EA043826B1 - COMBINATION THERAPY FOR THE TREATMENT OF TRIPLE NEGATIVE BREAST CANCER - Google Patents

Description

The present invention relates to the treatment of breast cancer.

State of the art

Triple negative breast cancer (TNBC), defined by the absence of estrogen receptor (ER) and progesterone receptor (PR) expression and the absence of overexpression and amplification of human epidermal growth factor receptor 2 (HER2), accounts for about 10-20% of all breast cancers glands. Patients with TNBC have an overall poorer prognosis compared with other types of breast cancer, with an increased likelihood of early recurrence and death (Bauer et al., 2007). Metastatic disease is characterized by a high incidence of metastases to the visceral and central nervous systems, with a median survival of approximately 1 year (Kassam et al., 2009). Therefore, new therapeutic strategies are urgently needed.

Recent advances in disease biology may provide opportunities to classify this heterogeneous entity into molecular subtypes with distinct drivers (Bareche et al., 2018). In particular, patients with breast cancer and terminal BRCA1 and BRCA2 mutations benefit from treatment using a class of targeted agents called poly(ADP-ribose) polymerase (PARP) inhibitors, which target base excision repair (DNA repair machinery). ) and which cause artificial lethality in tumors deficient in DNA repair mechanisms such as homologous recombination. Indeed, two phase 3 trials involving patients with metastatic breast cancer with terminal BRCA1 or BRCA2 mutations reported positive results with the PARP inhibitors olaparib (Robson et al., 2017) and talazoparib (Litton et al., 2017). ) compared to standard chemotherapy. Based on these results, the US Food and Drug Administration approved olaparib for the treatment of metastatic breast cancer with terminal BRCA mutations.

Although the prevalence of BRCA1 and BRCA2 mutations is higher in TNBC (up to 24% in some groups) (Copson et al., 2018), the vast majority of patients with TNBC do not carry terminal BRCA1 or BRCA2 mutations and therefore may not benefit from PARP inhibitor treatment (O'Shaughnessy et al., 2014).

In the preclinical setting, combinatorial strategies show promise to sensitize BRCA-positive tumors to PARP inhibitors, and new data have been obtained for several bromodomain and extra-terminal domain (BET) inhibitors. BET proteins are epigenetic readers and exhibit high selectivity for acetylated lysine residues in histones and other proteins. They act as transcriptional regulators by associating with many gene promoters or enhancers. Early clinical trials of BET inhibitors (BETi) demonstrated limited single-agent activity in patients with hematologic malignancies (Berthon et al., 2016), NUT carcinoma (Stathis et al., 2016), and more recently, solid tumors (Aftimos et al., 2016). 2017). However, there is promise for BETi in combinations with other agents as they modulate resistance mechanisms and confer sensitivity to different agents. Several exploratory combination clinical trials using BETi are ongoing, including combinations with immune checkpoint monoclonal antibodies, androgen receptor antagonists, estrogen modulators, BCL2 inhibitors, and others.

However, it is currently unclear which BET inhibitors will synergize with a PARP inhibitor; what level of synergy is required; and which PARP inhibitor would be the best combination partner for each BET inhibitor to provide clinical benefit when administered to patients with TNBC. In addition to clinical benefit, the combination should also be safe and well tolerated at effective doses. It is not possible from the prior art to predict which combinations will exhibit the best overall profile.

Short description

The present invention discloses methods of treating triple-negative breast cancer by coadministering a BET protein bromodomain inhibitor of Formula Ia or Formula Ib, or a pharmaceutically acceptable salt or co-crystal thereof, and a second therapeutic agent to a subject in need thereof.

In some embodiments, the BET protein bromodomain inhibitor is administered concomitantly with the second therapeutic agent. In some embodiments, the BET protein bromodomain inhibitor is administered sequentially with a second therapeutic agent. In some embodiments, the BET protein bromodomain inhibitor is administered in the same pharmaceutical composition with a second therapeutic agent. In some embodiments, the BET protein bromodomain inhibitor and the second therapeutic agent are administered as separate compositions. In some embodiments, the BET protein bromodomain inhibitor and the second therapeutic agent are administered in combination with an immune checkpoint inhibitor.

In some embodiments, the second therapeutic agent is an agent used to treat breast cancer. In some embodiments, the breast cancer is TNBC.

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In some embodiments, the second therapeutic agent is a PARP inhibitor.

In some embodiments, a bromodomain inhibitor of BET proteins and a PARP inhibitor are administered in combination with an immune checkpoint inhibitor.

The BET protein bromodomain inhibitor used in the combination therapies of the present invention is a compound of formula Ia or formula Ib,

or a stereoisomer, tautomer, pharmaceutically acceptable salt or co-crystal thereof, wherein:

ring A and ring B may be optionally substituted with groups independently selected from hydrogen, deuterium, -NH ₂ , amino, (C ₄ -C ₆ ) heterocycle, (C ₄ -C ₆ ) carbocycle, halogen, -CN, -OH, -CF ₃ , (C1C ₆ )alkyl, (C1-C ₆ )thioalkyl, (C ₂ -C ₆ )alkenyl and (C1-C ₆ )alkoxy;

X is selected from -NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2O-, -CH2CH2NH-, -CH2CH2S-, -C(O)-, -C(O)CH2-, -C(O )CH2CH2-, -CH2C(O)-, -CH2CH2C(O)-, -C(O)NH-, -C(O)O-, -C(O)S-, -C(O)NHCH2-, -C(O)OCH ₂ -, -C(O)SCH ₂ -, where one or more hydrogen atoms can be independently replaced by deuterium, hydroxyl, methyl, halogen, -CF ₃ , ketone, and where the S atom can be oxidized to sulfoxide or sulfone;

R ₄ is selected from optionally substituted 3-7 membered carbocycles or heterocycles and

D1 is selected from the following 5-membered monocyclic heterocycles:

which are optionally substituted with deuterium, (C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy, amino, halogen, amide, -CF ₃ , -CN, -N ₃ , (C1-C ₄ )ketone, -S (O)-(C ₁ -C ₄ )alkyl, -SO ₂ -(C ₁ -C ₄ )alkyl, -(C1-C ₄ )thioalkyl, -COOH and/or ester, each of which may be optionally substituted hydrogen, F, Cl, Br, -OH, -NH ₂ , -NHMe, -OMe, -SMe, oxo and/or thio-oxo.

