reagent Beijing Co., Ltd). The plates were washed with PBS and scanned on a LI-COR Odyssey CLx imager (LI-COR) using the 700 nm channel.
  • LI-COR LI-COR Odyssey CLx imager
  • COMPOUND A and COMPOUND B were tested as single agents in the BRCA1 mutant breast cancer line MDA-MB-436 and in the in BRCA2 mutant ovarian cancer cell line PEO1; single agents ICso values were above 0.5 pM which is indicative of poor efficacy.
  • CCG CellTiter-Glo®
  • MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution ranging from 30 pM to 0.014 pM COMPOUND A, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib. After 7 days, cell viability was assessed with the CTG assay. Dose response curves were interpolated using GraphPad Prism 9, and the synergy of drug combinations using data from the cell viability assays was analyzed with ComBenefit 2.02. Niraparib synergized with COMPOUND A and decreased ECso values of COMPOUND A in MDA-MB-436 cells (FIG. 8A-8D and Table 12).
  • MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution ranging from 30 pM to 0.014 pM COMPOUND B, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib.
  • Cell viability was assessed as described above.
  • In vitro synergy of COMPOUND B and Niraparib reduces cell viability in MDA- MB-436 breast cancer cells.
  • PEO1 cells were treated with an 8x5 drug matrix, with an 8-point, 3 -fold dilution ranging from 30 pM to 0.014 pM COMPOUND A, and a 5-point, 3-fold dilution ranging from 5 pM to 0.062 pM Niraparib.
  • Cell viability was assessed as described above.
  • Niraparib synergized with COMPOUND A and decreased ECso values of COMPOUND A in PEO1 cells (FIG. 10A-10D and Table 14).
  • PEO1 cells were treated with an 8x5 drug matrix, with an 8-point, 3 -fold dilution ranging from 30 pM to 0.014 pM COMPOUND B, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib.
  • Cell viability was assessed as described above.
  • Niraparib synergized COMPOUND B and decreased ECso values of COMPOUND B in PEO1 cells (FIG. 11A- 11D and Table 15).
  • Compound 4 was examined in the MDA-MB-436 efficacy model for tumor growth inhibition and durability of efficacy when administered as monotherapy and in combination with niraparib (Compound 11).
  • Compound 4 was found to significantly inhibit tumor growth as a monotherapy and in combination with niraparib. Following drug administration for up to 78 days, only the combination treatments resulted in continuous stable disease or tumor regressions, as tumors became resistant to niraparib monotherapy on treatment. In combination with niraparib, the combination treatment improved the durability of response by preventing tumor growth and enhanced the number of complete responses observed.
  • the combination of Compound 4 and niraparib administered at 10, 30 or 100 mg/kg BID produced complete responses (no remaining tumor) in 20, 30, and 50% of mice, respectively.
  • Tumor growth inhibition (TGI) was calculated by [(TV controlfinal - TV treatedfinai)/(TV controlfinal - TV controlinitial) x 100], TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from study day 30 and were considered statistically significant if less than 0.05. A Mixed-effects model with Tukey’s Multiple Comparisons was utilized for day 78 and results were considered statistically significant if less than 0.05.
  • Mean tumor volume at dosing start was approximately 187 to 193 mm 3 , with ten mice randomized to each treatment group.
  • the study consisted of eight treatment groups. Mice were dosed orally, twice per day (BID), with Vehicle A or Compound 4 at 10, 30, or 100 mg/kg, or dosed once per day (QD) with Vehicle B or Compound 11 at 25 mg/kg, or the combination of Compound 4 at 10, 30, or 100 mg/kg BID and Compound 11 at 25 mg/kg QD.
  • the control groups consisted of Vehicle A (for Compound 4, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound 11, 0.5% 400 cps methylcellulose in sterile water). Compound 4 was administered first in the morning, followed two hours later with Compound 11, and the second dose of Compound 4 was provided 6-hours following the dose of Compound 11.
  • the study examined the efficacy of Compound 4 or Compound 11 as monotherapies as well as the efficacy of the combination of Compound 4 and Compound 11 as combination antitumor therapies. Furthermore, the study examined the durability of the treatment response and the clinical outcome of the treatment. The efficacy of each treatment group was compared to the vehicle control group alone while the durability of the treatment response for the combination groups were compared to Compound 11 alone.
  • the vehicle control group reached endpoint tumor volume on study day 30.
  • the treatments groups on day 30 were compared to the control group on day 30 to calculate the tumor growth inhibition (TGI), Table 16.
  • TGI tumor growth inhibition
  • each treatment group produced statistically significant TGI on day 30.
  • Compound 4 at 100 mg/kg BID combined with Compound 11 improved the TGI observed over that of Compound 11 alone.
  • Tumor volume endpoint for individual treatment groups was defined as 50% of mice within the treatment group harbor tumors greater than 2000 mm 3 .
  • Compound 4 administered at 10 and 30 mg/kg BID reached endpoint on Day 40 while Compound 4 administered at 100 mg/kg BID reached endpoint on Day 50.
  • Compound 11 administered at 25 mg/kg demonstrated tumor growth prevention beginning on study day 16 and subsequently resulted in shrinking tumors until study day 34.
  • treatment with Compound 11 alone was no longer effective and tumors from each mouse grew on treatment, FIG. 13A.
  • On day 75, one mouse from Compound 11 treatment was euthanized for reaching endpoint tumor volume.
  • compound 11 administered at 25 mg/kg QD resulted in a mean tumor volume of 1313 mm 3 and contained no mice with tumors that were responding to treatment.
  • the combination of Compound 11 and Compound 4 administered at 10, 30 or 100 mg/kg BID produced mean tumor volumes of 145, 82, and 63 mm 3 , respectively.
  • each combination group enhanced the response rate over Compound 11 alone.
  • the combination of Compound 11 and Compound 4 administered at 10, 30 or 100 mg/kg BID produced complete responses (no remaining tumor) in 20, 30, and 50% of mice, respectively, Table 17.
  • the combination of Compound 4 and Compound 11 produced a durable anti-tumor response and was significantly more effective than Compound 11 alone.
  • MDA-MB-436 tumor bearing animals were either dosed twice daily with vehicle 0.5% Methylcellulose with 0.5% Tween, BID, PO or Compound A 100 mg/kg BID, PO or once daily Niraparib 25 mg/kg QD, PO or Compound A lOOmg/kg in combination with Niraparib 25 mg/kg QD for 70 days.
  • TGI tumor growth inhibition
  • 134-T tumor fragments (4x4 mm) were implanted into flank of 5-7 weeks old NOD SCID gamma (NSG) mice.
  • NSG NOD SCID gamma
  • tumor bearing tumors of the HR- deficient 134-T ovarian cancer PDX model were treated with Niraparib (25mg/kg QD), 100 mg/kg Compound A alone BID or a combination of 30mg/kg BID or 100 mg/kg BID Compound A with Niraparib 25mg/kg (FIG. 15). While Compound A alone did not yield to significant tumor growth inhibition (18% TGI), Niraparib at 25 mg/kg resulted in 60% tumor growth inhibition. After 35 days of dosing, 82% tumor growth inhibition was observed with 30mg/kg BID dosing of Compound A and 90% tumor growth inhibition with lOOmg/kg BID dosing similar to the MDA-MB-436 model.
  • the efficacy of Compound of Example 1 in combination with Niraparib 25 mg/kg was also tested in 134-T PDX model.
  • tumor bearing tumors of the HR-deficient 134-T ovarian cancer PDX model were treated with Niraparib (25 mg/kg QD) alone or Niraparib (25 mg/kg QD) in combination with Example 1 at doses of 30 mg/kg BID, 100 mg/kg QD, or 100 mg/kg BID (FIG. 16).
  • Niraparib at 25 mg/kg resulted in 60% tumor growth inhibition.

Abstract

Disclosed herein are compounds of Formula (I): (I) that inhibit DNA Polymerase Theta (Polθ) activity, in particular inhibit Polθ activity by inhibiting ATP dependent helicase domain activity of Polθ. Also, disclosed are pharmaceutical compositions comprising such compounds and methods of treating and/or preventing diseases treatable by inhibition of Polθ such as cancer, including homologous recombination (HR) deficient cancers. Further disclosed herein are methods of using compounds of Formula (I) or Formula (II) that inhibit DNA Polymerase Theta (Polθ) activity in combination with a Poly ADP Ribose Polymerase (PARP) inhibitor. (II)

Description

THIADIAZOL YL DERIVATIVES
AS DNA POLYMERASE THETA INHIBITORS AND USES THEREOF
BACKGROUND OF THE INVENTION
Targeting DNA repair deficiencies has become a proven and effective strategy in cancer treatment. However, DNA repair deficient cancers often become dependent on backup DNA repair pathways, which present an “Achilles heel” that can be targeted to eliminate cancer cells, and is the basis of synthetic lethality. Synthetic lethality is exemplified by the success of poly (ADP-ribose) polymerase (PARP) inhibitors in treating BRCA-deficient breast and ovarian cancers (Audeh M. W., et al., Lancet (2010); 376 (9737): 245-51).
DNA damage repair processes are critical for genome maintenance and stability, among which, double strand breaks (DSBs) are predominantly repaired by the nonhomologous end joining (NHEJ) pathway in G1 phase of the cell cycle and by homologous recombination (HR) in S-G2 phases. A less addressed alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ) pathway, is commonly considered as a “backup” DSB repair pathway when NHEJ or HR are compromised. Numerous genetic studies have highlighted a role for DNA polymerase theta (Pol0, encoded by POLQ) in stimulating MMEJ in higher organisms (Chan S. H., et al., PLoS Genet. (2010); 6: el001005; Roerink S. F., et al., Genome research. (2014); 24: 954-962; Ceccaldi R., et. al., Nature (2015); 518: 258-62; and Mateos-Gomez P. A., et al., Nature (2015); 518: 254-57).
Pole is distinct among human DNA polymerases, exhibiting not only a C-terminal DNA polymerase domain but also an N-terminal helicase domain separated by a long and lesser- conserved central domain of unknown function beyond Rad51 binding (Seki eta. Al, 2003, Shima et al 2003; Yousefzadeh and Wood 2013). The N-terminal ATPase/helicase domain belongs to the HELQ class of SF2 helicase super family. In homologous recombination deficient (HRD) cells, Pol0 can carry out error-prone DNA synthesis at DNA damage sites through alt-EJ pathway. It has been shown that the helicase domain of Pol0 causes suppression of HR pathway through disruption of Rad51 nucleoprotein complex formation involved in initiation of the HR-dependent DNA repair reactions following ionizing radiation. This anti-recombinase activity of Pol0 promotes the alt-EJ pathway. In addition, the helicase domain of Pol0 contributes to microhomology-mediated strand annealing (Chan SH et al., PLoS Genet. (2010); 6: el001005; and Kawamura K et al., Int. J. Cancer (2004); 109: 9-16). Pole efficiently promotes end-joining in alt-EJ pathway by employing this annealing activity when ssDNA overhangs contain >2 bp of microhomology (Kent T., et al., Elife (2016); 5: el3740), and Kent T., et al., Nat. Struct. Mol. Biol. (2015); 22: 230-237). This reannealing activity is achieved through coupled actions of Rad51 interaction followed by ATPase- mediated displacement of Rad51 from DSB damage sites. Once annealed, the primer strand of DNA can be extended by the polymerase domain of PolO.
The expression of PolO is largely absent in normal cells but upregulated in breast, lung, and ovarian cancers (Ceccaldi R., et al., Nature (2015); 518, 258-62). Additionally, the increase of PolO expression correlates with poor prognosis in breast cancer (Lemee F et al ., Proc Nad Acad Sci USA. (2010) ; 107: 13390-5). It has been shown that cancer cells with deficiency in HR, NHEJ or ATM are highly dependent on PolO expression (Ceccaldi R., et al., Nature (2015); 518: 258-62, Mateos-Gomez PA et al., Nature (2015); 518: 254-57, and Wyatt D.W., et al., Mol. Cell (2016); 63: 662-73). Therefore, PolO is an attractive target for novel synthetic lethal therapy in cancers containing DNA repair defects.
SUMMARY OF THE INVENTION
Disclosed herein are certain thiadiazolyl derivatives that inhibit PolO activity, in particular inhibit PolO activity by inhibiting the ATP dependent helicase domain activity of PolO. Also, disclosed are pharmaceutical compositions comprising such compounds and methods of treating and/or preventing diseases treatable by inhibition of PolO such as cancer, including homologous recombination (HR) deficient cancers.
In one aspect, provided is a compound of Formula (I), or a pharmaceutically acceptable salt thereof:
Figure imgf000003_0001
wherein Z, X, R1, R3a, R3b, and R3c have the meanings provided herein below.
In related aspects, provided are pharmaceutical compositions comprising a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In another aspect, provided is a method for treating and/or preventing a disease characterized by overexpression of PolO in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or an embodiment thereof disclosed herein). In one embodiment, the patient is in recognized need of such treatment. In another embodiment, the compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition. In yet another embodiment, the disease is a cancer.
In still another aspect, provided is a method for treating and/or preventing a homologous recombinant (HR) deficient cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof (or an embodiment thereof disclosed herein). In one embodiment, the patient is in recognized need of such treatment. In another embodiment, the compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition.
In another aspect, provided is a method for inhibiting DNA repair by Pol0 in a cancer cell comprising contacting the cell with an effective amount of a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof. In one embodiment, the cancer is HR deficient cancer.
In yet another aspect, provided is a method for treating and/or prevening a cancer in a patient, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutical composition.
In still another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for inhibiting DNA repair by Pol0 in a cell. In one embodiment, the cell is HR deficient cell.
In another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a disease in a patient, wherein the disease is characterized by overexpression of PolO.
In yet another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a cancer in a patient, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
In still another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a HR deficient cancer in a patient.
In another aspect, provided is a compound of Formula (I) or Table 1 (or an embodiment thereof disclosed herein), or a pharmaceutically acceptable salt thereof for use in the treatment and/or prevention of a cancer that is resistant to poly(ADP-ribose) polymerase (PARP) inhibitor therapy in a patient. Examples of cancers resistant to P ARP -inhibitors include, but are not limited to, breast cancer, ovarian cancer, lung cancer, bladder cancer, liver cancer, head and neck cancer, pancreatic cancer, gastrointestinal cancer, and colorectal cancer.
In related aspects for the methods, uses and compositions above, the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer (data from large scale drop out screens in cancer cell lines indicate that some cell lines from the above cancers are dependent on polymerase theta for proliferation https ://depmap .org/portal/) .
In some embodiments, a HR-deficient cancer is breast cancer. Breast cancer includes, but is not limited to, lobular carcinoma in situ (LCIS), a ductal carcinoma in situ (DCIS), an invasive ductal carcinoma (IDC), inflammatory breast cancer, Paget disease of the nipple, Phyllodes tumor, Angiosarcoma, adenoid cystic carcinoma, low- grade adenosquamous carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, tubular carcinoma, metaplastic carcinoma, micropapillary carcinoma, mixed carcinoma, or another breast cancer, including but not limited to triple negative, HER positive, estrogen receptor positive, progesterone receptor positive, HER and estrogen receptor positive, HER and progesterone receptor positive, estrogen and progesterone receptor positive, and HER and estrogen and progesterone receptor positive. In other embodiments, HR-deficient cancer is ovarian cancer. Ovarian cancer includes, but is not limited to, epithelial ovarian carcinomas (EOC), maturing teratomas, dysgerminomas, endodermal sinus tumors, granulosa-theca tumors, Sertoli-Leydig cell tumors, and primary peritoneal arcinoma.
Also provided herein is combination therapy comprising methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a DNA polymerase theta (Pol0) inhibitor (e.g. a compound of Formula (I) or Formula (II)) and administering to the subject a therapeutically effective amount of a Poly ADP Ribose Polymerase (PARP) inhibitor, thereby treating the cancer in the subject.
In another aspect, provided is a method for treating and/or preventing a homologous recombinant (HR) deficient cancer in a patient in need thereof comprising contacting the cancer cells in the patient with an effective amount of a Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II)) and a Poly ADP Ribose Polymerase (PARP) inhibitor. An exemplary Pol0 polymerase domain inhibitor other than those defined by Formula (I) or Formula (II) is known as ART4215 and is developed by Artios Pharma and now in Phase l/2a clinical trials. See “A Study of ART4215 for the Treatment of Advanced or Metastatic Solid Tumors,” NCT04991480 at clinicaltrials.gov. Other Pol0 polymerase domain inhibitors, including ART558, are also reported. See Zatreanu D., et al. “Pol0 inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance,” NATURE COMMUNICATIONS, 2021. 12(1):3636.
Formula (II) has the structure
Figure imgf000006_0001
wherein Z, R1, R3a, R3b, and R3c have the meanings provided hereinbelow.
In some aspects, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a combination comprising a DNA polymerase theta (PolO) (e.g. a compound of Formula (I) or Formula (II)) and a Poly ADP Ribose Polymerase (PARP) inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject.
A compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy. A combination of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and a Poly ADP Ribose Polymerase (PARP) inhibitor, for use in therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1B depict excess over Bliss synergy of Compound 4 (also called Compound A) and Niraparib combinations in MDA-MB-436 cell line.
