Classifications

• • 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

• 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
• 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 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.
• 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.
• Formula (II) has the structure wherein Z, R 1 , R 3a , R 3b , and R 3c have the meanings provided hereinbelow.
• 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. 5 depicts excess over Bliss synergy of Compound 8 and Niraparib inhibitor combinations in MDA-MB-436.
• FIG. 7 depicts excess over Bliss synergy of Compound 10 and Niraparib inhibitor combinations in MDA-MB-436 cell line.
• 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. 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.
• 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.
• 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.
• 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.
• 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 "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.
• 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.
• 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.
• 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.
• 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,
• 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
• 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 Representative compounds of Formula (I) are listed in Table 1 below.
• 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
• 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
• 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
• Pol0 Inhibitors for combination therapy with a PARP inhibitor for combination therapy with a PARP inhibitor
• R 3a , R 3b , and R 3c are each independently H, Ci-4 alkyl, Ci-4haloalkyl, halo, Ci-4 alkoxy, or Ci-4 haloalkoxy;
• 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 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
• Embodiment 5 The method of embodiment 1 or embodiment 2, wherein the Pol0 inhibitor is a compound of Formula (II): wherein:
• Embodiment 8 The method of any one of embodiments 5 to 7, wherein R 3a is Ci-4 alkoxy, or Ci-4 haloalkoxy/
• 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 41 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 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.
• Step-3 2-chloro-5-methoxy-6-methyl-(4,4-bipyridine)-3-carboxylic acid
• 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
• 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-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-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-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
• 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
• 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-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-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-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
• 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
• 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.
• 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.
• 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.
• 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.
• 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.

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