In some embodiments, the BET protein bromodomain inhibitor for use in the combination therapies of the present invention is a compound of formula Ia. In some embodiments, the compound of formula Ia is 1benzyl-6-(3,5-dimethylisoxazol-4-yl)-N-methyl-1H-imidazo[4,5-b]pyridin-2-amine (compound I), which has the following formula:

In some embodiments, the BET protein bromodomain inhibitor of Formula Ia is a pharmaceutically acceptable salt or cocrystal of Compound I. In some embodiments, the BET protein bromodomain inhibitor is a mesylate salt/cocrystal of Compound I in crystalline Form I.

Brief description of graphic materials

In fig. 1 shows the effect of Compound I, talazoparib and the combination of Compound I and talazoparib on the viability of TNBC cell line HCC1937 (BRCA1 mutant).

In fig. 2 shows the effect of Compound I, olaparib, and the combination of Compound I and olaparib on the viability of TNBC cell line HCC1937 (BRCA1 mutant).

In fig. 3 shows the effect of Compound I, veliparib, and the combination of Compound I and veliparib on the viability of TNBC cell line HCC1937 (BRCA1 mutant).

In fig. 4 shows the effect of Compound I, olaparib, and the combination of Compound I and olaparib on the viability of TNBC cell line HCC1599 (BRCA2 mutant).

In fig. 5 shows the effect of Compound I, talazoparib and the combination of Compound I and talazoparib on the viability of TNBC cells, BT549 cell line (BRCA1 and BRCA2 wild type).

In fig. 6 shows the effect of Compound I, veliparib, and the combination of Compound I and veliparib on the viability of TNBC cells, BT549 cell line (BRCA1 and BRCA2 wild type).

In fig. 7 shows the effect of Compound I, olaparib, and the combination of Compound I and olaparib on the viability of TNBC cells, BT549 cell line (BRCA1 and BRCA2 wild type).

In fig. 8 shows the effect of Compound I, niraparib, and the combination of Compound I and niraparib on the viability of HCC-70 cells (BRCA-1 and BRCA-2 wild type).

In fig. 9 shows an X-ray powder diffraction (XRPD) pattern of the mesylate salt/cocrystal of Compound I.

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In fig. 10 shows a differential scanning calorimeter (DSC) thermogram for the mesylate salt/cocrystal of Compound I.

In fig. 11 shows thermogravimetric analysis (TGA) of the mesylate salt/cocrystal of the compound.

I.

In fig. 12A shows the induction of an immune response in a tumor in response to the combination of Compound I with enzalutamide in patients with mCRPC. Both samples before administration of compound I and after administration of compound I were obtained under conditions of continuous administration of enzalutamide. In fig. 12B shows some of the immune response genes whose expression levels are increased in the tumor.

Definitions.

As used herein, treatment or treatment refers to reducing the intensity of a disease or disorder or at least one noticeable symptom thereof. In another embodiment, treating or administering a treatment relates to reducing the intensity of at least one measurable physical parameter not necessarily noticeable to the patient. In yet another embodiment, treating or administering a treatment relates to slowing the progression of a disease or disorder, either physically, such as stabilizing a noticeable symptom, or physiologically, such as stabilizing a physical parameter, or both. In yet another embodiment, treating or administering a treatment relates to delaying the onset of a disease or disorder.

By optional or optional it is meant that the event or condition described below may or may not occur, and that the description includes cases in which the event or condition occurs and cases in which it does not occur. For example, optionally substituted aryl includes both aryl and substituted aryl, as defined below. Those skilled in the art will appreciate that any group containing one or more substituents is not intended to introduce any substitutions or substitution patterns into such groups that are sterically infeasible, chemically infeasible, and/or unstable by nature.

As used herein, the term hydrate refers to a crystalline form with water included in the crystal structure in a stoichiometric or non-stoichiometric amount.

As used herein, the term alkenyl refers to a straight or branched chain unsaturated hydrocarbon having at least one carbon-carbon double bond, such as a group of 2 to 8 straight or branched chain carbon atoms, referred to herein as (C ₂ -C ₈ )alkenyl. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, and 4-(2-methyl-3-butene)pentenyl.

As used herein, the term alkoxy refers to an alkyl group attached to an oxygen atom (-O-alkyl-). Alkoxy groups also include an alkenyl group attached to an oxygen atom (alkenyloxy group) or an alkynyl group attached to an oxygen atom (alkynyloxy group). Exemplary alkoxy groups include, but are not limited to, those with an alkyl, alkenyl, or alkynyl group of 1-8 carbon atoms, referred to herein as (C ₁ -C ₈ )alkoxy. Exemplary alkoxy groups include, but are not limited to, methoxy and ethoxy.

As used herein, the term alkyl refers to a straight or branched chain saturated hydrocarbon, such as a group of 1-8 straight or branched chain carbon atoms, referred to herein as (C1- _C8 )alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3- butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl.

As used herein, the term amide refers to -NRaC(O)(Rb) or -C(O)NRbRc, wherein Ra, Rb and Rc are each independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl , heterocyclyl and hydrogen. An amide can be attached to another group through a carbon atom, nitrogen atom, Ra, Rb or Rc. The amide may also be cyclic, for example Rb and Rc may be joined to form a 3-8 membered ring, such as a 5 or 6 membered ring. The term amide covers groups such as sulfonamide, urea, ureido, carbamate, carbamic acid and cyclic variants thereof. The term amide also includes an amide group attached to a carboxyl group, such as -amide-COOH or salts such as -amide-COONa, an amino group attached to a carboxyl group (for example, -amino-COOH or salts such as -amino-COONa ).

As used herein, the term amine or amino refers to the structure -NRdRe or -N(Rd)Re-, wherein Rd and Re are independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbamate, cycloalkyl, haloalkyl, heteroaryl, heterocycle and hydrogen . The amino group can be attached to the group of the parent molecule through a nitrogen atom. The amino group can also be cyclic,

- 3 043826 for example, any two of Rd and Re can be joined together or with N to form a 3-12 membered ring (eg morpholino or piperidinyl). The term amino also includes the corresponding quaternary ammonium salt of any amino group. Exemplary amino groups include alkylamino groups where at least one of Rd or Re is an alkyl group. In some embodiments, Rd and Re may each be optionally substituted with hydroxyl, halogen, alkoxy, ester, or amino.

As used herein, the term aryl refers to an aromatic system of one, two, or more carbocyclic rings. The aryl group may optionally be fused to one or more rings selected from aryls, cycloalkyls and heterocyclyls. The aryl groups of the present invention may be substituted by groups selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone. Exemplary aryl groups include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl and naphthyl, as well as carbocyclic moieties with fused benzenes such as 5,6,7,8-tetrahydronaphthyl. Exemplary aryl groups also include, without limitation, a monocyclic aromatic ring system in which the ring contains 6 carbon atoms, referred to herein as (C ₆ )aryl.