FIG. 2A-2B depict excess over Bliss synergy of Compound 5 (also called Compound B) and Niraparib inhibitor combinations in MDA-MB-436 cell line.
FIG. 3 depicts excess over Bliss synergy of Compound 6 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
FIG. 4 depicts excess over Bliss synergy of Compound 7 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
FIG. 5 depicts excess over Bliss synergy of Compound 8 and Niraparib inhibitor combinations in MDA-MB-436.
FIG. 6 depicts excess over Bliss synergy of Compound 9 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
FIG. 7 depicts excess over Bliss synergy of Compound 10 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
FIG. 8A, 8B, 8C, and 8D show the in vitro efficacy of Compound A and Niraparib in combination in MDA-MB-436 breast cancer cells.
FIG. 9A, 9B, 9C, and 9D show the in vitro efficacy of Compound B and Niraparib in combination in MDA-MB-436 breast cancer cells.
FIG. 10A, 10B, IOC, and 10D show the in vitro efficacy of Compound A and Niraparib in combination in PEO1 ovarian cancer cells. FIG. 11A, 11B, 11C, and 11D show the in vitro efficacy of Compound B and Niraparib in combination in PEO1 ovarian cancer cells.
FIG. 12 shows the efficacy of Compound 4 and Compound 11 in MDA-MB-436 mouse model.
FIG. 13A, 13B, 13C, and 13D show individual tumor growth curves for Compound 11 and for Compound 11 combinations with Compound 4.
FIG. 14 displays an efficacy study in BRCA1 mutant MDA-MB-436 model. The dotted line represents the mean starting tumor volume. One-way Anova test was applied to calculate statistics, *p<0.05.
FIG. 15 displays an efficacy study in BRCA1 mutant 134T Ovarian PDX model. One-way Anova test was applied to calculate statistics, *p<0.05.
FIG. 16 displays an efficacy study with Example 1 in BRCA1 mutant 134T Ovarian PDX model. One-way Anova test was applied to calculate statistics, *p<0.05.
DETAILED DESCRIPTION
Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The singular forms “a,” “an,” and “the” as used herein and in the appended claims include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology such as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
When needed, any definition herein may be used in combination with any other definition to describe a composite structural group. By convention, the trailing element of any such definition is that which attaches to the parent moiety. For example, the composite group alkoxyalkyl means that an alkoxy group is attached to the parent molecule through an alkyl group.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
DEFINITIONS:
Unless otherwise stated, the following terms used in the specification and claims are defined for the purposes of this Application and have the following meaning:
The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a saturated straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e. Ci-8 means one to eight carbons). Alkyl can include any number of carbons, such as Ci-2, Ci-3, Ci-4, C1-5, C1-6, C1-7, C1-8, C1-9, C1-10, C2-3, C2-4, C2-5, C2-6, C3-4, C3-5, C3-6, C4-5, C4-6 and C5-6. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
The term "alkylene" refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For instance, a straight chain alkylene can be the bivalent radical of -(CH2)n- where n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, hexylene, and the like.
The term "alkoxy" refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O-. As for an alkyl group, alkoxy groups can have any suitable number of carbon atoms, such as C1-6, and can be straight or branced. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc.
The term "heterocycloalkyl" refers to a saturated or partially unsatured monocyclic ring having the indicated number of ring vertices (e.g., a 3- to 7-membered ring) and having from one to five heteroatoms selected from N, O, and S as ring vertices. Partially unsaturated heterocycloalkyl groups have one or more double or triple bonds in the ring, but heterocycloalkyl group are not aromatic. Heterocycloalkyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 7, 4 to 7, or 5 to 7 ring members. Any suitable number of heteroatoms can be included in the heterocycloalkyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. A heterocycloalkyl group can be attached to the remainder of the molecule through a ring carbon or a heteroatom.
The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
The term "haloalkyl" refers to alkyl, as defined above, where some or all of the hydrogen atoms are replaced with halogen atoms. As for alkyl group, haloalkyl groups can have any suitable number of carbon atoms, such as Ci-6. For example, the term "Ci-4 haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like.
The term "haloalkoxy" refers to an alkoxy group where some or all of the hydrogen atoms are substituted with halogen atoms. As for an alkyl group, haloalkoxy groups can have any suitable number of carbon atoms, such as Ci-6, and can be straight or branced, and are substituted with 1, 2, 3, or more halogens. When all the hydrogens are replaced with a halogen, for example by fluorine, the compounds are per-substituted, for example, perfluorinated. Haloalkoxy includes, but is not limited to, trifluoromethoxy, 2,2,2, -trifluoroethoxy, perfluoroethoxy, etc.
As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S). The term "pharmaceutically acceptable salts" is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, 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. Examples of 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- occuring amines and the like, such as arginine, betaine, caffeine, choline, N,N’- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 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. When compounds of the present invention contain relatively basic functionalities, 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. Examples of pharmaceutically acceptable acid addition salts 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, benzene sulfonic, p-tolylsulfonic, citric, tartaric, methane sulfonic, and the like. Also included are 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.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the parent compounds. Additionally, prodrugs can be converted to the parent compounds by chemical or biochemical methods in an ex vivo environment.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical depiction is shown, 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%.
The compounds of the present invention 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 isotope in question. For example, the compounds may incorporate radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C), or non-radioactive isotopes, such as deuterium (2H) or carbon-13 (13C). Such isotopic variations can provide additional utilities to those described elsewhere within this application. For instance, 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. Additionally, isotopic variants of the compounds of the invention can have altered pharmacokinetic and pharmacodynamic characteristics which can contribute to enhanced safety, tolerability or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
The terms "patient" or "subject" are used interchangeably to refer to a human or a non-human animal (e.g., a mammal). In one embodiment, the patient is human.
The terms "administration," "administer" and the like, as they apply to, for example, a subject, cell, tissue, organ, or biological fluid, refer to contact of, for example, an Pol0 modulator, a pharmaceutical composition comprising same, or a diagnostic agent to the subject, cell, tissue, organ, or biological fluid. In the context of a cell, 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.
The terms "treat," "treating," "treatment" and the like refer to a course of action (such as administering a Pol0 modulator 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 subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject. Thus, 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 subject 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. For example, the patient has been diagonosed as having a disease linked to overexpression of Pol0 or a homologous recombination (HR)-deficient cancer.
The phrase "therapeutically effective amount" refers to the administration of an agent to a subject, 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 subject. 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 subject’s condition, and the like. By way of example, measurement of the serum level of an PolO modulator (or, e.g., a metabolite thereof) at a particular time post-administration may be indicative of whether a therapeutically effective amount has been used.
The terms "inhibitors" and "activators" refer to inhibitory or activating molecules, respectively, for example, for the activation of, e.g., a ligand, receptor, cofactor, gene, cell, tissue, or organ. Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, or cell. Activators are molecules that increase, activate, facilitate, enhance activation, sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity.
The terms "modulate," "modulation" and the like refer to the ability of a molecule (e.g., an activator or an inhibitor) to increase or decrease the function or activity of Pol0, either directly or indirectly. A modulator may act alone, or it may use a cofactor, e.g., a protein, metal ion, or small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules.
The "activity" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor; to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity; to the modulation of activities of other molecules; and the like. The term “proliferative activity” encompasses an activity that promotes, that is necessary for, or that is specifically associated with, for example, normal cell division, as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.
"Pharmaceutically acceptable carrier or excipient" means a carrier or an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier or an excipient that is acceptable for veterinary use as well as human pharmaceutical use. “A pharmaceutically acceptable carrier/excipient” as used in the specification and claims includes both one and more than one such excipient.
As used herein, a wavy line, ""w ", that intersects a single, double or triple bond in any chemical structure depicted herein, represent the point attachment of the single, double, or triple bond to the remainder of the molecule. Additionally, a bond extending to the center of a ring (e.g., a phenyl ring) is meant to indicate attachment at any of the available ring vertices. One of skill in the art will understand that multiple substituents shown as being attached to a ring will occupy ring vertices that provide stable compounds and are otherwise sterically compatible.
"About, " as used herein, is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term “about” should be understood to mean that range which would encompass ± 10%, preferably ± 5%, the recited value and the range is included.
"Disease" as used herein is intended to be generally synonymous, and is used interchangeably with, the terms "disorder, " "syndrome, " and "condition" (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
"Inhibiting", "reducing," or any variation of these terms in relation of Pol0, includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of about, at most about, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of Pol0 activity compared to its normal activity.
The term "homologous recombination" refers to the cellular process of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA.
The term "homologous recombination (HR) deficient 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 of one or more HR-assocated genes or presence of one or more mutations in one or more HR-assocated genes. Examples of HR-associated genes include BRCA1, BRCA2, RAD54, RAD51B, CtlP (Choline Transporter-Like 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 (Werner 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, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANJ (BRIP1), FANCL, FANCM, FANCN (RALB2), FANCP (SLX4), FANCS (BRCA1), RAD51C, and XPF.
The term "Pol0 overexpression" refers to the increased expression or activity of Pol0 in a diseased cell e.g., cancerous cell, relative to expression or activity of Pol0 in a normal cell (e.g., non-diseased cell of the same kind). The amount of Pol0 can be at least 2-fold, at least 3 -fold, at least 4- fold, at least 5- fold, at least 10-fold, or more relative to the Pol0 expression in a normal cell. Examples of Pol0 cancers include, but are not limited to, breast, ovarian, cervical, lung, colorectal, gastric, bladder and prostate cancers.
As used herein, “Poly ADP Ribose Polymerase (PARP) inhibitor” refers to an agent that inhibits PARP activity, including PARP1 and PARP2. Examples of PARP inhibitors include, but are not limited to, niraparib, rucaparib, olaparib, talazoparib, and veliparib.
COMPOUNDS:
In some aspects, provided herein is a compound of Formula (I):
Figure imgf000016_0001
wherein:
R1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
R3a, R3b, and R3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
Z is:
Figure imgf000016_0002
X is -CH2O-P(O)(ORa)(ORb), -CH2-O-C(O)-Ci-6 alkylene-CO2H,
-CH2-O-C(O)-CI-6 alkylene-O-P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-
P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-NRaRb, or — CH2— O~ C(O)— Ci-6 alkylene-heterocycloalkyl;
Ra and Rb are each independently H or Ci -6 alkyl; and each heterocycloalkyl has from 4 to 6 ring members and from 1 to 3 heteroatoms as ring vertices independently selected from N, O, and S; or a pharmaceutically acceptable salt thereof.
In some embodiments, X in Formula (I) or a subembodiment thereof is -CH2O- P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-CChH, or -CH2-O-C(O)-CI-6 alkylene- P(O)(ORa)(ORb).
In some embodiments, X in Formula (I) or a subembodiment thereof is -CH2O- P(O)(ORa)(ORb) or -CH2-O-C(O)-CI-6 alkylene-CO2H.
In some embodiments, X in Formula (I) or a subembodiment thereof is -CH2-O-C(O)-CI-6 alkylene-piperidinyl .
In some embodiments, X in Formula (I) or a subembodiment thereof is -CH2O- P(O)(ORa)(ORb).
In some embodiments, X in Formula (I) or a subembodiment thereof is -CH2-O-C(O)-CI-6 alkylene-CO2H. In some embodiments, X in Formula (I) or a subembodiment thereof is
Figure imgf000017_0001
In some embodiments, X in Formula (I) or a subembodiment thereof is
Figure imgf000017_0002
In some embodiments, X in Formula (I) or a subembodiment thereof is
Figure imgf000018_0001
In some embodiments, X in Formula (I) or a subembodiment thereof is
Figure imgf000018_0002
In some embodiments, X in Formula (I) or a subembodiment thereof is
Figure imgf000018_0003
In some embodiments, R1 in Formula (I) or a subembodiment thereof is Ci-4 alkyl. In some embodiments, R1 in Formula (I) or a subembodiment thereof is methyl.
In some embodiments, R3a in Formula (I) or a subembodiment thereof is Ci-4 alkoxy, or Ci-4 haloalkoxy. In some embodiments, R3a in Formula (I) or a subembodiment thereof is methoxy.
In some embodiments, R3b in Formula (I) or a subembodiment thereof is Ci-4 alkyl or halo. In some embodiments, R3b in Formula (I) or a subembodiment thereof is methyl or chloro. In some embodiments, R3b in Formula (I) or a subembodiment thereof is methyl. In some embodiments, R3b in Formula (I) or a subembodiment thereof is chloro. In some embodiments, R3c in Formula (I) or a subembodiment thereof is H or halo. In some embodiments, R3c in Formula (I) or a subembodiment thereof is H. In some embodiments, R3c in Formula (I) or a subembodiment thereof is fluoro. In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0001
In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0002
In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0003
In some embodiments, Z in Formula (I) or a subembodiment thereof is
OH
In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0004
In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0005
In some embodiments, Z in Formula (I) or a subembodiment thereof is
Figure imgf000019_0006
In some embodiments, Z in Formula (I) or a subembodiment thereof is In some embodiments, Z in Formula (I) or a subembodiment thereof is
In some embodiments, Z in Formula (I) or a subembodiment thereof is
In some embodiments, Z in Formula (I) or a subembodiment thereof is
In some embodiments, Z in Formula (I) or a subembodiment thereof is Representative compounds of Formula (I) are listed in Table 1 below.
Table 1.
Figure imgf000020_0001
Figure imgf000021_0003
In some embodiments, the compound or pharmaceutically acceptable salt thereof is a compound from Table 1.
The compounds of Formula (I) are depicted as a (Z) isomer with respect to the double bond between the thiadiazole moiety and the nitrogen in the amide group:
Figure imgf000021_0001
The Formula (I) also encompasses the compounds of Formula (la), an (E) isomer:
Figure imgf000021_0002
Assay The ability of compounds of the present disclosure to inhibit Pol0 can be measured as described in the biological assay below.
Pharmaceutical Composition
The compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, provided herein may be in the form of compositions suitable for administration to a subject. In general, such compositions are pharmaceutical compositions comprising a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain embodiments, the compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof is present in a therapeutically effective amount. The pharmaceutical compositions may be used in all the methods disclosed herein; thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject in order to practice the therapeutic methods and uses described herein.
The pharmaceutical compositions can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. Furthermore, the pharmaceutical compositions may be used 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.
The pharmaceutical compositions containing the active ingredient (e.g., a compound of Formula (I) or Table 1, a pharmaceutically acceptable salt thereof) may be in a form suitable for oral use, for example, as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads or elixirs. Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets, capsules and the like contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets, capsules, and the like. These excipients may be, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
The pharmaceutical compositions typically comprise a therapeutically effective amount of a compound of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipient. Suitable pharmaceutically acceptable excipients include, but are not limited to, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethyl or n-propyl, p- hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, fillers, bulking agents, detergents, buffers, vehicles, diluents, and/or adjuvants. For example, a suitable vehicle may be physiological saline solution or citrate buffered saline, possibly supplemented with other materials common in pharmaceutical compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Those skilled in the art will readily recognize a variety of buffers that can be used in the pharmaceutical compositions and dosage forms contemplated herein. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. As an example, the buffer components can be water soluble materials such as phosphoric acid, tartaric acids, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof. Acceptable buffering agents include, for example, a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), and N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).
All the compounds and pharmaceutical compositions provided herein can be used in all the methods provided herein. For example, the compounds and pharmaceutical compositions provided herein can be used in all the methods for treatment and/or prevention of all diseases or disorders provided herein. Thus, the compounds and pharmaceutical compositions provided herein are for use as a medicament.
Routes of Administration
Compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof and compositions containing the same may be administered in any appropriate manner. Suitable routes of administration include oral, parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracistemal, intraarticular, intraperitoneal, intracerebral (intraparenchymal) and intracerebroventricular), nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), buccal and inhalation. Depot injections, which are generally administered subcutaneously or intramuscularly, may also be utilized to administer the compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof over a defined period of time. Particular embodiments of the present invention contemplate oral administration.
Dosing
The compounds of Formula (I) or Table 1, or a pharmaceutically acceptable salt thereof provided herein may be administered to a subject in an amount that is dependent upon, for example, the goal of administration (e.g., the degree of resolution desired); the age, weight, sex, and health and physical condition of the subject to which the formulation is being administered; the route of administration; and the nature of the disease, disorder, condition or symptom thereof. The dosing regimen may also take into consideration the existence, nature, and extent of any adverse effects associated with the agent(s) being administered. Effective dosage amounts and dosage regimens can readily be determined from, for example, safety and dose-escalation trials, in vivo studies (e.g., animal models), and other methods known to the skilled artisan.
In general, dosing parameters dictate that the dosage amount be less than an amount that could be irreversibly toxic to the subject (the maximum tolerated dose (MTD)) and not less than an amount required to produce a measurable effect on the subject. Such amounts are determined by, for example, the pharmacokinetic and pharmacodynamic parameters associated with ADME, taking into consideration the route of administration and other factors.
An effective dose (ED) is the dose or amount of an agent that produces a therapeutic response or desired effect in some fraction of the subjects taking it. The “median effective dose” or EDso of an agent is the dose or amount of an agent that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although the ED50 is commonly used as a measure of reasonable expectance of an agent’s effect, it is not necessarily the dose that a clinician might deem appropriate taking into consideration all relevant factors. Thus, in some situations the effective amount is more than the calculated ED50, in other situations the effective amount is less than the calculated ED50, and in still other situations the effective amount is the same as the calculated EDso.