As used herein, the term arylalkyl refers to an alkyl group having at least one aryl substituent (eg, -aryl-alkyl-). Exemplary arylalkyl groups include, but are not limited to, arylalkyls having a monocyclic aromatic ring system in which the ring contains 6 carbon atoms, referred to herein as (C ₆ )arylalkyl.

As used herein, the term carbamate refers to the structure -RgOC(O)N(Rh)-, -RgOC(O)N(Rh)Ri- or -OC(O)NRhRi, in which each of Rg, Rh and Ri is independently selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, haloalkyl, heteroaryl, heterocyclyl and hydrogen. Exemplary carbamates include, but are not limited to, aryl carbamates or heteroaryl carbamates (eg, wherein at least one of Rg, Rh, and Ri is independently selected from aryl or heteroaryl such as pyridine, pyridazine, pyrimidine, and pyrazine).

As used herein, the term carbocycle refers to an aryl or cycloalkyl group.

As used herein, the term carboxy refers to -COOH or the corresponding salts of carboxylic acids (eg, -COONa). The term carboxy also includes carboxycarbonyl, eg a carboxy group attached to a carbonyl group, eg -C(O)-COOH, or salts such as -C(O)-COONa.

As used herein, the term cycloalkoxy refers to a cycloalkyl group attached to an oxygen atom.

As used herein, the term cycloalkyl refers to saturated or unsaturated cyclic, bicyclic hydrocarbon groups or bridged bicyclic groups of 3-12 carbon atoms or 3-8 carbon atoms, referred to herein as (C ₃ C ₈ )cycloalkyl, derived from cycloalkane. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclohexenes, cyclopentanes, and cyclopentenes. Cycloalkyl groups may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone , nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide or thioketone. Cycloalkyl groups can be fused with other saturated or unsaturated cycloalkyl, aryl or heterocyclyl groups.

As used herein, the term dicarboxylic acid refers to a group containing at least two carboxyl groups, such as saturated and unsaturated hydrocarbon-based dicarboxylic acids and their salts. Exemplary dicarboxylic acids include alkyl dicarboxylic acids. Dicarboxylic acids may be substituted with alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, nicloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl , ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide or thioketone. Dicarboxylic acids include, but are not limited to, succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, maleic acid, phthalic acid, aspartic acid, glutamic acid, malonic acid, fumaric acid, (+)/(-)-malic acid acid, (+)/(-) tartaric acid, isophthalic acid and terephthalic acid. Dicarboxylic acids further include derivatives of the corresponding carboxylic acids, such as anhydrides, imides, hydrazides (eg, succinic anhydride and succinimide).

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The term ester refers to the structure -C(O)O-, -C(O)O-Rj-, -RkC(O)O-Rj- or -RkC(O)O-, where the O atom is not connected to a hydrogen, a Rj and Rk may independently be selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, cycloalkyl, ether, haloalkyl, heteroaryl and heterocyclyl. Rk may be hydrogen, but Rj may not be hydrogen. The ester may be cyclic, for example a carbon atom and Rj, an oxygen atom and Rk, or Rj and Rk may be joined to form a 3-12 membered ring. Exemplary esters include, but are not limited to, alkyl esters wherein at least one of Rj or Rk is alkyl, such as -O-C(O)-alkyl, -C(O)-O-alkyl- and -C-alkyl. (O)-O-alkyl-. Exemplary esters also include aryl or heteroaryl esters, for example, wherein at least one of Rj or Rk is a heteroaryl group such as pyridine, pyridazine, pyrimidine and pyrazine, such as a nicotinate ester. Exemplary esters also include reverse esters having the structure -RkC(O)O-, where the oxygen atom is bonded to the parent molecule.

Exemplary reverse esters include succinate, D-argininate, L-argininate, L-lysinate and D-lysinate. Esters also include carboxylic acid anhydrides and acid halides.

As used herein, the terms halogen or halogen refer to F, Cl, Br or I.

As used herein, the term haloalkyl refers to an alkyl group substituted with one or more halogen atoms. Haloalkyl also includes alkenyl or alkynyl groups substituted with one or more halogen atoms.

As used herein, the term heteroaryl refers to an aromatic system of one, two or more rings containing one or more heteroatoms, for example 1-3 heteroatoms such as nitrogen, oxygen and sulfur. Heteroaryls may be substituted with one or more substituents, including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, picloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide or thioketone. Heteroaryls can also be fused to non-aromatic rings. Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl, pyramidylyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, furyl, phenyl, isoxazolyl and oxazolyl. Exemplary heteroaryl groups include, but are not limited to, a monocyclic aromatic ring, where the ring contains 2-5 carbon atoms and 1-3 heteroatoms, referred to herein as (C ₂ C ₅ )heteroaryl.

As used herein, the terms heteropicl, heterocyclyl or heterocyclic refer to saturated or unsaturated 3-, 4-, 5-, 6- or 7-membered rings containing one, two or three heteroatoms independently selected from nitrogen, oxygen and sulfur. Heterocycles can be aromatic (heteroaryls) or non-aromatic. Heterocycles may be substituted with one or more substituents, including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, nicloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide or thioketone. Heterocycles also include bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings are fused to one or two rings independently selected from aryls, cycloalkyl and heterocycles. Exemplary heterocycles include acridinyl, benzimidazolyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, biotinyl, cinnolinyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, furyl, homopiperidinyl, imidazolidinyl, imidazolinyl, imidazolyl, indolyl, isoquinolyl , isothiazolidinyl, isothiazolyl, isoxazolidinyl, isoxazolyl . , tetrahydrofuryl, tetrahydroisoquinolyl , tetrahydropyranyl, tetrahydroquinolyl, tetrazolyl, thiadiazolyl, thiazolidinyl, thiazolyl, thienyl, thiomorpholinyl, thiopyranylyl and triazolyl.

As used herein, the terms hydroxy and hydroxyl refer to -OH.

As used herein, the term hydroxyalkyl refers to a hydroxyl group attached to an alkyl group.

As used herein, the term hydroxyaryl refers to a hydroxyl group attached to an aryl group.

As used herein, the term ketone refers to the structure -C(O)-Rn (such as acetyl, -C(O)CH ₃ ) or -Rn-C(O)-Ro-. A ketone can be attached to another group via Rn or Ro. Rn or Ro may be alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or aryl, or Rn

- 5 043826 or Ro can be connected to form a 3-12 membered ring.

As used herein, the term phenyl refers to a 6-membered carbocyclic aromatic ring. The phenyl group can also be fused to a cyclohexane or cyclopentane ring. Phenyl may be substituted with one or more substituents, including alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide and thioketone.

As used herein, the term thioalkyl refers to an alkyl group attached to a sulfur atom (-S-alkyl-).

The alkyl, alkenyl, alkynyl, alkoxy, amino and amide groups may be optionally substituted with at least one group selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, carbonyl, carboxy, piano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, thioketone, ureide and N, or be interrupted or branched by it. The substituents may be branched to form a substituted or unsubstituted heterocycle or cycloalkyl.

As used herein, a suitable substitution on an optionally substituted substituent is one that does not render the compounds of the present invention or intermediates useful in their preparation obsolete. Examples of suitable substitutions include, but are not limited to: O^alkyl, alkenyl or alkynyl; C ₁ . ₆ aryl, C _2-5 heteroaryl; C ₃ . ₇ cycloalkyl; C ₁ . ₈ αlkoxy; C ₆ aryloxy; -CN; -HE; oxo; halogen, carboxy; amino, such as -NH( _{C1_8alkyl} ), -N( _{C1_8alkyl} ) ₂ , -NH(( _C6 )aryl) or -N( _{( C6} )aryl) ₂ ; formed; ketones such as -CO( _{C1_8 alkyl} ), -CO(( _C6 aryl)-esters such as _-CO2 ( _{C1_8} alkyl) and -CO2 ( _C6 aryl). One skilled in the art will be able to easily select a suitable substitution based on the stability as well as the pharmacological and synthetic activity of the compound of the present invention.

As used herein, the term pharmaceutically acceptable composition refers to a composition containing at least one compound disclosed herein, which is formulated with one or more pharmaceutically acceptable carriers.

As used herein, the term pharmaceutically acceptable carrier refers to a variety of solvents, dispersion media, coatings, isotonic and absorption retarding agents, and the like that are compatible with pharmaceutical administration. The use of such media and means for pharmaceutically active substances is widely known in the art. The compositions may also contain other active compounds providing auxiliary, additional or enhanced therapeutic functions.

The term triple negative breast cancer or TNBC is used herein to refer to breast cancer that is characterized by tumors with less than 10% estrogen receptor and progesterone receptor positive cells and lacking HER2 amplification, as well as patients who are not candidates for endocrine therapy (Dawood 2010). TNBC tends to be more aggressive than other types of breast cancer and is therefore more likely to spread beyond the breast and/or recur after treatment.

The term immunotherapy agent is used herein to refer to agents used to treat a disease by activating or suppressing the immune system.

The term immune checkpoint inhibitor is used herein to refer to therapeutic agents that target immune checkpoints.

Exemplary Embodiments of the Present Invention

As summarized above, the present invention provides methods for treating TNBC using combination therapy that includes administering a BET protein bromodomain inhibitor of Formula Ia or Formula Ib, or a pharmaceutically acceptable salt or co-crystal thereof, and a second therapeutic agent to a subject in need thereof.

In one embodiment, the present invention provides a method of treating TNBC comprising administering a BET protein bromodomain inhibitor of Formula Ia or Formula Ib

,r ₄ o=< aJ in J

N' ⁴ 'n'^{> n} (Formula Ib), or a stereoisomer, tautomer, pharmaceutically acceptable salt, or co-crystal, or hydrate thereof together with a second therapeutic agent, wherein:

- 6 043826 ring A and ring B may be optionally substituted with groups independently selected from hydrogen, deuterium, -NH2, amino, (C ₄ -C ₆ )heterocycle, (C ₄ -C ₆ )carbocycle, halogen, -CN, - OH, _{-CF 3} , ( _C1C6 )alkyl, ( _CgC6 )thioalkyl, ( _C1 - _C6 )alkenyl and (C1- _C6 )alkoxy;

X is selected from -NH-, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2O-, -CH2CH2NH-, -CH2CH2S-, -C(O)-, C(O)CH ₂ -, -C(O )CH ₂ CH ₂ -, -CH ₂ C(O)-, -CH ₂ CH ₂ C(O)-, -C(O)NH-, -C(O)O-, -C(O)S- , -C(O)NHCH ₂ -, C(O)OSH ₂ -, -C(O)SCH ₂ -, where one or more hydrogen atoms can be independently replaced by deuterium, hydroxyl, methyl, halogen, -CF3, ketone , and where the S atom can be oxidized to a sulfoxide or sulfone;

R4 is selected from optionally substituted 3-7 membered carbocycles or heterocycles and

D1 is selected from the following 5-membered monocyclic heterocycles:

which are optionally substituted with hydrogen, deuterium, (C1- _C4 )alkyl, ( _C1 - _C4 )alkoxy, amino, halogen, amide, _-CF3 , -CN, -N3, (C1- _C4 )ketone, -S (O)-(C ₁ -C ₄ )alkyl, -SO ₂ -(C ₁ -C ₄ )alkyl, -(C1C ₄ )thioalkyl, -COOH and/or ester, each of which may be optionally substituted with hydrogen, F, Cl, Br, -OH, -NH ₂ , -NHMe, -OMe, -SMe, oxo and/or thio-oxo.

The compounds of formulas Ia and Ib, including compound I, have been previously described in international patent publication WO 2015/002754, incorporated herein by reference in its entirety, and in particular for the description of compounds of formula Ia and formula Ib, including compounds I, their synthesis and demonstration of their activity as a bromodomain inhibitor of BET proteins.

In some embodiments, a BET protein bromodomain inhibitor of formula Ia or formula Ib is selected from

1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-N-ethyl-1H-imidazo[4,5-b]pyridin-2amine;

1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-b1-methyl-1 H-imidazo[4,5-b]pyridin-2amine;

N,1-dibenzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2amine;

1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-N-(pyridin-3-ylmethyl)-1 Nimidazo[4,5-b]pyridin-2-amine;

4-(1-benzyl-2-(pyrrolidin-1-yl)-1H-imidazo[4,5-b]pyridin-6-yl)-3,5dimethylisoxazole;

4-(2-(azetidin-1-yl)-1-(cyclopentylmethyl)-1H-imidazo[4,5-b]pyridin-6-yl)3,5-dimethylisoxazole;

1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine;

1-(cyclopentylmethyl)-6-(3,5-dimethylisoxazol-4-yl)-H-(tetrahydro-2H-pyran4-yl)-1H-imidazo[4,5-b]pyridin-2-amine;

4-amino-1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-benzo|D]imidazol-2(3H)one;

4-amino-6-(3,5-dimethylisoxazol-4-yl)-1-(4-methoxybenzyl)-1Hbenzo[d]imide azol-2(3H)-one;

4-amino-6-(3,5-dimethylisocazol-4-yl)-l-(l-phenylethyl)-lH-benzo[d]imidazol2(3N)-one;

4-amino-1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-3-methyl-1Hbenzo[d]imide azol-2(3H)-one or pharmaceutically acceptable salts or co-crystals thereof.