Combinations of Compounds of Formula (II) and Poly ADP Ribose Polymerase (PARP) inhibitors
The combination of agents described in this section may display a synergistic effect. The term “synergistic effect” as used herein, refers to action of two agents such as, for example, a DNA polymerase theta (Pol0) inhibitor (e.g. a compound of Formula (I) or Formula (II))and a Poly ADP Ribose Polymerase (PARP) inhibitor producing an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
As used herein, the term “synergy” refers to the effect achieved when the active ingredients, i.e., a Pole inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor used together is greater than the sum of the effects that results from using the compounds separately.
In some aspects, provided herein is a combination therapy comprising a therapeutically effective amount of a PolO inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibito. A “therapeutically effective amount” of a combination of agents (i.e., a Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor is an amount sufficient to provide an observable improvement over the baseline clinically observable signs and symptoms of the disorders treated with the combination. Observable improvements include those that can be visually ascertained by a clinician and biological tests, biopsies, and assays.
In some aspects, provided herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Pole inhibitor (e.g. a compound of Formula (I) or Formula (II))and administering to the subject a therapeutically effective amount of a PARP inhibitor, thereby treating the cancer in the subject.
In some aspects, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject a combination comprising a PolO inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor, together with at least a pharmaceutically acceptable carrier, thereby treating the cancer in the subject.
In some aspects, use of a combination of a PolO inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor for the manufacture of a medicament is provided. In another embodiment, use of a combination of a Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II))and a PARP inhibitor for the treatment of cancer is provided.
In some embodiments, the cancer is characterized as a homologous recombinant (HR) deficient cancer.
In some embodiments, the Pol0 inhibitor (e.g. a compound of Formula (I) or Formula (II))is an inhibitor of the ATPase domain of Pol0.
In some embodiments, the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
Pol0 Inhibitors for combination therapy with a PARP inhibitor
Pol0 inhibitors suitable for the combination therapy treatment with PARP inhibitors described in this section are compounds of Formula (II)
Figure imgf000026_0001
wherein:
R1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
R3a, R3b, and R3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
Z is:
Figure imgf000026_0002
or a pharmaceutically acceptable salt thereof.
In some embodiments, R1 in Formula II and subembodiments thereof is Ci-4 alkyl. In some embodiments, R1 in Formula (II) and subembodiments thereof is methyl.
In some embodiments, R3a in Formula (II) and subembodiments thereof is Ci-4 alkoxy, or Ci-4 haloalkoxy. In some embodiments, R3a in Formula (II) and subembodiments thereof is methoxy. In some embodiments, R3b in Formula (II) and subembodiments thereof is Ci-4 alkyl or halo. In some embodiments, R3b in Formula (II) and subembodiments thereof is methyl or chloro.
In some embodiments, R3b in Formula (II) and subembodiments thereof is methyl. In some embodiments, R3b in Formula (II) and subembodiments thereof is chloro.
In some embodiments, R3c in Formula (II) and subembodiments thereof is H or halo. In some embodiments, R3c in Formula (II) and subembodiments thereof is H. In some embodiments, R3c in Formula (II) and subembodiments thereof is fluoro.
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0001
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0002
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0003
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0004
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0005
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000027_0006
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0001
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0002
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0003
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0004
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0005
In some embodiments, Z in Formula (II) and subembodiments thereof is
Figure imgf000028_0006
In some embodiments, the Pol0 inhibitor of Formula (II) is
Figure imgf000028_0007
Compound 4 (also called Compound A) Compound 5 (also called Compound B)
Figure imgf000029_0001
Compound 8 Compound 9
Figure imgf000029_0002
Compound 10.
Compound 4 and Compound A are used herein interchangeably. Compound 5 and Compound B are used herein interchangeably.
In some embodiments, the PolO inhibitor of Formula (II) is Compound 4:
Figure imgf000029_0003
Compound 4 or a pharmaceutically acceptable salt thereof.
In some embodiments, the PolO inhibitor of Formula (II) is Compound 5 :
Figure imgf000029_0004
Compound 5 or a pharmaceutically acceptable salt thereof. In some embodiments, the Pol0 inhibitor for combination therapy is ART558 having the structure:
Figure imgf000030_0001
In some embodiments, the Pol0 inhibitor for combination therapy is ART4215.
PARP Inhibitors for combination therapy with Pol0 Inhibitors
The combination therapy described herein provides PARP inhibitors for use with a Pol0 inhibitor^ .g. a compound of Formula (I) or Formula (II)). A number of agents with PARP inhibitory activity and methods of making the same are known in the art. Each of these embraced by this disclosure. In some embodiments, the PARP inhibitor is
Figure imgf000030_0002
Niraparib (also called Compound 11 herein)
Figure imgf000030_0003
Rucaparib (also called Compound 12 herein)
Figure imgf000030_0004
Olaparib (also called Compound 13 herein)
Figure imgf000031_0001
Talazoparib (also called Compound 14 herein)
Figure imgf000031_0002
Veliparib (also called Compound 15 herein) or a pharmaceutically acceptable salt or hydrate thereof.
The preparation and activity of niraparib are described in US 8,071,579; US 8,071623; US 8,143,241; US 8,426,185; US 8,859,562; and US 11,091,459, the entire contents of which are hereby incorporated by reference in their entirety.
The preparation and activity of rucaparib are described in US 6,495,541; US 7,351,701; and US 7,531,530, the entire contents of which are hereby incorporated by reference in their entirety.
The preparation and activity of olaparib are described in US 7,151,102; US 7,449,464; US 7,981,889; and US 8,071,579, the entire contents of which are hereby incorporated by reference in their entirety.
The preparation and activity of talazoparib are described in US 8,012,976; US 8,420,650; and US 8,735,392, the entire contents of which are hereby incorporated by reference in their entirety.
In some embodiments, the PARP inhibitor is niraparib tosylate monohydrate.
In some embodiments, the PARP inhibitor is
Figure imgf000031_0003
or pharmaceutically acceptable salt or hydrate thereof.
Select Combination Therapy Embodiments
Embodiment 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Pol0 inhibitor, or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
Embodiment 2. The method of embodiment 1, wherein the Pol0 inhibitor is an inhbitior of ATPase domain of Pol0.
Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the Pol0 inhibitor is a compound of Formula (I) as defined herein, or a pharmaceutically acceptable salt thereof.
Embodiment 4. The method of embodiment 3, wherein the Pol0 inhibitor is the compound of Example 1, having the structure:
Figure imgf000032_0001
5 or a pharmaceutically acceptable salt thereof.
Embodiment 5. The method of embodiment 1 or embodiment 2, wherein the Pol0 inhibitor is a compound of Formula (II):
Figure imgf000032_0002
wherein:
R1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Cwhaloalkyl, or Ci-4 haloalkoxy;
R3a, R3b, and R3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
Figure imgf000033_0001
or a pharmaceutically acceptable salt thereof.
Embodiment 6. The method of embodiment 5, wherein R1 is Ci-4 alkyl.
Embodiment 7. The method of embodiment 5, wherein R1 is methyl.
Embodiment 8. The method of any one of embodiments 5 to 7, wherein R3a is Ci-4 alkoxy, or Ci-4 haloalkoxy/
Embodiment 9. The method of any one of embodiments 5 to 7, wherein R3a is methoxy.
Embodiment 10. The method of any one of embodiments 5 to 9, wherein R3b is Ci-4 alkyl or halo.
Embodiment 11. The method of any one of embodiments 5 to 9, wherein R3b is methyl or chloro.
Embodiment 12. The method of any one of embodiments 5 to 9, wherein R3b is methyl.
Embodiment 13. The method of any one of embodiments 5 to 9, wherein R3b is chloro.
Embodiment 14. The method of any one of embodiments 5 to 13, wherein R3c is H or halo.
Embodiment 15. The method of any one of embodiments 5 to 13, wherein R3c is H.
Embodiment 16. The method of any one of embodiments 5 to 13, wherein R3c is fluoro.
Embodiment 17. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000033_0002
Embodiment 18. The method of any one of embodiments 5 to 16 wherein Z is
Figure imgf000034_0001
Embodiment 19. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0002
Embodiment 20. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0003
Embodiment 21. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0004
Embodiment 22. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0005
Embodiment 23. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0006
Embodiment 24. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0007
Embodiment 25. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0008
Embodiment 26. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000034_0009
Embodiment 27. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000035_0001
Embodiment 28. The method of any one of embodiments 5 to 16, wherein Z is
Figure imgf000035_0002
Embodiment 29. The method of embodiment 5, wherein the Pol0 inhibitor of Formula
Figure imgf000035_0003
Compound 10, or a pharmaceutically acceptable salt thereof. Embodiment 30. The method of embodiment 5, wherein the Pol0 inhibitor of Formula
(II) is Compound 4:
Figure imgf000036_0001
Compound 4 or a pharmaceutically acceptable salt thereof. Embodiment 31. The method of embodiment 5, wherein the Pol0 inhibitor of Formula
(II) is Compound 5 :
Figure imgf000036_0002
Compound 5 or a pharmaceutically acceptable salt thereof.
Embodiment 32. The method of any one of embodiments 1-31, wherein the PARP inhibitor is
Figure imgf000036_0003
Compound 14 Compound 15
Figure imgf000037_0001
or a pharmaceutically acceptable salt or hydrate thereof.
Embodiment 33. The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 11 :
Figure imgf000037_0002
Compound 11 or a pharmaceutically acceptable salt thereof.
Embodiment 34. The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 12:
Figure imgf000037_0003
Compound 12 or a pharmaceutically acceptable salt thereof. Embodiment 35. The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 13:
Figure imgf000037_0004
Compound 13 or a pharmaceutically acceptable salt thereof. Embodiment 36. The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 14:
Figure imgf000038_0001
Compound 14 or a pharmaceutically acceptable salt thereof.
Embodiment 37. The method of any one of embodiments 1-31, wherein the PARP inhibitor is Compound 15:
Figure imgf000038_0002
Compound 15 or a pharmaceutically acceptable salt thereof.
Embodiment 38. The method of any one of embodiments 1-31, wherein the PARP inhibitor is:
Figure imgf000038_0003
pharmaceutically acceptable salt thereof.
Embodiment 39. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of Compound 4
Figure imgf000038_0004
Compound 4 or a pharmaceutically acceptable salt thereof, and administering to the subject a therapeutically effective amount of a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
Embodiment 40. The method of embodiment 39, wherein the PARP inhibitor is
Compound 11 :
Figure imgf000039_0001
Compound 11 or a pharmaceutically acceptable salt thereof.
Embodiment 41. The method of embodiment 39, wherein the PARP inhibitor is
Compound 12:
Figure imgf000039_0002
Compound 12 or a pharmaceutically acceptable salt thereof.
Embodiment 42. The method of embodiment 39, wherein the PARP inhibitor is
Compound 13:
Figure imgf000039_0003
Compound 13 or a pharmaceutically acceptable salt thereof. Embodiment 43. The method of embodiment 39, wherein the PARP inhibitor is
Compound 14:
Figure imgf000040_0001
Compound 14 or a pharmaceutically acceptable salt thereof.
Embodiment 44. The method of embodiment 39, wherein the PARP inhibitor is
Compound 15:
Figure imgf000040_0002
Compound 15 or a pharmaceutically acceptable salt thereof.
Embodiment 45. The method of embodiment 39, wherein the PARP inhibitor is:
Figure imgf000040_0003
or pharmaceutically acceptable salts thereof.
Embodiment 46. The method of any one of embodiments 1-45, wherein the cancer is a homologous recombinant (HR) deficient cancer.
Embodiment 47. The method of any one of embodiments 1-46, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein.
Embodiment 48. The method of any of embodiments 1-47, wherein the cancer is a solid tumor. Embodiment 49. The method of any one of embodiments 1-47, wherein the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.
Embodiment 50. The method of any one of embodiments 1-49, wherein the PolO inhibitor and the PARP inhibitor are in separate dosage forms.
Embodiment 51. The method of any one of embodiments 1-49, wherein the PolO inhibitor and the PARP inhibitor are in the same dosage form.
Embodiment 52. A combination comprising a PolO inhibitor of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, and a PARP inhibitor, or a pharmaceutically acceptable salt thereof.
Embodiment 53. The combination of embodiment 52, wherein the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof.
Embodiment 54. The combination of embodiment 52 or 53, wherein the Pol0 inhibitor is Compound 4 or Compound 5, and the PARP inhibitor is compound 11, compound 12, compound 13, compound 14, or compound 15 or a pharmaceutically acceptable salt thereof.
Embodiment 55. A Pol0 inhibitor of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, for use in treating cancer, wherein the Pol0 inhibitor is to be administered simultaneously or sequentially with a PARP inhibitor.
Embodiment 56. The Pol0 inhibitor of Formula (I) or Formula (II) for use of embodiment 55, wherein the PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, AZD5305, or AZD9574, or a pharmaceutically acceptable salt thereof.
Embodiment 57. The use of embodiment 56, wherein the Pol0 inhibitor of Formula (II) is Compound 4 or Compound 5. Embodiment 58. The use of embodiment 56, wherein the Pol0 inhibitor of Formula (I) is compound of Example 1.
Embodiment 59. Use of a Pol0 inhibitor of Formula (I) or Formula (II) in the manufacture of a medicament for treating cancer, wherein the Pol0 inhibitor is to be administered simultaneously or sequentially with a PARP inhibitor.
Embodiment 60. The use of embodiment 59, wherein PARP inhibitor is Compound 11, Compound 12, Compound 13, Compound 14, Compound 15, AZD5305, or AZD9574 or a pharmaceutically acceptable salt thereof.
Embodiment 61. The use of embodiment 60, wherein the Pol0 inhibitor is Compound 4, or a pharmaceutically acceptable salt thereof.
Embodiment 61. The use of embodiment 60, wherein the Pol0 inhibitor is compound of Example 1, or a pharmaceutically acceptable salt thereof.
Examples
The following examples and references (intermediates) are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed or that they are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate data and the like of a nature described therein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for.
Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (°C), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: THF= tetrahydrofuran; DIEA = diisopropylethylamine; EtOAc = ethyl acetate; NMP = N- methylpyridine, TFA = trifluoroacetic acid; DCM = dichloromethane; Cs2CC>3= cesium carbonate; XPhos Pd G3 = 2-dicyclohexylphosphino-2',4',6'-triisopropyl-l,l'-biphenyl)(2-(2'- amino-l,l'-biphenyl))palladium-(II) methanesulfonate; LiCl = lithium chloride; POCh = phosphoryl chloride; PE = petroleum ether; DMSO = dimethylsulfoxide; HC1 = hydrochloric acid; Na2SC>4 = sodium sulfate; DMF = dimethylformamide; NaOH = sodium hydroxide; K2CO3 = potassium carbonate; MeCN= acetonitrile; BOC= tert-butoxy carbonyl; MTBE = methyl tert-butyl ether; MeOH = methanol; NaHC’O? = sodium bicarbonate; NaBEECN = sodium cyanoborohydride; EtOH = ethanol; PC15= phosphorus pentachloride; NH-iOAc = ammonium acetate; Et2O = ether; HO Ac = acetic acid; AC2O = acetic anhydride; z-PrOH = isopropanol; NCS = N-chlorosuccinimide; K3PO4 = potassium phosphate; Pd(dtbpf)Ch =1,1'- Bis(di-tert-butylphosphino)ferrocene)dichloro-palladium(II); Zn(CN)2= Zinc cyanide; Pd(PPh3)4 =tetrakis(triphenylphosphine)palladium(0); Et?N = triethylamine; CuCN = copper cyanide; t-BuONO = tert-butyl nitrite; HATU = l-(bis(dimethylamino)methylene)-lH-l,2,3- triazolo(4,5-b)pyridinium 3-oxid hexafluorophosphate; DBU= l,8-diazabicyclo(5.4.0)undec- 7-ene; LiAlH4 = lithium aluminium hydride; NH3 = ammonia; H2SO4 = sulfuric acid; H2O2 = hydrogen peroxide; EDCI = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; HOBT = 1 -hydroxybenzotriazole hydrate; DHP = dihydropyran; TsOH = p- Toluenesulfonic acid; FA = formic acid; TCFH = N,N,N,N’- tetramethylchloroformamidinium hexafluorophosphate ; NMI = N-methylimidazole;
Pd(dppf)Ch = (l,l’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II); Pd(dppf)Ch- DCM = (l,l’-Bis(diphenylphosphino)ferrocene)dichloropalladium(II), complex with dichloromethane; Mel = methyliodide; TBS-C1 = tert-Butyldimethylsilyl chloride ; TBAF = Tetrabutylammonium fluoride; DIBAL-H = Diisobutylaluminum hydride; LDA = Lithium diisopropylamide ..
Synthetic Examples: Compounds of Formula (I)
Example 1
(R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3- carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate
Figure imgf000043_0001
Step-1: 2-chloro-5-methoxypyridin-4-ylboronic acid
Figure imgf000044_0001
A stirred solution of 2-chloro-5 -methoxypyridine (10.0 g, 69.65 mmol) in THF (500 mL) was added LDA (14.9 g, 139.30 mmol) dropwise at -78 °C under N2 atmosphere. The resulting mixture was stirred at -78 °C for 2 h. Then Triisopropyl borate (26.2 g, 139.30 mmol) was added to the above mixture at -78 °C. The resulting mixture was stirred at -78 °C for 2 h. Then the resulting mixture was stirred at room temperature for 16 h. The resulting mixture was quenched with HC1 (2 N) and stirred at room temperature for 30 min. The resulting mixture was extracted with ethyl acetate. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. 2-chloro-5- methoxypyridin-4-ylboronic acid (9 g, 68.9%) as a brown solid. MS (ESI) calc’d for (C6H7BCINO3) (M+l)+, 188.0; found 188.0.
Step-2: methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3 -carboxylate
Figure imgf000044_0002
To a degassed solution of methyl 4-chloro-6-methylpyridine-3-carboxylate (700 mg, 3.77 mmol) and 2-chloro-5-methoxypyridin-4-ylboronic acid (918 mg, 4.90 mmol) in dioxane (6 mL) and H2O (2 mL) were added Pd(dppf)Ch (275 mg, 0.37 mmol) and K2CO3 (1563 mg, 11.31 mmol) under nitrogen atmosphere. The resulting mixture was stirred at 80 °C for 16 h under nitrogen atmosphere. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography with 0-60% ethyl acetate in petroleum ether to afford methyl 2-chloro-5- methoxy-6-methyl-(4,4-bipyridine)-3-carboxylate (220 mg, 19.9%) as a white solid. MS (ESI) calc’d for (C14H13CIN2O3) (M+l)+, 293.1; found 293.1.
Step-3 : 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid
Figure imgf000045_0001
To a stirred solution of methyl 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylate (220 mg, 0.75 mmol) in THF (2 mL) and water (2 mL) were added LiOH.LLO (126 mg, 3.01 mmol). The resulting mixture was stirred at room temperature for 2 h. The mixture was acidified to pH 3 with citric acid. The resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2-chloro-5-methoxy-6-methyl-(4,4- bipyridine) -3 -carboxylic acid (160 mg, 76.3%) as a white solid. MS (ESI) calc’d for (C13H11CIN2O3) (M+l)+, 279.0; found, 279.0.
Step-4: Synthesis of (R)-O-((l,4-dioxan-2-yl)methyl) S-methyl carbonodithioate
Figure imgf000045_0002
To a stirred solution of (R)-(l,4-dioxan-2-yl)methanol (200.0 mg, 1.69 mmol) in Tetrahydrofuran (5 mL) was added NaH (136.0 mg, 3.40 mmol) at 0 °C under nitrogen atmosphere. The resulting solution was stirred at 0 °C for 0.5 h. To the above solution was added CS2 (193.0 mg, 2.54 mmol) at 0 °C under nitrogen atmosphere. The resulting mixture was then stirred at 0 °C for 0.5 h. To the above solution was added Mel (360.0 mg, 2.54 mmol) at 0 °C under nitrogen atmosphere. The resulting mixture was then stirred at 0 °C for 0.5 h. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum to afford (R)-O-((l,4-dioxan-2-yl)methyl) S- methyl carbonodithioate (360.0 mg, crude) as a yellow oil, the crude product was used in the next step without further purification.
Step-5: Synthesis of (R)-O-((l,4-dioxan-2-yl)methyl) hydrazinecarbothioate
Figure imgf000045_0003
To a stirred solution of (R)-O-((l,4-dioxan-2-yl)methyl) S-methyl carbonodithioate (360.0 mg, 1.73 mmol) in Methanol (4 mL) were sequentially added hydrazine hydrate (96.0 mg, 1.90 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 0.5 h. The solvents were removed under vacuum to afford (R)-O-((l,4-dioxan-2-yl)methyl) hydrazinecarbothioate (400.0 mg, crude) as a yellow oil, which was used in the next step without further purification. MS (ESI) calculated for (C6H12N2O3S) (M+l)+, 193.1; found, 193.2.
Step-6: Synthesis of (R)-5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-amine
Figure imgf000046_0001
To a stirred solution of (R)-O-((l,4-dioxan-2-yl)methyl) hydrazinecarbothioate (400.0 mg, 2.08 mmol) in Methanol (4 mL) were sequentially added TEA (0.58 mL, 4.16 mmol) and cyanic bromide (242.0 mg, 2.29 mmol) at 23 °C. The resulting solution was stirred at 25 °C for 1 h. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum . The resulting residue was dissolved in DCM (1 mL) and purified by Combi Flash which applied to a 20 g silica gel column that was eluted with 0-15% ethyl acetate in petroleum ether within 25 min to afford (R)-5-((l,4- dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-amine (140.0 mg, 30%) as a white solid. MS (ESI) calculated for (C7H11N3O3S) (M+l)+, 218.1; found, 218.2.
Step-7: Synthesis of (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000046_0002
To a stirred solution of (R)-5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-amine (100.0 mg, 0.46 mmol) in acetonitrile (1 mL) were added Intermediate H (128.0 mg, 0.46 mmol) and NMI (189.0 mg, 2.30 mmol). To the above solution was added TCFH (129.3 mg, 0.46 mmol) in acetonitrile (1 mL). The resulting solution was stirred at 25 °C under nitrogen for 1 hr. The solvents were removed under vacuum and purified directly. The resulting residue was dissolved in DMF (1 mL) which was applied to a 25 g Cl 8 column and purified by Combi Flash , eluted with 5-80% acetonitrile in water within 25 min to afford (R)-N-(5-((l,4- dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-(4,4'- bipyridine)-3 -carboxamide (26.6 mg, 11.9%) as a white solid. MS (ESI) calculated for (C20H20CIN5O5S) (M+l)+, 478.1; found, 478.2. *HNMR (400 MHz, DMSO-r/e) 5 12.91 (s, 1H), 8.82 (s, 1H), 8.17 (s, 1H), 7.53 (s, 1H), 7.42 (s, 1H), 4.45 - 4.33 (m, 2H), 3.95 - 3.87 (m, 1H), 3.83 - 3.73 (m, 2H), 3.63 - 3.57 (m, 5H), 3.55 - 3.45 (m, 1H), 3.42 - 3.34 (m, 1H), 2.59 (s, 3H).
Step 8: Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate
Figure imgf000047_0001
The reaction vessel was charged with solid (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4- thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (40 g, 84 mmol). N-Methyl -2 -pyrrolidone (NMP) (300 mL) was added to the reaction vessel and the mixture was stirred at 100 rpm. Cesium carbonate (40.9 g, 126 mmol), potassium iodide (6.95 g, 41.8 mmol) was added followed by di-tert-butyl (chloromethyl) phosphate (24.4 g, 94 mmol). N-Methyl-2 -pyrrolidone (NMP) (100 mL) was added (this was used to rinse any residual solid/reagent off the side of the reaction flask). The temperature was set to 40 °C, stirring was increased to 250 rpm and the reaction was allowed to stir overnight. After 22 h the reaction was quenched with 10 volume of de-ionized water (400 mL) followed by 20 volume of EtOAc (800 mL) and the resulting mixture was stirred for 10 min. The aqueous layer was removed and the organic layer was washed once more with 10 vol de-ionized water. The mixture was allowed to stir for an additional 10 min followed by 10 min of standing. The organic layer was washed with 10 vol 15% brine for 10 mins and allowed to separate for 10 min. The aqueous layer was removed and the organic layer was collected (no drying agent) and concentrated under vacuum. The crude mixture was purified by silica gel column chromatography (gradient 0-5% MeOH in DCM, 33 min, 330 g column). (Two columns were used to purify all the material, 50% of the crude on each column.) to get the desired product (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate (49 g, 70.0 mmol, 84 % yield) as a light brown viscous oil. LCMS (ES) calc’d for C29H39CIN5O9PS [M+H]+, 700.2; found 700.1.
Step 9: Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate
Figure imgf000048_0001
To a stirred suspension of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6- methyl-[4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl di-tert-butyl phosphate (49 g, 61.6 mmol) in water (200 m ) at 35 °C was added formic acid (150 m ) dropwise over 5 min. The mixture was allowed to stir at 35 °C for 2.5 h. The reaction was concentrated under vacuum (bath temperature 40 °C) then methanol (100 mb) was added and the mixture was concentrated once more. To the residue was added EtOH (lb) and the mixture was vigorously stirred at 60 °C for 1 hr and then raised to 80 °C for 30 mins. The temperature was then reduced to 40 °C and the mixture was allowed to stir at that temperature for 3 h. then left overnight with continued stirring. The solid was collected and slurried with MeOH (200 mb) with stirring at room temperature for 1 h. The suspension was filtered to give the crude product (29.6 g) which was further purified by slurrying in methanol (600 mb) under reflux, cooling to 35 °C and filtration (then repeat this process a second time) to afford (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'- bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate (20.6 g, 35 mmol, 57 % yield) as an off white solid. The stereochemistry of this compound was confirmed by X-ray crystallography. ECMS (ES) calc’d for C21H23CIN5O9PS [M+H]+, 588.1; found 588.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 5 9.28 (s, 1H), 8.15 (s, 1H), 7.42 (s, 1H), 7.36 - 7.24 (m, 1H), 5.79 (d, J = 9.3 Hz, 2H), 4.47 - 4.32 (m, 2H), 3.95 - 3.87 (m, 1H), 3.82 - 3.73 (m, 2H), 3.67 (s, 1H), 3.64 (s, 3H), 3.63 - 3.57 (m, 1H), 3.53 - 3.44 (m, 1H), 3.37 (dd, J = 11.5, 10.0 Hz, 1H), 2.57 (s, 3H).
Figure imgf000049_0001
((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3-carbonyl)imino)-5-(((lr,4r)-4- hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate
Step-1 : 2-chloro-3-fluoro-5-methoxypyridine
Figure imgf000049_0002
To a solution of 6-chloro-5-fluoropyridin-3-ol (20.0 g, 135.60 mmol) in acetone (150 mL) were added Mel (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) at 25 °C under nitrogen atmosphere. The resulting solution was stirred at 25 °C for 16 h under nitrogen before concentrated under vacuum. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 330 g silica gel column and eluted with 0-22% ethyl acetate in petroleum ether within 45 min to afford 2- chloro-3-fhroro-5-methoxypyridine (16.0 g, 80%) as a colorless oil. MS (ESI) calc’d for (C6H5C1FNO) (M+l)+, 162.0; found 162.0.
Step-2: 2-chloro-3-fluoro-4-iodo-5 -methoxypyridine
Figure imgf000049_0003
To a degassed solution of 2-chloro-3-fluoro-5 -methoxypyridine (16.0 g, 99.00 mmol) in dry Tetrahydrofuran (160 mL) was added n-butyllithium (44 mL, 110.00 mmol, 2.5 N in hexane) dropwise at -60 °C and stirred at -60 °C for 1 h under nitrogen atmosphere. Then iodine (27.6 g, 109.00 mmol) was added to the above mixture at -60 °C. The resulting solution was stirred at -60 - 20 °C for 2 hr. The reaction mixture was quenched by the addition of saturated sodium thiosulfate aqueous solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 330 g silica gel column and eluted with 0-50% ethyl acetate in petroleum ether within 40 min to afford 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid MS (ESI) calc’d for (CelLCIFINO) (M+l)+, 287.9; found, 287.9.
Step-3 : methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate
Figure imgf000050_0001
To a degassed solution of methyl 6-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)nicotinate (7.2 g, 26.10 mmol) and 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (5.0 g, 17.39 mmol) in dry 1,4-Dioxane (50 mL) were added Water (10 m ), (1,1'- Bis(diphenylphosphino)ferrocene)dichloropalladium (II), complex with dichloromethane (4.2 g , 5.15 mmol) and K2CO3 (7.2 g, 52.20 mmol) at 25 °C under nitrogen atmosphere. The resulting solution was stirred at 25 °C for 2 h under nitrogen atmosphere. The suspension was fdtered. The fdtrate was collected and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 120 g silica gel column and eluted with 0-46% ethyl acetate in petroleum ether within 45 min to afford methyl 2'- chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (2.8 g, 53%) as a white solid. MS (ESI) calc’d for (C14H12CIFN2O3) (M+l)+, 311.1; found, 311.1.
Step-4: methyl 3 '-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3 -carboxylate
Figure imgf000050_0002
To a degassed solution of methyl 2'-chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)- 3-carboxylate (500.0 mg, 1.61 mmol) in DME (5 mL) were added K2CO3 (667.0 mg, 4.83 mmol), Pd(dppf)Ch (235.0 mg, 0.32 mmol) at 25 °C under nitrogen atmosphere. Then 2,4,6- trimethyl-l,3,5,2,4,6-trioxatriborinane (222.0 mg, 1.77 mmol) was added to the above mixture at 25 °C. The resulting solution was stirred at 120 °C for 1 h under nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting residue was dissolved in DCM (4 mb) and purified by Combi Flash (Biotage Isolera Prime) which applied to a 40 g silica gel column that was eluted with 0-8% methanol in dichloromethane within 40 min to afford methyl 3'-fluoro-5'-methoxy-2',6- dimethyl-(4,4'-bipyridine)-3-carboxylate (340.0 mg, 69 %) as a brown solid MS (ESI) calc’d for (C15H15FN2O3) (M+l)+, 291.1; found, 291.1.
Step-5 : 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid
Figure imgf000051_0001
To a stirred solution of methyl 3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3- carboxylate (300.0 mg, 1.03 mmol) in Methanol (3 mb) were added NaOH (165.0 mg, 4.13 mmol) and Water (3 mb) at 25 °C. The resulting solution was stirred at 25 °C for 2 hr. The organic solvent was removed under vacuum. The aqueous layer was acidified with Citric acid to pH -4 and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 3'-fhioro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxylic acid (160 mg, crude) MS (ESI) calc’d for (C14H13FN2O3) (M+l)+, 277.1; found, 277.1.
Step-6 : methyl ( 1 r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane- 1 -carboxylate
Figure imgf000051_0002
To a solution of methyl (lr,4r)-4-hydroxycyclohexane-l -carboxylate (500 mg, 3.145 mmol) and imidazole (642 mg, 9.441 mmol) in DCM (10 mb) was added TBS-C1 (712 mg, 4.715 mmol) at 0 °C. The resulting mixture was stirred at room temperature for 8 h. The reaction mixture was then quenched by the addition of water and extracted with ethyl acetate. The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate and fdtered. The fdtrate was concentrated under vacuum. The residue was purified by flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to afford methyl (lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-l-carboxylate (900 mg, 88%) as a white solid. MS (ESI) calc’d for (Ci^OsSi) (M+l)+, 273.2; found 272.0.
Step-7: ((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol
Figure imgf000052_0001
To a solution of methyl (lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexane-l -carboxylate (900 mg, 3.297 mmol) in THF (30 m ) was added LiAlHi (250 mg, 6.579 mmol) in portions at 0~5°C. The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel with 0-10% methanol in dichloromethane to afford ((lr,4r)-4-((tert- butyldimethylsilyl)oxy)cyclohexyl)methanol (690 mg, 77%) as a white solid. MS (ESI) calc’d for (CisftsChSi) (M+l)+, 245.0; found, 245.0.
Step-8: O-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate
Figure imgf000052_0002
To a solution of((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methanol (690 mg, 2.816 mmol) in THF (20 mb) was added NaH (225 mg, 9.375 mmol, 60%) in portions at 0 °C and stirred at 0 °C for 30 min. Then CS2 (321 mg, 4.224 mmol) was added to the above mixture and stirred at 0 °C for 10 min, Then Mel (600 mg, 4.225 mmol) was added to the above mixture at 5 °C. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum, The residue was purified by flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to afford O-(((lr,4r)-4-((tert- butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate (700 mg , 78%) as a colorless oil. MS (ESI) calc’d for (CisH3o02S2Si) (M+l)+, 335.0, found 335.0.
Step-9 : O-((( 1 r,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) hydrazinecarbothioate
Figure imgf000053_0001
To a solution of O-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) S-methyl carbonodithioate (700 mg, 2.089 mmol) in MeOH (10 mb) was added hydrazine (130 mg, 4.062 mmol, 80%). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford O-(((lr,4r)-4-((tert- butyldimethylsilyl)oxy)cyclohexyl)methyl) hydrazinecarbothioate (630 mg, 90%) as red oil. MS (ESI) calc’d for (CuHso^CESSi) (M+l)+, 319.0, found 319.1.
Step-10: 5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4-thiadiazol-2- amine
Figure imgf000053_0002
To a solution of O-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methyl) hydrazinecarbothioate (630 mg, 1.975 mmol) in MeOH (10.00 m ) were added TEA (402 mg, 3.981 mmol) and BrCN (232 mg, 2.189 mmol). The resulting mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel with 0-50% ethyl acetate in petroleum ether to afford 5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4-thiadiazol-2-amine (300 mg ,48%) as a red solid. MS (ESI) calc’d for (CisHiyNiChSSi) (M+l)+, 344.1, found 344.0.