In some embodiments, the present invention provides a method of treating TMBC, comprising administering to a subject in need thereof a BET protein bromodomain inhibitor selected from 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-N-methyl-1H- imidazo[4,5-b]pyridin-2-amine (compound I), 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2- amine and pharmaceutically acceptable salts or co-crystals thereof, concurrently with another therapeutic agent.

In one embodiment, the second therapeutic agent is a PARP inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib, talazoparib, rucaparib, veliparib, niraparib, pamiparib, CEP9722, and E7016.

In one embodiment, the second therapeutic agent is olaparib.

In one embodiment, the second therapeutic agent is talazoparib.

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In one embodiment, the subject has previously been treated with breast cancer therapy.

In one embodiment, the subject has previously been treated with chemotherapy.

In one embodiment, the subject has previously been treated with an inhibitor

PARP.

In one embodiment, the subject has previously been treated with a PARP inhibitor in combination with an immunotherapy agent.

In one embodiment, the subject has previously been treated with a combination of PARP with an immune checkpoint inhibitor.

In one embodiment, the subject has previously experienced disease progression while treated with a PARP inhibitor.

In one embodiment, the subject has previously experienced disease progression when treated with a PARP inhibitor in combination with an immunotherapy agent.

In one embodiment, the subject has previously been treated with a combination therapy containing Abraxane as one of the therapeutic agents.

In one embodiment, the subject has previously been treated with immunotherapy.

In one embodiment, the subject has previously experienced disease progression while being treated with immunotherapy.

In one embodiment, the subject showed no evidence of disease progression during platinum treatment in either the neoadjuvant or metastatic setting. For subjects receiving platinum in the neoadjuvant setting, at least 12 months must elapse between the last dose of platinum-based treatment and study entry.

In one embodiment, the subject has previously been treated with a combination therapy containing Tecentriq as one of the therapeutic agents.

In one embodiment, the BET protein bromodomain inhibitor is a pharmaceutically acceptable salt or cocrystal of Compound I. In one embodiment, the BET protein bromodomain inhibitor is a mesylate salt or cocrystal of Compound I.

In one embodiment, the subject is a human.

In one embodiment, a subject with breast cancer has one or both terminal mutations of BRCA1 and BRCA2.

In one embodiment, a subject with TNBC has one or both terminal BRCA1 and BRCA2 mutations.

In one embodiment, the subject with breast cancer does not carry terminal BRCA1 or BRCA2 mutations.

In one embodiment, the subject with TNBC does not carry terminal BRCA1 or BRCA2 mutations.

In one embodiment, a subject with breast cancer has somatic BRCA1 and BRCA2 mutations.

In one embodiment, a subject with TNBC has somatic BRCA1 and BRCA2 mutations.

In one embodiment, a subject with breast cancer has somatic mutations in either BRCA1 or BRCA2.

In one embodiment, a subject with TNBC has somatic mutations in either BRCA1 or BRCA2.

In one embodiment, a subject with breast cancer has mutations or changes that affect the expression of the BRCA1 and/or BRCA2 genes, including methylation of the BRCA1 or BRCA2 gene promoter that prevents its expression.

In one embodiment, a subject with TNBC has mutations or changes that affect the expression of the BRCA1 and/or BRCA2 genes, including methylation of the BRCA1 or BRCA2 gene promoter that prevents its expression.

In one embodiment, the subject with breast cancer has one or more somatic mutations of genes involved in homologous recombination (HR) or non-homologous end joining (NHEJ), including

ATM, SNEK2, NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, SNK1,

FANCD2, FANCA, FANCC, FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIP1.

In one embodiment, the subject with TNBC has one or more somatic mutations of genes involved in homologous recombination (HR) or non-homologous end joining (NHEJ), including

ATM, SNEC2,

NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, CHK1, FANCD2, FANCA, FANCC,

FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIPE

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In one embodiment, the subject with breast cancer has one or more terminal mutations of genes involved in homologous recombination (HR) or non-homologous end joining (NHEJ), including

ATM, SNEK2, NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, SNK1,

FANCD2, FANCA, FANCC, FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIP1.

In one embodiment, the subject with TNBC has one or more terminal mutations of genes involved in homologous recombination (HR) or non-homologous end joining (NHEJ), including

ATM, SNEC2, NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, CHK1, FANCD2,

FANCA, FANCC, FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIP1.

In one embodiment, the subject has a tumor characterized by homologous recombination (HR) deficiency.

In one embodiment, the subject has a tumor characterized by homologous recombination deficiency (HRD).

In one embodiment, a compound selected from 1-benzyl-6-(3,5-dimethylisoxazol-4yl)-N-methyl-1H-imidazo[4,5-b]pyridin-2-amine (Compound I), 1- benzyl-6-(3,5-dimethylisoxazol-4-yl)1H-imidazo[4,5-b]pyridin-2-amine and pharmaceutically acceptable salts or co-crystals thereof are dosed with a PARP inhibitor, which does not result in dose-limiting thrombocytopenia.

In one embodiment, a compound selected from 1-benzyl-6-(3,5-dimethylisoxazol-4yl)-N-methyl-1H-imidazo[4,5-b]pyridin-2-amine (compound I) and 1- benzyl-6-(3,5-dimethylisoxazol-4yl)-1H-imidazo[4,5-b]pyridin-2-amine and pharmaceutically acceptable salts or co-crystals thereof are dosed with talazoparib, which does not result in thrombocytopenia as dose-limiting toxicity.

In one embodiment, the bromodomain inhibitor of BET proteins described herein can be administered concomitantly with another therapeutic agent. Concurrent means that the bromodomain inhibitor of BET proteins described herein and another therapeutic agent are administered over a time interval of several seconds (e.g., 15 seconds, 30 seconds, 45 seconds, 60 seconds or less), several minutes (e.g., 1 min, 2 min, 5 min or less, 10 min or less, 15 min or less), or 1-12 hours. When administered concomitantly, the BET bromodomain inhibitor and the other therapeutic agent may be administered in two or more administrations and may be contained in separate compositions or dosage forms, which may be contained in the same or different packages or packages.

In one embodiment, the BET protein bromodomain inhibitor described herein and the PARP inhibitor (PARPi) can be administered according to the same or different schedules.

In one embodiment, Compound I, described herein, and talazoparib can be administered according to the same or different schedules, including:

connection I - permanent + PARPi - permanent;

connection 1-3 weeks on, one week off + PARPi - permanent;

connection 1-2 weeks on, two weeks off + PARPi - permanent;

connection 1-3 weeks on, one week off + PARPi - 3 weeks on, one week off;

connection 1-2 weeks on, two weeks off + PARPi - 3 weeks on, one week off;

connection I - constantly + PARPi - 3 weeks of admission, one week break or connection I - constantly + PARPi - 2 weeks of admission, two weeks of break.