Step-11 : N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4- thiadiazol-2-yl)-3'-fhioro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000053_0003
To a solution of 3 '-fhioro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3 -carboxylic acid (160.0 mg, 0.57 mmol, Current Example, Step 5) in dry Acetonitrile (4 mL) were added 5-(((lr,4r)- 4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)- 1 ,3,4-thiadiazol-2-amine (199.0 mg, 0.58 mmol, Current Example, Step 10), 1 -methyl- 1 //-imidazole (238.0 mg, 2.90 mmol) at 25 °C. Then TCFH (162.0 mg, 0.57 mmol) in acetonitrile (2 mL) was added to the above mixture at 25 °C. The resulting solution was stirred at 25 °C for 1 hr. The resulting mixture was applied to a 40 g Cl 8 column and purified by Combi Flash (Biotage Isolera Prime), eluted with 5-30% acetonitrile in water within 40 min to afford N-(5-(((lr,4r)-4-((tert- butyldimethylsilyl)oxy)cyclohexyl)methoxy)- 1 ,3 ,4-thiadiazol-2-yl)-3 '-fluoro-5 '-methoxy- 2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide (136.0 mg, 37%) as a yellow solid. MS (ESI) calc’d for (C29H4oFN504SSi) (M+l)+, 602.3; found, 602.3.
Step-12: 3'-fhioro-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'- methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000054_0001
To a stirred solution of N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)- l,3,4-thiadiazol-2-yl)-3'-fluoro-5'-methoxy-2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide (100.0 mg, 0.17 mmol) in THF (2 mL) was added TBAF (174.0 mg, 0.66 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 16 h. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum. The resulting residue was dissolved in DMF (3 mL) which was applied to a 40 g Cl 8 column and purified by Combi Flash (Biotage Isolera Prime), eluted with 5-40% acetonitrile in water within 40 min to afford 3'-fluoro-N-(5-(((lr,4r)-4- hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy-2',6-dimethyl-(4,4'- bipyridine)-3 -carboxamide (14.0 mg, 17%) as a white solid MS (ESI) calc’d for
(C23H26FN5O4S) (M+l)+, 488.2; found, 488.2. *HNMR (400 MHz, DMSO-tfc) 5 12.9 (br, 1H), 8.92 (s, 1H), 8.12 (s, 1H), 7.31 (s, 1H), 4.52 - 4.51 (m, 1H), 4.17 (d, J= 6.0 Hz, 2H), 3.66 (s, 3H), 3.38 - 3.28 (m, 1H), 2.56 (s, 3H), 2.40 (s, 3H), 1.88 - 1.80 (m, 2H), 1.79 - 1.71 (m, 3H), 1.16 - 1.06 (m, 4H). Step 13: Synthesis of di-tert-butyl (((Z)-2-((3'-fhroro-5'-methoxy-2',6-dimethyl-[4,4'- bipyridine]-3-carbonyl)imino)-5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol- 3(2H)-yl)methyl) phosphate
Figure imgf000055_0001
To a solution of 3'-fluoro-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2- yl)-5'-methoxy- 2',6-dimethyl-[4,4'-bipyridine]-3-carboxamide (500 mg, 1.026 mmol) in N,N-Dimethylformamide (DMF) (10 mL) was added K2CO3 (425 mg, 3.08 mmol), potassium iodide (170 mg, 1.026 mmol), followed by dropwise addition of di-tert-butyl (chloromethyl)phosphate (0.442 mL, 1.538 mmol). The reaction vessel was sealed and the reaction mixture was heated to 40 °C under nitrogen. After 24 h, the reaction mixture was quenched with ice-water (30 mL) and the aqueous mixture was extracted with EtOAc (3 x 20 mL). The combined organics were washed with water (20 mL) and brine (20 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure. The residue was dissolved in minimal EtOAc and purified by silica gel chromatography (40 g Redisep Gold column; DCM isocratic then gradient of 0-90% DCM in 4: 1 EtOAc/EtOH; flow rate 40 mL/min) to afford di-tert-butyl (((Z)-2-((3'-fhroro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3- carbonyl)imino)-5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)- l,3,4-thiadiazol-3(2H)- yl)methyl) phosphate (505 mg, 0.711 mmol, 69.4 % yield) as a yellow foam. MS(ES)+ m/e calc’d for C32H45FN5O8PS [M+H]+, 710.3; found 710.3. *HNMR (400 MHz, DMSOd6) 5 9.41 (s, 1H), 8.18 (s, 1H), 7.27 (s, 1H), 5.89 - 5.74 (m, 2H), 4.58 - 4.51 (m, 1H), 4.21 (d, J = 6.4 Hz, 2H), 3.70 (s, 3H), 3.39 - 3.34 (m, 1H), 2.58 (s, 3H), 2.42 (d, J = 2.9 Hz, 3H), 1.84 (br d, J = 9.3 Hz, 2H), 1.74 (br d, J = 8.3 Hz, 3H), 1.38 (d, J = 1.5 Hz, 18H), 1.17 - 0.99 (m, 4H). 31P NMR (162 MHz, DMSOd6) 5 -11.88 (t, J = 11.2 Hz, IP).
Step 14: Synthesis of ((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3- carbonyl)imino)-5 -((( 1 r,4r)-4-hydroxycyclohexyl)methoxy)- 1 ,3 ,4-thiadiazol-3 (2H)-yl)methyl dihydrogen phosphate
Figure imgf000056_0001
To a solution of di-z -but l (((Z)-2-((3'-fluoro-5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3- carbonyl)imino)-5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4- thiadiazol-3(2H)- yl)methyl) phosphate (452 mg, 0.637 mmol) in dichloromethane (6 mL) was added 2,2,2- trifluoroacetic acid (0.244 mL, 3.18 mmol). After 10 min, no product was observed by LCMS analysis so additional 2,2,2-trifluoroacetic acid (0.244 mL, 3.18 mmol) was added and stirring continued at room temperature. After 6 h, the reaction mixture was concentrated under reduced pressure. The residue was dried under high vacuum for 1 h and then dissolved in acetonitrile (10 mL). The resulting yellow solution was allowed to stand for 72 h. LCMS indicated 52% desired product and 42% trifluoroacetylated-desired product. To remove the trifluoroacetate, the residue was dissolved in methanol (3 mL) and treated with K2CO3 (176 mg, 1.274 mmol). The mixture was stirred at room temperature. LCMS analysis showed no reaction so an additional portion of K2CO3 (88 mg, 0.637 mmol, 1 eq) was added. The reaction was stirred a total of 2 h then concentrated under reduced pressure. The residue was dissolved in DMSO (5 mL), filtered through an Acrodisc frit and purified by reverse-phase column-chromatography (XSELECT CSH C-18 column; 150mm x 30mm; gradient of 15- 55% of 0.1% v/v Formic Acid-Acetonitrile in 0.1% v/v Formic Acid-water). The semi-solid crude product was treated with MeOH (10 mL) and MeCN (10 mL) and the mixture was concentrated under reduced pressure and the resulting residue was slurried in acetonitrile (10 mL) for 7 d and the resulting precipitate was collected by filtration to afford ((Z)-2-((3'- fluoro- 5'-methoxy-2',6-dimethyl-[4,4'-bipyridine]-3-carbonyl)imino)-5-(((lr,4r)-4- hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-3(2H)-yl)methyl dihydrogen phosphate (218 mg, 0.365 mmol, 57.3 % yield) as a white crystalline solid. MS(ES)+ m/e calc’d for C24H29FN5O8PS [M+H]+, 598.1; found 598.1. *HNMR (400 MHz, DMSO-d6) 5 9.39 (s, 1H), 8.18 (s, 1H), 7.27 (s, 1H), 5.74 - 5.66 (m, 2H), 4.20 (d, J = 6.4 Hz, 2H), 3.71 (s, 3H), 3.43 - 3.26 (m, 1H), 2.58 (s, 3H), 2.44 - 2.40 (m, 3H), 1.84 (br d, J = 11.7 Hz, 2H), 1.74 (br d, J = 11.2 Hz, 3H), 1.20 - 1.00 (m, 4H); 31P NMR (162 MHz, DMSO-de) 5 -3.12 (t, J = 9.3 Hz, IP). Example 3
(R,Z)-4-((5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-
3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methoxy)-4-oxobutanoic acid
Figure imgf000057_0001
Step 1: Synthesis of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl tert-butyl succinate
Figure imgf000057_0002
To a solution of (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-5'- methoxy-6-methyl-[4,4'-bipyridine]-3-carboxamide (500 mg, 1.046 mmol, Example 1, Step 7) in N,N-Dimethylformamide (DMF) (8 m ) was added K2CO3 (434 mg, 3.14 mmol), potassium iodide (174 mg, 1.046 mmol), followed by tert-butyl (chloromethyl) succinate (524 mg, 2.354 mmol). The reaction mixture was heated to 50 °C in a sealed vial with stirring under nitrogen. After 3 h, an additional portion of tert-butyl (chloromethyl) succinate (262 mg, 1.18 mmol) was added. After a total of 16 h the reaction temperature was increased to 60 °C and after a further 6 h, additional portions of K2CO3 (217 mg, 1.57 mmol) and tertbutyl (chloromethyl) succinate (262 mg, 1.18 mmol) were added and the reaction mixture was cooled back to 50 °C. After an additional 18 h, the reaction mixture was quenched with water (30 m ) and brine (15 mb) and the aqueous mixture was extracted with DCM (3 x 20 mb). The combined organics were washed with water (20 mb) and brine (20 mb), dried over Na2SC>4, filtered and concentrated under reduced pressure. The residue was dissolved in DCM and purified by silica gel chromatography (40 g Redisep Gold column; DCM isocratic then MeOH in DCM gradient; flow rate 40 then 50 mb/min) to provide (R,Z)-(5-((l,4- dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3- carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methyl tertbutyl succinate (510 mg, 0.768 mmol, 73.4 % yield) as a yellow oil. MS(ES)+ m/e calc’d for C29H34CIN5O9S [M+H]+, 664.2; found 664. 1, which was used without further purification.
Step 2: Synthesis of (R,Z)-4-((5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6- methyl-[4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methoxy)-4- oxobutanoic acid
Figure imgf000058_0001
To a solution of (R,Z)-(5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'-methoxy-6-methyl- [4,4'-bipyridine] -3 -carbonyl)imino)- 1 ,3 ,4-thiadiazol-3 (2H)-yl)methyl tert-butyl succinate (502 mg, 0.756 mmol) in Dichloromethane (DCM) (6 mL) was added 2,2,2-trifluoroacetic acid (2 mL, 26.1 mmol). After 1 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (4 mL) and purified by reverse-phase columnchromatography (XSELECT CSH C18 column with 150mm x 30mm, i.d. 5pm; gradient of 15-55% of 0. 1% v/v Formic Acid-Acetonitrile in 0. 1% v/v Formic Acid-water). The crude product was then treated with MeCN (10 mL) and MeOH (10 mL) and concentrated under reduced pressure. The precipitate was slurried in diethyl ether (10 mL) for 2 h then collected by filtration and dried to afford (R,Z)-4-((5-((l,4-dioxan-2-yl)methoxy)-2-((2'-chloro-5'- methoxy-6-methyl-[4,4'-bipyridine]-3-carbonyl)imino)-l,3,4-thiadiazol-3(2H)-yl)methoxy)- 4-oxobutanoic acid (221 mg, 0.363 mmol, 48.1 % yield) as a crystalline white solid. MS(ES)+ m/e calc’d for C25H26CIN5O9S [M+H]+, 608.1; found 608.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) 5 12.26 (br s, 1H), 9.15 (s, 1H), 8.14 (s, 1H), 7.44 (s, 1H), 7.29 (s, 1H), 5.91 (s, 2H), 4.40 - 4.31 (m, 2H), 3.93 -3.86 (m, 1H), 3.80 - 3.72 (m, 2H), 3.68 - 3.57 (m, 5H), 3.51 - 3.43 (m, 1H), 3.37 (dd, J = 11.2, 9.8 Hz, 1H), 2.62 - 2.51 (m, 7H).
Synthetic Examples: Compounds of Formula (II)
Compound 4 (Compound A)
(R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-5'-methoxy-6-methyl-
(4, 4'-bipyridine)-3 -carboxamide
Figure imgf000059_0001
Compound 4 was prepared as described in Example 1, Steps 1 to 7.
Compound 5 (Compound B)
3'-fluoro-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy- 2',6-dimethyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000059_0002
Compound 5 was prepared as described in Example 2, Steps 1 to 12.
Compound 6
(R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl- (4, 4'-bipyridine)-3 -carboxamide
Figure imgf000059_0003
Step-1 : 2-bromo-4-iodo-5 -methoxypyridine
Figure imgf000059_0004
To a stirred solution of methanol (380.0 mg, 11.84 mmol) in N,N-Dimethylformamide (15 mb) was added NaH (230.0 mg, 5.68 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 0.5 h. Then 2-bromo-5-fluoro-4-iodopyridine (1.4 g, 4.74 mmol) was added to the above mixture at 0 °C. The resulting solution was then stirred at 25 °C for 1 h. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was dissolved in dichloromethane (5 mL) and purified by flash chromatography (Biotage Isolera Prime) which applied to a 80.0 g silica gel column that was eluted with 0-20% ethyl acetate in petroleum ether within 25 min to afford 2-bromo-4-iodo-5-methoxypyridine (1.2 g, 54% yield) as a white solid. MS (ESI) calc’d for (CeHsBrINO) (M+l)+, 313.9; found 313.9.
Step-2: methyl 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate
Figure imgf000060_0001
To a stirred solution of 2-bromo-4-iodo-5 -methoxypyridine (200.0 mg, 0.64 mmol) and methyl 6-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)nicotinate (265.0 mg, 0.96 mmol) in 1,4-dioxane (2 mL) were sequentially added water (0.4 mL), potassium carbonate (264.0 mg, 1.91 mmol) and l,r-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (52.0 mg, 0.06 mmol) at 23 °C. The resulting solution was stirred at 80 °C for 2 hr under nitrogen. The suspension was fdtered. The filtrate was collected and concentrated under vacuum. The resulting residue was dissolved in acetonitrile (3 mL) which was applied to a 40.0 g C18 column and purified by flash chromatography (Biotage Isolera Prime), eluted with 5-45% acetonitrile in water within 30 min to afford methyl 2'-bromo-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (80.0 mg, 36% yield) as a colorless oil. MS (ESI) calc’d for (CuHisBrl s) (M+l)+, 337.0, found 337.0. Tl NMR (400 MHz, DMSO-de) 5 8.88 (s, 1H), 8.23 (s, 1H), 7.59 (s, 1H), 7.37 (s, 1H), 3.78 (s, 3H), 3.32 (s, 3H), 2.58 (s, 3H).
Step-3 : 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid
Figure imgf000060_0002
To a stirred solution of methyl 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (175.0 mg, 0.52 mmol) in methanol (0.3 mL) were added water (0.3 mL) and sodium hydroxide (83.0 mg, 2.08 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 2 hr under nitrogen. The organic solvent was removed under vacuum. The aqueous layer was acidified with sat. citric acid solution to pH -6 and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2'-bromo-5'-methoxy-6-methyl-(4,4'- bipyridine) -3 -carboxylic acid (100.0 mg, 58% yield) as a yellow solid. MS (ESI) calc’d for (Ci3HnBrN2O3) (M+l)+, 323.0, found 323.1. 1HNMR (400 MHz, DMSO-de) 5 13.00 (s, 1H), 8.89 (s, 1H), 8.22 (s, 1H), 7.54 (s, 1H), 7.30 (s, 1H), 3.78 (s, 3H), 2.56 (s, 3H).
Step 4: (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-bromo-5'-methoxy-6- methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000061_0001
To a stirred solution of 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3 -carboxylic acid (140.0 mg, 0.43 mmol) in Acetonitrile (1 mL) were sequentially added (R)-5-((l,4-dioxan-2- yl)methoxy)-l,3,4-thiadiazol-2-amine (113.0 mg, 0.52 mmol, Example 1, Step 6) and 1- methyl-lH-imidazole (178.0 mg, 2.16 mmol) at 25 °C. Then TCFH (122.0 mg, 0.43 mmol) in Acetonitrile (1 mL) was added to the above mixture at 25 °C. The resulting solution was stirred at 25 °C for 2 h. The resulting solution (2 mL) which was applied to a 20 g C 18 column and purified by flash chromatography (Biotage Isolera Prime), eluted with 5-32% acetonitrile in water within 30 min to afford (R)-N-(5-((l,4-dioxan-2-yl)methoxy)-l,3,4- thiadiazol-2-yl)-2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (67.1 mg, 29%) as a white solid. MS (ESI) calc’d for (C2oH2oBrN50sS) (M+l)+, 522.0, 524.0, found 522.0, 524.0. 1H NMR (4OO MHz, DMSO-d6) 5 12.89 (s, 1H), 8.81 (s, 1H), 8.17 (s, 1H), 7.63 (s, 1H), 7.42 (s, 1H), 4.46 - 4.34 (m, 2H), 3.94 - 3.90 (m, 1H), 3.89 - 3.80 (m, 2H), 3.71 - 3.57 (m, 5H), 3.55 - 3.42 (m, 1H), 3.38 - 3.35 (m, 1H), 2.59 (s, 3H).
Compound 7
N-(5-(((R)-l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6- methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000062_0001
Step-1 : 2-chloro-3-fluoro-5-methoxypyridine
Figure imgf000062_0002
To a solution of 6-chloro-5-fluoropyridin-3-ol (20.0 g, 135.60 mmol) in Acetone (150 mL) were added Mel (17 mL, 271.00 mmol) and K2CO3 (37.5 g, 271.00 mmol) at 25 °C under nitrogen atmosphere. The resulting solution was stirred at 25 °C for 16 h under nitrogen before concentrated under vacuum. The reaction mixture was quenched with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 330 g silica gel column and eluted with 0-22% ethyl acetate in petroleum ether within 45 min to afford 2- chloro-3-fhroro-5-methoxypyridine (16.0 g, 80%) as a colorless oil. MS (ESI) calc’d for (C6H5C1FNO) (M+l)+, 162.0; found 162.0.
Step-2: 2-chloro-3-fluoro-4-iodo-5 -methoxypyridine
Figure imgf000062_0003
To a degassed solution of 2-chloro-3-fluoro-5 -methoxypyridine (16.0 g, 99.00 mmol) in dry Tetrahydrofuran (160 mL) was added n-butyllithium (44 mL, 110.00 mmol, 2.5 N in hexane) dropwise at -60 °C and stirred at -60 °C for 1 hr under nitrogen atmosphere. Then iodine (27.6 g, 109.00 mmol) was added to the above mixture at -60 °C. The resulting solution was stirred at -60 - 20 °C for 2 hr. The reaction mixture was quenched by the addition of saturated sodium thiosulfate aqueous solution and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 330 g silica gel column and eluted with 0-50% ethyl acetate in petroleum ether within 40 min to afford 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (22.0 g, 73%) as a white solid MS (ESI) calc’d for (CelLCIFINO) (M+l)+, 287.9; found, 287.9. Step-3 : methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate
Figure imgf000063_0001
To a degassed solution of methyl 6-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)nicotinate (7.2 g, 26.10 mmol) and 2-chloro-3-fluoro-4-iodo-5-methoxypyridine (5.0 g, 17.39 mmol) in dry 1,4-Dioxane (50 mL) were added Water (10 mL), (1,T- Bis(diphenylphosphino)ferrocene)dichloropalladium (II), complex with dichloromethane (4.2 g , 5.15 mmol) and K2CO3 (7.2 g, 52.20 mmol) at 25 °C under nitrogen atmosphere. The resulting solution was stirred at 25 °C for 2 h under nitrogen atmosphere. The suspension was filtered. The filtrate was collected and concentrated under vacuum. The resulting residue was purified by Combi Flash (Biotage Isolera Prime) which applied to 120 g silica gel column and eluted with 0-46% ethyl acetate in petroleum ether within 45 min to afford methyl 2'- chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylate (2.8 g, 53%) as a white solid. MS (ESI) calc’d for (C14H12CIFN2O3) (M+l)+, 311.1; found, 311.1.
Step-4: 2'-chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxylic acid
Figure imgf000063_0002
To a stirred solution of methyl 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3- carboxylate (2.8 g, 9.17 mmol) in Methanol (10 mb) were added NaOH (1.4 g, 36.70 mmol) and Water (10 mL) at 25 °C. The resulting solution was stirred at 25 °C for 2 h before diluted with water. The organic solvent was removed under vacuum. The aqueous layer was acidified with Citric acid to pH ~5 and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 2'-chloro-3'-fhioro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3 -carboxylic acid (1.3 g, crude) as a yellow oil.MS (ESI) calc’d for (C13H10CIFN2O3) (M+l)+, 297.0, found 297.0.
Step 5: N-(5-(((R)-l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000064_0001
To a stirred solution of 2'-chloro-3'-fluoro-5'-methoxy-6-methyl-(4,4'-bipyridine)-3- carboxylic acid (150.0 mg, 0.51 mmol) in Acetonitrile (1 mb) were added (R)-5-((l,4- dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-amine (110.0 mg, 0.51 mmol, Example 1, Step 6) and 1 -methylimidazole (208.0 mg, 2.53 mmol). To the above was added TCFH (142.0 mg, 0.51 mmol) in Acetonitrile (1 mL) at 30 °C. The resulting mixture was stirred at 30 °C for 1 hr. The suspension was filtered. The filter cake was collected and dried under vacuum. The residue was dissolved in DMF (1 mL) and purified by prep-HPLC with the following conditions: (Column: XBridge Shield RP18 OBD Column, 30* 150 mm, 5pm; Mobile Phase A: Water(10 mmol/L NH4HCO3), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 20% B to 35% B in 8 min, 35% B; Wave Length: 254 nm; RTl(min): 7.7) to afford N-(5- (((R)-l,4-dioxan-2-yl)methoxy)-l,3,4-thiadiazol-2-yl)-2'-chloro-3'-fluoro-5'-methoxy-6- methyl-(4,4'-bipyridine)-3-carboxamide (66.0 mg, 26%) as a white solid. MS (ESI) calc’d for (C20H19CIFN5O5S) (M+l)+, 496.1; found, 496.2. 1H NMR (4OO MHz, DMSO-d6) 5 13.05 (s, 1H), 8.98 (s, 1H), 8.18 (s, 1H), 7.46 (s, 1H), 4.43 - 4.38 (m, 2H), 3.97 - 3.86 (m, 1H), 3.82 - 3.76 (m, 2H), 3.74 (s, 3H), 3.69 - 3.57 (m, 2H), 3.54 - 3.44 (m, 1H), 3.41 - 3.36 (m, 1H), 2.60 (s, 3H).
Compound 8
2'-bromo-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy-6- methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000064_0002
Step-1: 2'-bromo-N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)- 1,3,4- thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide:
Figure imgf000065_0001
To a stirred solution of 2'-bromo-5'-methoxy-6-methyl-(4,4'-bipyridine)-3 -carboxylic acid (150.0 mg, 0.46 mmol, Compound 6, Step 3) in Acetonitrile (1 mb) were added 5-(((lr,4r)-4- ((tert-butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4-thiadiazol-2-amine (159.0 mg, 0.46 mmol, Example 2, Step 10) and 1 -methylimidazole (191.0 mg, 2.32 mmol). To the above was added a solution of TCFH (130.0 mg, 0.46 mmol) in Acetonitrile (0.5 m ) at 25 °C. The resulting mixture was stirred at 25 °C for 2 h. The suspension was fdtered. The filter cake was collected and dried under vacuum to afford 2'-bromo-N-(5-(((lr,4r)-4-((tert- butyldimethylsilyl)oxy)cyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl- (4, 4'-bipyridine)-3 -carboxamide (180.0 mg, 60%) as a white solid. MS (ESI) calc’d for (C28H38BrN5O4SSi) (M+l)+, 648.2, 650.2; found, 648.2, 650.2.
Step-2: 2'-bromo-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'- methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide:
Figure imgf000065_0002
To a stirred solution of 2'-bromo-N-(5-(((lr,4r)-4-((tert-butyldimethylsilyl)oxy)cyclohexyl) methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (180.0 mg, 0.28 mmol) in Tetrahydrofuran (2 mb) was added TBAF (363.0 mg, 1.39 mmol) at 25 °C. The resulting solution was stirred at 25 °C for 2 h. The reaction was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was dissolved in DMF (2 mb) and was purified by prep-HPEC with the following conditions: (Column: Sunfire prep C18 column, 30* 150 mm, 5pm; Mobile Phase A: ACN, Mobile Phase B: Water(0.05%TFA ); Flow rate: 60 mb/min; Gradient: 30% B to 38% B in 8 min, 38% B to 38% B in 10 min, 38% B; Wave Eength: 254/220 nm; RTl(min): 8.52) to afford 2'-bromo-N-(5-(((lr,4r)-4-hydroxycyclohexyl)methoxy)-l,3,4-thiadiazol-2- yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (63.0 mg, 42%) as a white solid. MS (ESI) calc’d for (C22H24BrN5O4S) (M+l)+, 534.1, 536.1; found, 534.1, 536.1. 1H NMR (400 MHz, DMSO-d6) 5 12.86 (s, 1H), 8.80 (s, 1H), 8.17 (s, 1H), 7.63 (s, 1H), 7.42 (s, 1H), 4.52 (d, J = 4.4 Hz, 1H), 4.22 (d, J = 6.4 Hz, 2H), 3.63 (s, 3H), 3.37 - 3.33 (m, 1H), 2.59 (s, 3H), 1.90 - 1.80 (m, 2H), 1.80 - 1.68 (m, 3H), 1.21 - 1.04 (m, 4H).
Compound 9
2'-chloro-N-(5-(((lS,2R)-2-hydroxycyclopentyl)methoxy)-l,3,4-thiadiazol-2-yl)-5'-methoxy- 6-methyl-(4,4'-bipyridine)-3-carboxamide
Figure imgf000066_0001
Step-1: Synthesis of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-l -carboxylate.
Figure imgf000066_0002
To a stirred solution of ethyl 2-hydroxycyclopentane-l -carboxylate (4.8 g, 30.3 mmol) in N,N-Dimethylformamide (DMF) (50 mL) was added imidazole (3.10 g, 45.5 mmol), DMAP (0.185 g, 1.517 mmol) and TBDMS-C1 (5.49 g, 36.4 mmol) under nitrogen atmosphere at room temperature. The resulting reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with ice-water (200 mL) and extracted with methyl tertiary butyl ether (200 mL x 2). The combined organic layer was washed with saturated sodium bicarbonate solution (30 mL) and the with brine (30 mL). The organic layer was dried over sodium sulphate and evaporated under vacuum to get the crude product as a colorless oil. The crude residue was pre-absorbed on silica, loaded on the biotage prepacked column (40g) and eluted at 20% of Ethyl acetate in petroleum ether for 60 min. The appropriate fractions were collected and concentrated under vacuum to afford ethyl 2-((tert- butyldimethylsilyl)oxy)cyclopentane-l -carboxylate (7 g, 25.7 mmol, 85 % yield) as a colourless oil. MS (ESI) calculated for Ci^sOsSi, (M)+ 272.18; found, GCMS m/z = 215.1 (M-57) (mixture of diastereomers). 1H-NMR (400 MHz, CDC13): 5 4.52-4.38 (m, 1H), 4.25- 4.00 (m, 2H), 2.79-2.62 (m, 1H), 2.25-2.00 (m, 1H), 1.96-1.82 (m, 1H), 1.81-1.65 (m, 3H), 1.64-1.52 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H).
Step-2: Synthesis of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol.
Figure imgf000067_0001
To a stirred solution of ethyl 2-((tert-butyldimethylsilyl)oxy)cyclopentane-l -carboxylate (5.2 g, 19.09 mmol) in Tetrahydrofuran (100 mb) was added DIBAL-H (IM in THF) (28.6 mb, 28.6 mmol) drop wise at -78 °C under nitrogen atmosphere. The resulting reaction mixture was stirred at -78 °C for 15 min and then slowly warmed to 0 °C and stirred for 1 h. The reaction mixture was quenched using and 2M solution of sodium potassium tartrate (60 mb) at 0 °C and stirred for 20 min at room temperature. After 20 min, the reaction mixture was extracted with ethyl acetate (100 mb x 2). The emulsion was formed which was passed through celite. The organic layer was separated, washed with brine (20 mb), dried over sodium sulphate and concentrated under vacuum to get crude product as a colourless gum. The crude residue was pre-absorbed on silica using 40 mb DCM, 10 g of silica (60-120 mesh), loaded on the biotage pre-pack 30 g snap and eluted at 20% of Ethyl acetate in petroleum ether for 45 min with flow rate 30 mL/min. The appropriate fractions were collected and concentrated under vacuum to afford (2-((tert- butyldimethylsilyl)oxy)cyclopentyl)methanol (1.8 g, 40.8 % yield) as a colorless oil. 2.4 g of starting material was also recovered. MS (ESI) calculated for CnEheChSi, (M)+ 230.17; found, GCMS m/z = 173.1 (M-57) (99.56%). 1H-NMR (400 MHz, DMSO- 6): 5 4.45 (t, J = 5.2 Hz, 1H), 3.95 (q, J = 5.6 Hz, 1H), 3.38-3.30 (m, 1H), 3.28-3.19 (m, 1H), 1.95-1.56 (m, 4H), 1.55-1.39 (m, 2H), 1.32-1.17 (m, 1H), 0.85 (s, 9H), 0.03 (s, 6H)
Step-3: Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl) S-methyl carbonodithioate .
Figure imgf000067_0002
To a stirred solution of (2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methanol (3 g, 13.02 mmol) in Tetrahydrofuran (THF) (50 mL) under nitrogen at 0 °C was added sodium hydride (1.041 g, 26.0 mmol) in portions over 3 min. After addition, the reaction mixture was stirred at room temperature for 30 min. After 30 min, to the above reaction mixture were added carbon disulfide (1.570 mL, 26.0 mmol) followed by methyl iodide (0.814 mL, 13.02 mmol) at room temperature. The reaction mixture was stirred at room temperature for an additional 30 min. The reaction was quenched with cold water and extracted with ethyl acetate (50 mL x2). The combined organic layer was washed with brine solution. The organic layer was dried over sodium sulphate, fdtered, and concentrated under vacuum to afford 0-((2-((tert- butyldimethylsilyl)oxy)cyclopentyl)methyl) S-methyl carbonodithioate (4.45 g) as a yellow oil. MS (ESI) calculated for (Ci4H28O2S2Si) (M-CH3)', 305.11; found, 305.2. 1H-NMR (400 MHz, DMSO- 6): 54.62-4.41 (m, 2H), 4.06-3.98 (m, 1H), 2.56 (s, 3H), 2.35-2.12 (m, 1H), 1.91-1.65 (m, 3H), 1.65-1.35 (m, 2H), 1.33-1.20 (m, 1H), 0.84 (s, 9H), 0.04 (s, 6H).
Step-4: Synthesis of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazine carbothioate.
Figure imgf000068_0001
To a stirred solution of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl) S-methyl carbonodithioate (4.45 g, 13.88 mmol) in methanol (50 mL) was added hydrazine hydrate (1.069 g, 13.88 mmol) at room temperature. The reaction mixture was stirred for 2 h at room temperature. The organic solvent was removed under vacuum. The residue was diluted with water. The aqueous layer was extracted with ethyl acetate (100 mL X 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford 0-((2-((tert- butyldimethylsilyl)oxy)cyclopentyl)methyl)hydrazine carbothioate (3.4 g, 77 % yield) as a yellow liquid. MS (ESI) calculated for (Ci3H28N2C>2SSi) (M+l)+, 305.17; found, 305.2.
Step-5: Synthesis of rac-5-(((lS,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)- l,3,4-thiadiazol-2-amine and rac-5-(((lR,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl) methoxy)- 1 ,3,4-thiadiazol-2-amine .
Figure imgf000068_0002
To a stirred solution of O-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methyl) hydrazinecarbothioate (3.4 g, 11.16 mmol), in Ethanol (30 mL) were added triethylamine (1.556 mL, 11.16 mmol) followed by Cyanogen bromide (1.183 g, 11.16 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The organic solvent was removed under vacuum. The residue was diluted with water. The aqueous layer was extracted with ethyl acetate (50 mL X 2). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum to get the crude product as orange solid.
The crude product was pre-absorbed on silica using 20 mb DCM 10 g of silica (60-120 mesh), loaded on the biotage pre-pack 45 g column, and eluted with 50% of Ethyl aetate in petroleum ether for 45 min with flow rate of 30 mL/min. The appropriate fractions were collected and concentrated under vacuum to afford 5-((2-((tert- butyldimethylsilyl)oxy)cyclopentyl) methoxy)- 1, 3, 4-thiadiazol-2 -amine (1.7 g, 46 % yield) as orange solid. MS (ESI) calculated for (Ci-iFHNiChSSi) (M+l)+, 330.54; found, 330.1.
Diastereomeric separation of 5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl) methoxy)- 1 ,3 ,4-thiadiazol-2 -amine :
5-((2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-l,3,4-thiadiazol-2-amine was purified by prep-HPLC for diastereomeric separation using the following conditions: (Column: YMC- C8 (19 x 250mm) 5pm; Mobile Phase A: lOmM ABC in MQ water, Mobile Phase B: acetonitrile 50%; RTl(min): 4.78; RT2(min): 4.94;) to afford major isomer (rac-5- ((( 1 S,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)- 1 ,3,4-thiadiazol-2-amine) (0.82 g, 22% yield) as an off-white solid with the first peak with shorter retention time and minor isomer (rac-5-(((lR,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl) methoxy)-l,3,4- thiadiazol-2 -amine) (0.4 g, 10.8%) as an off-white solid with the second peak with longer retention time. rac-5-(((lS,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl)methoxy)-l,3,4-thiadiazol-2- amine: MS (ESI) calculated for (Ci^NiChSSi) (M+l)+, 330.17; found, 330.2. 1H-NMR (400 MHz, DMSO- 6): 5 6.73 (s, 2H), 4.17 (d, J = 6.8 Hz, 2H), 3.98 (q, J = 5.6 Hz, 1H), 2.15-2.05 (m, 1H), 1.86-1.74 (m, 2H), 1.70-1.59 (m, 1H), 1.58-1.40 (m, 2H), 1.31-1.20 (m, 1H), 0.82 (s, 9H), 0.007 (s, 6H). rac-5-(((lR,2R)-2-((tert-butyldimethylsilyl)oxy)cyclopentyl) methoxy)- 1,3, 4-thiadiazol-2- amine: MS (ESI) calculated for (C14H2 -NiChSSi) (M+l)+, 330.17; found, 330.2. 1H-NMR (400 MHz, DMSO- 6): 5 6.70 (s, 2H), 4.32-4.18 (m, 3H), 2.26-2.15 (m, 1H), 1.80-1.64 (m, 3H), 1.62-1.50 (m, 2H), 1.42-1.30 (m, 1H), 0.84 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).