In certain embodiments, a compound selected from Compound I and 1-benzyl-6(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine, for use in combination therapies of the present invention is administered at a dose of 25-200 mg/day. In some embodiments, a compound selected from Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4yl)-1H-imidazo[4,5-b]pyridin-2-amine is administered to a subject at a dose of 36 -144 mg/day. In some embodiments, a compound selected from Compound I and 1-benzyl-6-(3,5dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine, for use in combination therapies of of the present invention is administered to a subject at a dose of 48 mg -96 mg/day. In some embodiments, a compound selected from Compound I and 1-benzyl-6-(3,5dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine, for use in combination therapies of of the present invention is administered to a subject at a dose of 48 mg, 60 mg, 72 mg or 96 mg/day. In any of the embodiments described herein, a compound selected from Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2- amine, can be administered in combination with 0.25 mg - 1 mg talazoparib. In some embodiments, 36-144 mg of Compound I is administered in combination with 0.25-1 mg talazoparib.

In certain embodiments, a compound selected from pharmaceutically acceptable salts or cocrystals of Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5

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L]pyridin-2-amine may be administered in the combination therapies of the present invention at a dosage level that provides human exposure similar to that of 25-200 mg/day of the corresponding free base. In certain embodiments, a compound selected from pharmaceutically acceptable salts or cocrystals of Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine may be administered in the combination therapies of the present invention at a dosage level that provides human exposure similar to the amount of 36-144 mg/day of the corresponding free base. In certain embodiments, a compound selected from pharmaceutically acceptable salts or cocrystals of Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1Himidazo[4,5-b]pyridin-2-amine may be administered in the combination therapies of the present invention at a dosage level that provides human exposure similar to the amount of 48 mg to 96 mg/day of the corresponding free base. In any of the embodiments described herein, a compound selected from pharmaceutically acceptable salts or cocrystals of Compound I and 1-benzyl-6-(3,5-dimethylisoxazol-4-yl)-1H-imidazo[4,5b]pyridine -2-amine, can be administered in combination with 0.25 mg - 1 mg talazoparib.

Links.

Aftimos R, Bechter O, Awada A, Jungels S, Dumez N, Huyvaert N, Costermans J, Lee C, Meeus MA, Burkard U, Musa H, Zhao Y, Schoffski P. Phase I first-in-man trial of a novel bromodomain and extra-terminal domain (BET) inhibitor (BI 894999) in patients (Pts) with advanced solid tumors. J Clin Oncol 35, 2017 (suppl; absti 2504)

Bareche Y, Venet D, Ignatiadis M, Aftimos P, Piccart M, Rothe F, Sotiriou C.

Unravelling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis. Ann Oncol. 2018 Jan 22

Bauer, KR, Brown M, Cress RD, Parise CA, Caggiano V. Descriptive analysis of estrogen receptor (ER) negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype : a population-based study from the California cancer Registry. Cancer. 2007 May 1; 109(9): 1721-8

Berthon C, Raffoux E, Thomas X, Vey N, Gomez-Roca C, Yee K, Taussig DC, Rezai

K, Roumier C, Herait P, Kahatt C, Quesnel B, Michallet M, Recher C, Lokiec F, Preudhomme C, Dombret H. Bromodomain inhibitor OTX015 in patients with acute leukaemia: a dose-escalation, phase 1 study. Lancet Haematol. 2016 Apr;3(4):e 186-95

Copson ER, Maishman TC, Tapper WJ, Cutiess RI, Greville-Heygate S, Altman DG,

Eccles B, Gerty S, Durcan LT, Jones L, Evans DG, Thompson AM, Pharoah P, Easton DF,

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Dunning AM, Hanby A, Lakhani S, Eeles R, Gilbert FJ, Hamed H, Hodgson S, Simmonds P, Stanton L, Eccles DM. Germline BRCA mutation and outcome in young-onset breast cancer (POSH): a prospective cohort study. Lancet Oncol. 2018 Feb;19(2): 169-180. doi: 10.1016/S 1470-2045(17)30891-4

Dawood S, Triple-Negative Breast Cancer. Drugs (2010) 70(17):2247–2258

Kassam F, Emight K, Dent R, Dranitsaris G, Myers J, Flynn C, Fralick M, Kumar R, Clemons M. Survival outcomes for patients with metastatic triple-negative breast cancer: implications for clinical practice and trial design. Clin Breast Cancer. 2009 Feb;9( 1):29-33

Litton J, Rugo HS, Ettl J, Hurvitz S, Goncalves A, Lee K-H, Fehrenbacher L, Yerushalmi R, Mina LA, Martin M, Roche H, Im Y-H, Quek RGW, Tudor IC, Hannah AL, Eiermann W, Blum JL . EMBRACA: A phase 3 trial comparing talazoparib, an oral PARP inhibitor, to physician's choice of therapy in patients with advanced breast cancer and a germline BACAmutation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl) Abstract nr GS6-07

O'Shaughnessy J, Schwartzberg L, Danso MA, Miller KD, Rugo HS, Neubauer M, Robert N, Hellerstedt B, Saleh M, Richards P, Specht JM, Yardley DA, Carlson RW, Finn RS, Charpentier E, Garcia-Ribas I, Winer EP. Phase III study of iniparib plus gemcitabine and carboplatin versus gemcitabine and carboplatin in patients with metastatic triple-negative breast cancer. J Clin Oncol. 2014 Dec 1;32(34):3840-7

Robson M, Im SA, Senkus E, Xu B, Domchek SM, Masuda N, Delaloge S, Li W, Tung N, Armstrong A, Wu W, Goessl C, Runswick S, Conte P. Olaparib for Metastatic Breast Cancer in Patients with a Germline BRCA Mutation. N Engl J Med. 2017 Aug 10;377(6):523-533

Stathis A, Zucca E, Bekradda M, Gomez-Roca C, Delord JP, de La Motte Rouge T, Uro-Coste E, de Braud F, Pelosi G, French CA. Clinical Response of Carcinomas Harboring the BRD4-NUT Oncoprotein to the Targeted Bromodomain Inhibitor OTX015/MK-8628. Cancer Discov. 2016 May;6(5):492-500

Examples

Tissue culture media and reagents were obtained from ThermoFisher Scientific. Talazoparib, olaparib, niraparib, and veliparib were obtained from Selleck Chemicals.

Example 1. Synthesis of compound I.