Step-6: Synthesis of (lS,2R)-2-(((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol and ( 1 R,2S)-2-(((5 -amino- 1 ,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan- 1 -ol
Figure imgf000070_0001
To a stirred solution of rac-5-(((lS,2R)-2-((tert- butyldimethylsilyl)oxy)cyclopentyl)methoxy)-l,3,4-thiadiazol-2-amine (5.6 g, 16.99 mmol) in dichloromethane (50 mL) was added trifluoroacetic acid (19.64 mL, 255 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with saturated bicarbonate solution (100 mL) and extracted with ethyl acetate (100 mL x 6). The combined organic layer was dried over sodium sulphate and evaporated under vacuum to get the crude product as an off-white solid.
The crude product was pre-absorbed on silica using 50 mL DCM 20 g of silica (60-120 mesh), loaded on the biotage pre-pack 100 g column, and eluted with 10% of Methanol in dichloromethane for 30 min with a flow rate of 60 mL/min. The appropriate fractions were collected and concentrated under vacuum to afford rac-(lS,2R)-2-(((5-amino-l,3,4- thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol (2.5 g) as off-white solid (mixture of enantiomers). rac-(lS,2R)-2-(((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol (2.5 g) was separated by prep-chiral SFC with the following conditions: (Column: Chiralcel OXH (30 x 250 mm) 5pm; Mobile Phase A: CO2, Mobile Phase B: MeOH; Flow rate: 70 mL/min; Gradient: isocratic 30% B; Column Temperature(°C): 35; Back Pressure(bar): 110; Wave Length: 254 nm; RTl(min): 5.74; RT2(min): 7.31; Sample Solvent: MeOH (40 mL);
Injection Volume: 0.8 mL; Number of runs: 71) to afford 5-(((lS,2R)-2-((tert- butyldimethylsilyl)oxy)cyclopentyl)methoxy)-l,3,4-thiadiazol-2-amine (1 g, 26.0 % yield) as a off-white solid with the first peak on chiral SFC with shorter retention time and (lR,2S)-2- (((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol (1 g, 27.0 % yield) as a off- white solid with the second peak on chiral SFC with longer retention time. The absolute stereochemistry was not determined.
(lS,2R)-2-(((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol: MS (ESI) calculated for (C8H13N3O2S) (M+l)+, 216.08; found, 216.0. 1H-NMR (400 MHz, DMSO- 6): 5 6.73 (s, 2H), 4.69 (d, J = 4.4 Hz, 1H), 4.26 (dd, J = 6.0 Hz, 10.0 Hz, 1H), 4.12 (dd, J = 7.2 Hz, 10 Hz, 1H), 3.82 (quintet, J = 5.6 Hz, 1H), 2.13-2.02 (m, 1H), 1.90-1.71 (m, 2H), 1.70- 1.59 (m, 1H), 1.58-1.40 (m, 2H), 1.34-1.22 (m, 1H). (lR,2S)-2-(((5-amino-l,3,4-thiadiazol-2-yl)oxy)methyl)cyclopentan-l-ol: MS (ESI) calculated for (C8H13N3O2S) (M+l)+, 216.08; found, 216.0. 1H-NMR (400 MHz, DMSO- 6): 5 6.73 (s, 2H), 4.69 (d, J = 4.4 Hz, 1H), 4.26 (dd, J = 6.0 Hz, 10.0 Hz, 1H), 4.12 (dd, J = 7.6 Hz, 10.4 Hz, 1H), 3.85-3.78 (m, 1H), 2.12-2.02 (m, 1H), 1.90-1.70 (m, 2H), 1.70-1.60 (m, 1H), 1.59-1.40 (m, 2H), 1.34-1.22 (m, 1H).
Step-10: Synthesis of 2'-chloro-N-(5-(((lS,2R)-2-hydroxycyclopentyl)methoxy)- 1,3,4- thiadiazol-2-yl)-5'-methoxy-6-methyl-(4,4'-bipyridine)-3-carboxamide (Compound 9)
Figure imgf000071_0001
To a stirred solution of 2'-chloro-5'-methoxy-6-methyl-[4,4'-bipyridine]-3-carboxylic acid (0.8 g, 2.87 mmol, Intermediate H) in Acetonitrile (15 m ) and N,N-Dimethylformamide (DMF) (2.5 mL) was added (lR,2S)-2-(((5-amino-l,3,4-thiadiazol-2- yl)oxy)methyl)cyclopentan-l-ol (0.618 g, 2.87 mmol), 1-methyl-lH-imidazole (0.943 mL, 11.48 mmol) and Chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (1.208 g, 4.31 mmol) at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with water (100 mL) and extracted with Ethyl acetate (50 mL x 2). The organic phases were combined and washed with brine solution. The organic layer was dried over anhydrous Na2SC>4 and fdtered. The fdtrate was concentrated under reduced pressure to get the crude product as an off-white solid. The crude product was mixed with another batch of 180 mg material. The combined crude was pre-absorbed on silica using 20 mL DCM and 5g of silica (60-120 mesh), loaded on the pre-packed biotage 45g column and eluted at 10% of methanol in dichloromethane for 60 min with flow rate 30 mL/min. The appropriate fractions were collected and concentrated under vacuum to afford Isomer 1 (600 mg, 43.6 % yield) as a white solid (Compound 9). The stereochemistry was determined using X-ray crystallography. MS (ESI) calculated for (C21H22CIN5O4S) (M+l)+, 476.12; found, 476.0. 1H-NMR (4OO MHz, DMSO- 6): 5 12.88 (brs, 1H), 8.80 (s, 1H), 8.17 (s, 1H), 7.55 (s, 1H), 7.44 (s, 1H), 4.71 (d, J = 4.4 Hz, 1H), 4.42 (dd, J = 6.0 Hz, 10 Hz, 1H), 4.28 (dd, J = 7.2 Hz, 10 Hz, 1H), 3.86 (quintet, J = 5.2 Hz, 1H), 3.63 (s, 3H), 2.59 (s, 3H), 2.20-2.10 (m, 1H), 1.91-1.72 (m, 2H), 1.71-1.60 (m, 1H), 1.60-1.43 (m, 2H). 1.38-1.28 (m, 1H). Compound 10
(R)-2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-l,3,4-thiadiazol-2- yl)-(4,4'-bipyridine)-3-carboxamide
Figure imgf000072_0001
Step- 1 : 5 -((tetrahydrofuran-3-yl)methoxy)- 1 ,3 ,4-thiadiazol-2 -amine
Figure imgf000072_0002
To a solution of (tetrahydrofuran-3-yl)methanol (1.0 g, 9.79 mmol) in THF (5 mL) was added NaH (0.5 g, 14.69 mmol, 60%) in portions at 0 °C and stirred at 0 °C for 30 min under nitrogen atmosphere. To the above solution was added 5-bromo-l,3,4-thiadiazol-2-amine (2.1 g, 11.75 mmol) at 0 °C under nitrogen. The resulting solution was then stirred at 0 °C for 1 hr. The reaction mixture was quenched by the addition of water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, fdtered, and concentrated under vacuum. The resulting residue was dissolved in DCM (3 mL) and was applied to a 20 g silica gel column that was eluted with 0-50% ethyl acetate in petroleum ether within 30 min to afford 5-((tetrahydrofuran-3-yl)methoxy)-l,3,4-thiadiazol- 2 -amine (120.0 mg, 69 %) as a yellow solid, MS (ESI) calc’d for (C7H11N3O2S) (M+l)+, 202.1; found, 202.1.
Step-2: Synthesis of 2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)- l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide
Figure imgf000072_0003
To a solution of 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3 -carboxylic acid (100.0 mg, 0.36 mmol, Example 1, Step 3) in Acetonitrile (2 mL) were added 5-((tetrahydrofuran-3- yl)methoxy)-l,3,4-thiadiazol-2-amine (72.0 mg, 0.36 mmol) and 1 -methylimidazole (147.0 mg, 1.79 mmol) at 20 °C under nitrogen. To the above solution was added TCFH (100.0 mg, 0.36 mmol) in Acetonitrile (2 mL) at 20 °C under nitrogen. The resulting mixture was then stirred at 20 °C for 1 hr. The resulting residue was dissolved in DMF (1 mL) which was applied to a 20 g Cl 8 column and purified by Combi Flash (Biotage Isolera Prime), eluted with 5—55% acetonitrile in water within 30 min to afford racemic 2'-chloro-5'-methoxy-6- methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3- carboxamide (67.4 mg, 39%) as a yellow solid. MS (ESI) calc’d for (C20H20CIN5O4S) (M+l)+, 462.1; found 462.1. 1HNMR (400 MHz, DMSO- e) 5 12.87 (s, 1H), 8.81 (s, 1H), 8.17 (s, 1H), 7.54 (s, 1H), 7.43 (s, 1H), 4.44 - 4.30 (m, 2H), 3.82 - 3.72 (m, 2H), 3.71 - 3.60 (m, 1H), 3.64 (s, 3H), 3.53 - 3.50 (m, 1H), 2.74 - 2.75 (m, 1H), 2.59 (s, 3H), 2.02 - 1.99 (m, 1H), 1.66 - 1.64 (m, 1H).
Step-3: Separatino of (R)-2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3- yl)methoxy)-l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide
Racemic 2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)- 1,3,4- thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (67.4 mg) was separated by prep-chiral HPLC with the following conditions: (Column: CHIRALPAK IF, 2*25 cm, 5 pm; Mobile Phase A: MtBE(0.1% FA)-HPLC, Mobile Phase B: EtOH-HPLC; Flow rate: 11 mL/min; Gradient: 50% B to 50% B in 34 min; Wave Length: 220/254 nm; RTl(min): 18.66; RT2(min): 27.68; Sample Solvent: MeOH: DCM=1: 1; Injection Volume: 1 mL; Number Of Runs: 2) to afford (R)-2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)- 1, 3, 4-thiadiazol-2-yl)-(4,4'-bipyridine)-3 -carboxamide (25.6 mg, 38%) as a white solid with retention time on chiral-HPLC and (S)-2'-chloro-5'-methoxy-6-methyl-N-(5- ((tetrahydrofuran-3-yl)methoxy)-l,3,4-thiadiazol-2-yl)-(4,4'-bipyridine)-3-carboxamide (25.3 mg, 37%) as a white solid with retention time on chiral-HPLC. The absolute stereochemistry was determined using vibrational circular dichroism spectroscopy.
(R)-2'-chloro-5'-methoxy-6-methyl-N-(5-((tetrahydrofuran-3-yl)methoxy)-l,3,4-thiadiazol-2- yl)-(4,4'-bipyridine)-3-carboxamide: MS (ESI) calc’d for (C20H20CIN5O4S) (M+l)+, 462.1; found, 462.1. 1HNMR (400 MHz, DMSO- r,) 5 12.91 (s, 1H), 8.82 (s, 1H), 8.17 (s, 1H), 7.52 (s, 1H), 7.41 (s, 1H), 4.43 - 4.28 (m, 2H), 3.76 - 3.73 (m, 2H), 3.65 - 3.63 (m, 4H), 3.53 - 3.51 (m, 1H), 2.73 - 2.70 (m, 1H), 2.58 (s, 3H), 2.01 - 2.05 (m, 1H), 1.65 - 1.68 (m, 1H).
Biological Assay
Pol Theta ATPase activity The ability of the compounds of Formula (II) to inhibit ATPase activity of Pol theta (1-899) was determined using the assay described below.
Pol Theta ATPase activity was determined by measuring the rate of ATP turn over in a NADH oxidation-coupled enzymatic assay. 10-point dilution series of compounds were used in a 384 well format for the inhibition assays. Pol theta (1-899) (10 nM) in assay buffer (20 mM Tris HC1 (pH 7.80), 80 mM KC1, 10 mM MgCh, 1 mM DTT, 0.01% BSA, 0.01% Tween, 5% glycerol) was transferred to the test wells (20 pL), except the low control wells (20 pL of assay buffer was added to the low control wells). The plate was then incubated at room temperature for 15 min. An equal volume (20 pL) of 100 pM ATP, 300 nM dTso (single-stranded DNA (ssDNA) containing 50 thymine bases), 300 pM NADH, 6 mM PEP, 10 U/mL lactate dehydrogenase and 20 U/mL pyruvate kinase in assay buffer was added to all the test wells. The plate was then centrifuged at 1000 rpm for 1 min. The reaction was monitored for 30 min by measuring absorbance (X= 340 nm) in a Tecan Spark multimode plate reader every minute. The high control (DMSO with enzyme) with low absorbance intensity represents no inhibition of ATPase reaction while the low control (DMSO with buffer) with high absorbance intensity represents full inhibition of ATPase activity. Slope of the reaction progress curves were used to calculate the rate of ATP hydrolysis. The rates were used to determine the percent inhibition using a four-parameter inhibition model to generate ICso, Hill slope and max inhibition.
The IC50 of the compounds 4-10 are disclosed in Table 2 below:
Table 2.
Figure imgf000074_0001
IC50: 10 uM > (+) > 1 uM ; 1 uM > (++) > 500 nM;
500 nM > (+++) > 200 nM; 200 nM > (++++)
DLD-1 BRCA2-/- Cell Viability Assay
Cell viability assay was conducted for certain compounds. DLD-1 BRCA2-/- cells (Horizon Discovery) were plated in 200 pl growth medium at 500 cells/well in 96-well flat-bottomed plates. After an overnight incubation at 37°C, 5% CO2, compounds were added to cells across a concentration range. Cells were incubated for a further 7 days at 37°C, 5% CO2, to allow for 4-5 population doublings. Cells were then fixed with 4% paraformaldehyde and stained with Hoechst to allow cells to be imaged on the Incell 2200 reader. The cell count data were normalised to control wells containing DMSO (high control) and 100 pM doxorubicin (low control) before analysis using a 4-parameter logistic curve for calculation of IC50 and pICso.
The pro-drugs are converted by endogenous alkaline phosphatases or esterases present in the assay to give the free parent compounds of equivalent potency to dosing parent compound alone (see Table 3).
Table 3.
Figure imgf000075_0001
Solubility & PK Assay
The solubility of Compound A and Compound B in FASSIF (Fasted State Simulated Intestinal Fluid) are 17 ug/mL and 37 ug/mL, respectively. The FASSIF solubility for the prodrugs (Examples 1, 2, and 3) is greater than 1 mg/mL.
Pharmacokinetics data of the prodrug compounds of Example 1 and Example 2 and the parent compound (Compound A) were measured. Wistar-Han rats were dosed orally in 1% methyl cellulose formulation with each compound at doses as listed in Table 4 below. Blood samples were collected up to 24 hours and samples were analyzed by LC-MS/MS for Compound A concentration in all three groups. Both prodrugs demonstrated significant improvement in exposures of Compound A when compared to dosing of Compound A alone. Dose normalized AUC improvement was roughly 15X with Example 1 and 8.5X with Example 3 when compared to dosing of Compound A only. Similar improvements in Cmax were noted with prodrug approach.
Table 4
Figure imgf000075_0002
Figure imgf000076_0001
Combination Therapy: Assessing Combination Synergy Index with Compounds of Formula (II) and a PARP inhibitor
A 15 -day colony formation assay was performed in BRCA1 mutant MDA-MB-436 cell line. Combinations included double titrations of seven different PolQ inhibitor compounds (Compound 4, 5, 6, 7,8, 9 and 10) with the PARP inhibitor, Niraparib.
Optimal cell seeding was determined by assessing the growth of colonies over a range of seeding densities in a 6-well format to identify conditions that permitted growth for 15 days. Cells were then plated at the optimal seeding density (1000 cells per well) and treated with a double titration of a 9-point three-fold dilution series of the PolQ inhibitor compounds and a 3-point three-fold dilution series of Niraparib. This double titration was compared to 9-point 3 -fold dilution series of the PolQ inhibitor compounds single agent or a 3-point three-fold dilution series of Niraparib single agent alone or to 0.1% DMSO. Concentrations tested for the PolQ inhibitors alone or in combination ranged from 4.6 nM to 30,000 nM and Niraparib alone or in combination ranged from 0.56 nM to 5 nM. Plates were incubated for 15 days at 37 °C in 5% CO2. Media containing compounds were replenished at 8 days of the treatment. After 15 days of treatment, cells were fixed with 95% ethanol solution and stained with 0.25% (w/v) crystal violet staining solution (Sinopharm Chemical Reagent Beijing Co., Ltd). The plates were washed with PBS and scanned on a LI-COR Odyssey CLx imager (LI-COR) using the 700 nm channel.
To assess combination synergy index, Combenefit software tool was applied using classical Bliss synergy model for the combinations of compound doses across the dose range tested. Di Veroli et al., “An interactive platform for the analysis and visualization of drug combinations,” Bioinformatics. 2016;32(18):2866-2868. Synergistic growth effect was observed with majority of PolQ inhibitor compounds in combination with Niraparib across several combination concentrations as assessed by Combenefit (Bliss model) in MDA-MB- 436 cell line (Table 5-Table 11; FIG. 1A-FIG. 7). Scores >10 (highlighted black) were considered synergistic. Table 5-Table 11: Bliss Synergy of PolQ inhibitor compound and Niraparib combinations in MDA-MB-436 cell line.
Table 5A
Figure imgf000077_0001
Table 5B
Figure imgf000077_0002
Table 6A
Figure imgf000077_0003
Table 6B
Figure imgf000077_0004
Table 7
Figure imgf000078_0001
Table 8
Figure imgf000078_0002
Table 9
Figure imgf000078_0003
Table 10
Figure imgf000078_0004
Table 11
Figure imgf000078_0005
In Vitro Efficacy of Parent Compounds and Niraparib Combination
In vitro efficacy was assayed as % of cell viability in a 7-days CellTiter-Glo® (CTG) assay CTG. COMPOUND A and COMPOUND B were tested as single agents in the BRCA1 mutant breast cancer line MDA-MB-436 and in the in BRCA2 mutant ovarian cancer cell line PEO1; single agents ICso values were above 0.5 pM which is indicative of poor efficacy. When used in combination with Niraparib, both COMPOUND A and COMPOUND B showed synergy in MDA-MB-436 as well as in PEO1 cells, as indicated by the decreases ECso values.
A. In vitro efficacy of COMPOUND A and Niraparib combination in MDA-MB-436 breast cancer cells
MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution ranging from 30 pM to 0.014 pM COMPOUND A, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib. After 7 days, cell viability was assessed with the CTG assay. Dose response curves were interpolated using GraphPad Prism 9, and the synergy of drug combinations using data from the cell viability assays was analyzed with ComBenefit 2.02. Niraparib synergized with COMPOUND A and decreased ECso values of COMPOUND A in MDA-MB-436 cells (FIG. 8A-8D and Table 12).
Table 12. In vitro synergy of COMPOUND A and Niraparib reduces cell viability in MDA- MB-436 breast cancer cells.
Figure imgf000079_0001
B. In vitro efficacy of COMPOUND B and Niraparib combination in MDA-MB-436 breast cancer cells
MDA-MB-436 cells were treated with an 8x5 drug matrix, with an 8-point, 3-fold dilution ranging from 30 pM to 0.014 pM COMPOUND B, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib. Cell viability was assessed as described above. Niraparib synergized with COMPOUND B and decreases ECso values of COMPOUND B in MDA- MB-436 cells (FIG. 9A-9D and Table 13). Table 13. In vitro synergy of COMPOUND B and Niraparib reduces cell viability in MDA- MB-436 breast cancer cells.
Figure imgf000080_0001
C. In vitro efficacy of COMPOUND A and Niraparib combination in PEO1 ovarian cancer cells
PEO1 cells were treated with an 8x5 drug matrix, with an 8-point, 3 -fold dilution ranging from 30 pM to 0.014 pM COMPOUND A, and a 5-point, 3-fold dilution ranging from 5 pM to 0.062 pM Niraparib. Cell viability was assessed as described above. Niraparib synergized with COMPOUND A and decreased ECso values of COMPOUND A in PEO1 cells (FIG. 10A-10D and Table 14).
Table 14. In vitro synergy of COMPOUND A and Niraparib reduces cell viability in PEO1 ovarian cancer cells.
Figure imgf000080_0002
D. In vitro efficacy of COMPOUND B and Niraparib combination in PEO1 ovarian cancer cells
PEO1 cells were treated with an 8x5 drug matrix, with an 8-point, 3 -fold dilution ranging from 30 pM to 0.014 pM COMPOUND B, and a 5-point, 3-fold dilution ranging from 100 nM to 1.2 nM Niraparib. Cell viability was assessed as described above. Niraparib synergized COMPOUND B and decreased ECso values of COMPOUND B in PEO1 cells (FIG. 11A- 11D and Table 15).
Table 15. In vitro synergy of COMPOUND B and Niraparib reduces cell viability in PEO1 ovarian cancer cells.
Figure imgf000080_0003
Figure imgf000081_0001
Efficacy of Compound 4 and Compound 11 in MDA-MB-436 CDX Model
Summary
Compound 4 was examined in the MDA-MB-436 efficacy model for tumor growth inhibition and durability of efficacy when administered as monotherapy and in combination with niraparib (Compound 11). Compound 4 was found to significantly inhibit tumor growth as a monotherapy and in combination with niraparib. Following drug administration for up to 78 days, only the combination treatments resulted in continuous stable disease or tumor regressions, as tumors became resistant to niraparib monotherapy on treatment. In combination with niraparib, the combination treatment improved the durability of response by preventing tumor growth and enhanced the number of complete responses observed. The combination of Compound 4 and niraparib administered at 10, 30 or 100 mg/kg BID produced complete responses (no remaining tumor) in 20, 30, and 50% of mice, respectively.
Study Design & Results
The effect of Compound 4 and Compound 11 as single-agent anti-tumor agents and in combination was assessed in the MDA-MB-436 human breast cancer cell line xenograft model. Cells were expanded in DMEM medium with 10% fetal bovine serum. Ten million cells in log growth phase were resuspended in DMEM containing 50% Matrigel and implanted subcutaneously into the flank of each recipient female NOD SCID mouse. Mice were housed in microisolator cages with soft wood bedding. The environment was maintained by providing a 12-hour light cycle, temperature of 23 ± 3 °C, and 40-70% relative humidity.
Tumor Volume (TV) was calculated using the following formula: TV (mm3) = (width x width x length)/2. Tumor growth inhibition (TGI) was calculated by [(TV controlfinal - TV treatedfinai)/(TV controlfinal - TV controlinitial) x 100], TV was analyzed for statistical significance utilizing GraphPad Prism version 9.1.0. Repeated Measures 2-Way ANOVA with Tukey’s Multiple Comparisons was utilized, and P-values were presented from study day 30 and were considered statistically significant if less than 0.05. A Mixed-effects model with Tukey’s Multiple Comparisons was utilized for day 78 and results were considered statistically significant if less than 0.05. Mean tumor volume at dosing start was approximately 187 to 193 mm3, with ten mice randomized to each treatment group. The study consisted of eight treatment groups. Mice were dosed orally, twice per day (BID), with Vehicle A or Compound 4 at 10, 30, or 100 mg/kg, or dosed once per day (QD) with Vehicle B or Compound 11 at 25 mg/kg, or the combination of Compound 4 at 10, 30, or 100 mg/kg BID and Compound 11 at 25 mg/kg QD. The control groups consisted of Vehicle A (for Compound 4, 0.5% 400 cps methylcellulose with 0.5% Tween-80 in sterile water) and Vehicle B (for Compound 11, 0.5% 400 cps methylcellulose in sterile water). Compound 4 was administered first in the morning, followed two hours later with Compound 11, and the second dose of Compound 4 was provided 6-hours following the dose of Compound 11.
The study examined the efficacy of Compound 4 or Compound 11 as monotherapies as well as the efficacy of the combination of Compound 4 and Compound 11 as combination antitumor therapies. Furthermore, the study examined the durability of the treatment response and the clinical outcome of the treatment. The efficacy of each treatment group was compared to the vehicle control group alone while the durability of the treatment response for the combination groups were compared to Compound 11 alone.
The vehicle control group reached endpoint tumor volume on study day 30. The treatments groups on day 30 were compared to the control group on day 30 to calculate the tumor growth inhibition (TGI), Table 16. Compared to the vehicle group, each treatment group produced statistically significant TGI on day 30. Compared to Compound 11 alone on Day 30, Compound 4 at 100 mg/kg BID combined with Compound 11 improved the TGI observed over that of Compound 11 alone.
The study continued for the treatment groups until study day 78 or until the group reached tumor volume endpoint, FIG. 12. Tumor volume endpoint for individual treatment groups was defined as 50% of mice within the treatment group harbor tumors greater than 2000 mm3. Compound 4 administered at 10 and 30 mg/kg BID reached endpoint on Day 40 while Compound 4 administered at 100 mg/kg BID reached endpoint on Day 50. Compound 11 administered at 25 mg/kg demonstrated tumor growth prevention beginning on study day 16 and subsequently resulted in shrinking tumors until study day 34. Following day 34, treatment with Compound 11 alone was no longer effective and tumors from each mouse grew on treatment, FIG. 13A. On day 75, one mouse from Compound 11 treatment was euthanized for reaching endpoint tumor volume. On day 78, the study was completed, and the efficacy of the combination of Compound 4 and Compound 11 was compared to that of Compound 11 alone.
On day 78, compound 11 administered at 25 mg/kg QD resulted in a mean tumor volume of 1313 mm3 and contained no mice with tumors that were responding to treatment. The combination of Compound 11 and Compound 4 administered at 10, 30 or 100 mg/kg BID produced mean tumor volumes of 145, 82, and 63 mm3, respectively. In addition, each combination group enhanced the response rate over Compound 11 alone. The combination of Compound 11 and Compound 4 administered at 10, 30 or 100 mg/kg BID produced complete responses (no remaining tumor) in 20, 30, and 50% of mice, respectively, Table 17. The combination of Compound 4 and Compound 11 produced a durable anti-tumor response and was significantly more effective than Compound 11 alone.
Table 16. Summary of Efficacy of Compound 4 and Compound 11 in MDA-MB-436 on study day 30
Figure imgf000083_0001
Table 17. Summary of Day 78 tumor volumes and response rate for Compound 11 and for Compound 11 combinations with Compound 4
Figure imgf000083_0002
Figure imgf000084_0001
Compound A regressed tumors when combined with Niraparib in HR deficient human cell line xenograft model MDA-MB-436
The effect of Compound A on tumor growth in vivo was assessed in mice bearing BRCA1 mutant MDA-MB-436 cell line xenografts (FIG. 14). Ten million(10e7) MDA-MB-436 viable cells were implanted with 50% Matrigel in the back flank of the 5-7 weeks old NSG mice (Jax). When the tumor volume reach to around 200 mm3 size, the animals were randomized into efficacy study. MDA-MB-436 tumor bearing animals were either dosed twice daily with vehicle 0.5% Methylcellulose with 0.5% Tween, BID, PO or Compound A 100 mg/kg BID, PO or once daily Niraparib 25 mg/kg QD, PO or Compound A lOOmg/kg in combination with Niraparib 25 mg/kg QD for 70 days. No significant tumor growth inhibition (TGI) was observed with lOOmg/kg BID dose of Compound A alone, whereas Niraparib at 25mg/kg QD dose (corresponding 200mg clinical dose) demonstrated 72% tumor growth inhibition. Strikingly, when Niraparib 25mg/kg was dosed in combination with 100 mg/kg Compound A BID, complete tumor regression was observed with TGI 104%. These in vivo studies suggest Compound A is synergistic in combination with Niraparib to achieve better efficacy.
To confirm the combination benefit of Compound A and Niraparib, the efficacy in vivo was tested using a PARP -inhibitor progressed ovarian carcinoma patient-derived xenograft (PDX) model, 134-T, generated from an HR-deficient (BRCA1 Frame-shift (FS) mutation) highgrade serous ovarian carcinoma patient. This model was developed from a patient who was heavily treated with and progressed over lines of chemotherapy including Carboplatin, Paclitaxel, and Doxorubucin, and Bevacizumab and lastly PARP inhibitor, Talozaparib in the clinic.
134-T tumor fragments (4x4 mm) were implanted into flank of 5-7 weeks old NOD SCID gamma (NSG) mice. When tumors reach to 150mm3 size, tumor bearing tumors of the HR- deficient 134-T ovarian cancer PDX model were treated with Niraparib (25mg/kg QD), 100 mg/kg Compound A alone BID or a combination of 30mg/kg BID or 100 mg/kg BID Compound A with Niraparib 25mg/kg (FIG. 15). While Compound A alone did not yield to significant tumor growth inhibition (18% TGI), Niraparib at 25 mg/kg resulted in 60% tumor growth inhibition. After 35 days of dosing, 82% tumor growth inhibition was observed with 30mg/kg BID dosing of Compound A and 90% tumor growth inhibition with lOOmg/kg BID dosing similar to the MDA-MB-436 model.
Efficacy study with Compound of Example 1 in BRCA1 mutant 134T Ovarian PDX model
The efficacy of Compound of Example 1 in combination with Niraparib 25 mg/kg was also tested in 134-T PDX model. When tumors reached 150 mm3 size, tumor bearing tumors of the HR-deficient 134-T ovarian cancer PDX model were treated with Niraparib (25 mg/kg QD) alone or Niraparib (25 mg/kg QD) in combination with Example 1 at doses of 30 mg/kg BID, 100 mg/kg QD, or 100 mg/kg BID (FIG. 16). Niraparib at 25 mg/kg resulted in 60% tumor growth inhibition. While the combination with 30 mg/kg BID of Example 1 dosing resulted in 75% TGI and 100 mg/kg QD of Example 1 dosing resulted in 77% TGI, the combination with 100 mg/kg BID of Example 1 dosing leads to superior efficacy with 94% TGI in 134-T PDX model.
Particular embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Upon reading the foregoing, description, variations of the disclosed embodiments may become apparent to individuals working in the art, and it is expected that those skilled artisans may employ such variations as appropriate. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:
1 A compound of Formula (I):
Figure imgf000086_0001
wherein:
R1 is H, Ci-4 alkyl, Ci-4 alkoxy, halo, Ci-4 haloalkyl, or Ci-4 haloalkoxy;
R3a, R3b, and R3c are each independently H, Ci-4 alkyl, Ci-4 haloalkyl, halo, Ci-4 alkoxy, or
Ci-4 haloalkoxy;
Z is:
Figure imgf000086_0002
X is -CH2O-P(O)(ORa)(ORb), -CH2-O-C(O)-Ci-6alkylene-CO2H,
-CH2-O-C(O)-CI-6 alkylene-O-P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-
P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-NR:iRb, or — CH2— 0~ C(0)— Ci-6 alkylene-heterocycloalkyl;
Ra and Rb are each independently H or Ci-6 alkyl; and each heterocycloalkyl has from 4 to 6 ring members and from 1 to 3 heteroatoms as ring vertices independently selected from N, O, and S; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is -CH2O-P(O)(ORa)(ORb), -CH2-O-C(O)-CI-6 alkylene-CCFH, or -CH2-O-C(O)- Ci-6 alkylene-P(O)(ORa)(ORb).
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is -CH2O-P(O)(ORa)(ORb) or -CH2-O-C(O)-Ci-6alkylene-CO2H.
Q
4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is -CH2O-P(O)(ORa)(ORb).
5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is -CH2-O-C(O)-CI-6 alkylene-CChH.
6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is
Figure imgf000087_0001
7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is
Figure imgf000087_0002
8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is
Figure imgf000087_0003
9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein X is
Figure imgf000088_0001
10. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R1 is Ci-4 alkyl.
11. The compound of any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, wherein R1 is methyl.
12. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R3a is Ci-4 alkoxy, or Ci-4 haloalkoxy.
13. The compound of any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein R3a is methoxy.
14. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R3b is Ci-4 alkyl or halo.
15. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R3b is methyl or chloro.
16. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R3b is methyl.
17. The compound of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein R3b is chloro.
18. The compound of any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R3c is H or halo.
19. The compound of any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R3c is H.
20. The compounds of any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein R3c is fluoro.
21. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein Z is
Figure imgf000089_0001
22. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein Z is
°0°
23. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein Z is
Figure imgf000089_0002
24. The compound of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, wherein Z is
Figure imgf000089_0003
25. The compound of claim 1, selected from Table 1.
26. A pharmaceutical composition comprising a compound of any one of claims 1 to 25, and at least one pharmaceutically acceptable excipient.
27. A method for treating a disease characterized by overexpression of PolO in a patient comprising administering to the patient a therapeutically effective amount of a compound of any one of claims 1 to 25, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 26.
28. The method of claim 27, wherein the patient is in recognized need of such treatment and the disease is a cancer.
29. A method of treating a homologous recombinant (HR) deficient cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of any one of claims 1 to 25, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 26.
30. The method of claim 29, wherein the patient is in recognized need of such treatment.
31. A method for treating a cancer in a patient, wherein the cancer is characterized by a reduction or absence of BRCA gene expression, the absence of the BRCA gene, absence of BRCA protein, or reduced function of BRCA protein, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1 to 25, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 26.
32. The method of any one of claims 27 to 31, wherein the cancer is lymphoma, rhabdoid tumor, multiple myeloma, uterine cancer, gastric cancer, peripheral nervous system cancer, rhabdomyosarcoma, bone cancer, colorectal cancer, mesothelioma, breast cancer, ovarian cancer, lung cancer, fibroblast cancer, central nervous system cancer, urinary tract cancer, upper aerodigestive cancer, leukemia, kidney cancer, skin cancer, esophageal cancer, and pancreatic cancer.
33. The method of any one of claims 27 to 32, further comprising administering to the patient a therapeutically effective amount of a PARP inhibitor or a pharmaceutically accpetable salt thereof.
34. The method of claim 33, wherein the PARP inhibitor is niraparib.
35. The method of claim 34, wherein the PARP inhibitor is niraparib tosylate monohydrate.
36. The method of claim 33, wherein the PARP inhibitor is olaparib.
37. A Pole inhibitor for use in treating cancer, wherein the PolO inhibitor is a compound of any one of claims 1 to 25, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 26.
38. Use of a PolO inhibitor in the manufacture of a medicament for treating cancer, wherein the PolO inhibitor is a compound of any one of claims 1 to 25, or a pharmaceutically accpetable salt thereof, or a pharmaceutical composition of claim 26.
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