Stage A. Synthesis of 5-bromo-N ³ -(phenylmethylene)pyridine-2,3-diamine (compound B) Benzaldehyde ^Br AcOH L II _Ν MeOH, stage 1, Η ₂ Ν

A 89-94% B

Starting material A was dissolved in methanol and acetic acid. The solution was cooled to 0-5°C and benzaldehyde was added dropwise. Immediately after completion of the reaction, industrial water and NaHCO ₃ solution were added dropwise, maintaining a low temperature (0-5°C). The solid was filtered and washed with 1:1 methanol/water and then dried to give Compound B in 94% yield and +99% purity as determined by HPLC. 'H-NMR (DMSO-de): δ 8.75 (1H), 8.04 (2H), 7.93 (1H), 7.65 (1H), 7.50-7.60 (3H).

Stage B. Synthesis of N ³ -benzyl-5-bromopyridine-2,3-diamine (compound C)

Compound B was dissolved in ethanol and NaHB4 was added in portions, maintaining the temperature in the range of 15-25°C. The reaction mixture was stirred for 8-15 hours until the reaction was complete, which was monitored by HPLC. HCl solution was added, bringing the pH to 6-7, and then technical water, maintaining the temperature in the range of 15-25°C. The mixture was stirred for 1-5 hours, filtered and washed with ethanol/water.

After drying at ~60°C for 15-20 hours, compound C was obtained. ¹ H-NMR (DMSO-d ₆ ): δ 7.2-7.4 (6H), 6.55 (1H), 5.70 -5.83 (3H), 4.30 (2H).

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Step C. Synthesis of N ³ -benzyl-5-(3,5-dimethyl-1,2-oxazol-4-yl)pyridin-2,3-dampane (compound D)

Compound C, Compound G and tribasic potassium phosphate trihydrate were mixed, and then 1,4-dioxane and industrial water were added. The resulting mixture was thoroughly purged with nitrogen. Tetrakis(triphenylphosphine)palladium(0) was added and the mixture was heated to >90° C. until the ratio of Compound C to Compound D was no more than 1%. After cooling, the reaction mixture was filtered, the solid was washed with 1,4-dioxane and then concentrated. Process water was added and the mixture was stirred until the amount of compound D remaining in the mother liquors was no more than 0.5%. Compound D was isolated by filtration and washed successively with 1,4-dioxane/water and t-butyl methyl ether. The wet filter cake was stirred in methylene chloride and silica gel. After stirring, the mixture was filtered and then concentrated. The mixture was cooled and tert-butyl methyl ether was added. The product was isolated by filtration and dried until the levels of methylene chloride, t-butyl methyl ether and moisture were no more than 0.5%. 1H-NMR (DMSO-d6): δ 7.30-7.45 (4H), 7.20-7.25 (2H), 6.35 (1H), 5.65-5.80 (3H), 4.30-4.40 (2H), 2.15 (3H), 1.95 (3H).

Step D. Synthesis of 1-benzyl-6-(3,5-dimethyl-1,2-oxazol-4-yl)-3H-imidazo[4,5-b]pyridin-2-one (compound E)

Carbonyldiimidazole as a solid was added to a stirred mixture of Compound D and dimethyl sulfoxide. The mixture was heated until the ratio of compound D to compound E was no more than 0.5%. The mixture was cooled and industrial water was added over several hours. The resulting mixture was stirred at ambient temperature for at least 2 hours. The product was isolated by filtration and washed with industrial water. Before drying using heat and vacuum, it was checked that the dimethyl sulfoxide content was no more than 0.5%. Drying was completed when the moisture level was no more than 0.5% to give Compound E. 1H-NMR (DMSO- _d6 ): δ 11.85(1H), 7.90(1H), 7.20-7. 45 (6H), 5.05 (2H), 3.57 (3H), 2.35 (3H), 2.15 (3H).

Step E. Synthesis of 4-[1-benzyl-2-chloro-1H-imidazo[4,5-b]pyridin-6-yl]-3,5-dimethyl-1,2-oxazole (compound F)

Compound E and phosphorus oxychloride were mixed and then treated with diisopropylethylamine (DIPEA), which can be added dropwise. The resulting mixture was heated for several hours, cooled, and samples were taken to verify completion of the reaction. If the ratio of compound E to compound F was no more than 0.5%, the reaction was completed. Otherwise, the reaction mixture was heated for additional time and samples were taken for testing as described above. After completion of the reaction, the mixture was concentrated and then cooled. Ethyl acetate was added and the mixture was concentrated in vacuo several times. Ethyl acetate (EtOAc) was added to the concentrate, the mixture was cooled and then added to an aqueous sodium bicarbonate solution. The organic phase was separated and the organic layer was washed with aqueous sodium bicarbonate and then with water. The organic phase was concentrated, ethyl acetate was added and the mixture was concentrated to ensure that the moisture level was no more than 0.2%. The mixture in ethyl acetate was decolorized with activated carbon. The mixture was concentrated and n-heptane was added. The product was isolated by filtration and dried under vacuum. Drying was completed when the content of residual moisture, ethyl acetate and n-heptane was no more than 0.5%. 1H NMR (MeOH-d4): δ 8.40 (1H), 7.90 (1H), 7.25-7.45 (5H), 5.65 (2H), 2.37 (3H), 2, 22 (3H).

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Step F. Synthesis of 1-benzyl-6-(3,5-dimethyl-1,2-oxazol-4-yl)-N-methyl-1H-imidazo[4,5-b]pyridin-2amine (compound I)

F Compound I

Compound F was mixed with methylamine in tetrahydrofuran (THF) and stirred at ambient temperature until the ratio of Compound F to Compound I was no more than 0.1%, as determined by HPLC. After completion of the reaction, the mixture was concentrated in vacuo, industrial water was added, and the product was isolated by filtration. The filter cake was washed with industrial water. The wet filter cake was dissolved in hydrochloric acid and the resulting solution was washed with methylene chloride to remove impurities. The aqueous solution was neutralized with sodium hydroxide solution and compound I was isolated by filtration, washed with industrial water and dried under vacuum. If it is necessary to remove any remaining hydrochloric acid, the dried material can be dissolved in ethanol, treated with a solution of sodium hydroxide in ethanol, and then process water added to precipitate the product. Compound I was isolated by filtration, washed with technical water and dried. ¹ H-NMR (DMSO-d6): δ 7.96 (d, 1H, J=2.0 Hz), 7.42 (d, 1H, J=2.0 Hz), 7.37 (q, 1H , J=4.2 Hz), 7.32 (m, 2H), 7.26 (m, 1H), 7.24 (m, 2H), 5.30 (s, 2H), 3.00 (d , 3H, 4.5 Hz), 2.34 (s, 3H), 2.16 (s, 3H). ¹³ C-NMR (DMSO-d6): δ 164.8, 158.4, 157.7, 156.0, 141.1, 136.4, 128.6 (2C), 127.5, 127.4, 127.2 (2C), 115.8, 114.2 (2C), 44.5, 29.3, 11.2, 10.3.

Example 2: Crystalline form of compound I mesylate.

Approximately 5 g of compound I was dissolved in ethanol (115 ml) and a solution of methanesulfonic acid in ethanol (10 ml, 158.7 mg/ml) was added at a molar ratio of 1:1. The mixture was shaken at 50°C for 2 hours before concentrating to half volume and stirring overnight. The resulting solid (mesylate salt/form I cocrystal of compound I) was isolated, dried and characterized.

The Form I mesylate salt/cocrystal of Compound I was also prepared using other solvents and solvent mixtures, including acetone and acetonitrile.

The mesylate salt/cocrystal of Form I of Compound I was characterized by XRPD containing the following peaks at values expressed in degrees of 2-theta angle at 8.4±0.2, 10.6±0.2, 11.7±0.2, 14 .5±0.2, 15.3±0.2, 16.9±0.2, 18.2±0.2, 19.0±0.2, 19.9±0.2, 20.5 ±0.2, 22.6±0.2, 23.8±0.2, 24.5±0.2, and 27.6±0.2, as determined by diffractometer using a Cu-Ka radiation tube (Fig. 9).

The Form I mesylate salt/cocrystal of Compound I exhibited a DSC curve containing an endothermic peak at approximately 207° C. (FIG. 10).

The Form I mesylate salt/cocrystal of Compound I was characterized by TGA to give the composition shown in FIG. 10 thermogram confirming that Form I of Compound I is the anhydrous form.

Example 3 Compound I and talazoparib in HCC1937 cells (BRCA1 mutant).

Synergistic inhibition of HCC1937 cell viability by combination of compound I with talazoparib.

HCC1937 (CRL-2336) cells were seeded at a density of 1000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS and penicillin/streptomycin and incubated for 24 h at 37°C, 5% _CO2 . The medium was replaced with RPMI-1640 medium containing 10% FBS with variable doses of either Compound I or talazoparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO ₂ for 7 days. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 30-90 minutes. Fluorescence at 380-400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 1, the addition of Compound I to talazoparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.5.

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Example 4 Compound I and olaparib in HCC1937 cells (BRCA1 mutant).

Synergistic inhibition of HCC1937 cell viability by compound combination

I with olaparib.

HCC1937 (CRL-2336) cells were seeded at a density of 1000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS and penicillin/streptomycin and incubated for 24 h at 37°C, 5% _CO2 . The medium was replaced with RPMI-1640 medium containing 10% FBS with variable doses of either Compound I or olaparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO2 for 7 days. Cells were re-treated as described above on day 3 or 4. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 30-90 minutes. Fluorescence at 380-400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 2, the addition of Compound I to olaparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.4.

Example 5 Compound I and veliparib in HCC1937 cells (BRCA1 mutant).

Synergistic inhibition of HCC1937 cell viability by combination of compound I with veliparib.

HCC1937 (CRL-2336) cells were seeded at a density of 10,000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS and penicillin/streptomycin and incubated for 24 h at 37°C, 5% _CO2 . The medium was replaced with RPMI-1640 medium containing 10% FBS with variable doses of either Compound I or veliparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO2 for 7 days. Cells were re-treated as described above on day 3 or 4. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 30-90 minutes. Fluorescence at 380-400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 3, the addition of Compound I to veliparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.1.

Example 6 Compound I and olaparib in HCC1599 cells (BRCA2 mutant).

Confluent HCC1599 (CRL-2331) cells were diluted 1:2 and seeded at 50 μl/well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS and penicillin/streptomycin. 50 μl/well of RPMI-1640 medium containing 10% FBS with variable doses of either Compound I or olaparib as monotherapy or a combination of both drugs was added to the cells and incubated at 37°C, 5% CO2 for 3 days. Wells were used in triplicate for each concentration, and wells containing only 0.2% DMSO medium were used as a control. To measure cell viability, 20 µl of MTS tetrazolium compound (CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 3 hours. Optical density at 490 nm was read using a 96-well plate reader (MultiSkan GO), and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software. (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging CI values for effective doses (ED) 50, 75 and 90. As shown in FIG. 4, the addition of Compound I to olaparib resulted in increased inhibition of cell viability compared to either monotherapy.

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Example 7 Compound I and talazoparib in BT549 cells (BRCA1/2 wild type).

Synergistic inhibition of BT549 cell viability by combination of compound I with talazoparib.

BT-549 (HTV-122) cells were seeded at a density of 1000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS, 0.023 IU/ml insulin and penicillin/streptomycin, and incubated for 24 h at 37°C, 5% CO ₂ . The medium was replaced with RPMI1640 medium containing 10% FBS, 0.023 IU/ml insulin with variable doses of either Compound I or talazoparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO ₂ for 7 days. Cells were re-treated as described above on day 3 or 4. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 30-90 minutes. Fluorescence at 380-400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 5, the addition of Compound I to talazoparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.2.

Example 8 Compound I and veliparib in BT549 cells (BRCA1/2 wild type).

Synergistic inhibition of BT549 cell viability by combination of compound I with veliparib.

BT-549 (HTV-122) cells were seeded at a density of 1000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS, 0.023 IU/ml insulin and penicillin/streptomycin, and incubated for 24 h at 37°C, 5% CO2. The medium was replaced with RPMI1640 medium containing 10% FBS, 0.023 IU/ml insulin with variable doses of either Compound I or olaparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO2 for 7 days. Cells were re-treated as described above on day 3 or 4. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO2 in for an additional 30-90 minutes. Fluorescence at 380-400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 6, the addition of Compound I to veliparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.2.

Example 9 Compound I and olaparib in BT549 cells (BRCA1/2 wild type).

Synergistic inhibition of BT549 cell viability by combination of compound I with olaparibb.

BT-549 (HTV-122) cells were seeded at a density of 1000 cells per well in 96-well flat-bottom plates in RPMI-1640 medium containing 10% FBS, 0.023 IU/ml insulin and penicillin/streptomycin, and incubated for 24 h at 37°C, 5% CO2. The medium was replaced with RPMI-1640 medium containing 10% FBS, 0.023 IU/ml insulin with variable doses of either Compound I or veliparib as monotherapy or a combination of both drugs, and incubated at 37°C, 5% CO2 for 7 days . Cells were re-treated as described above on day 3 or 4. Wells were used in triplicate for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO ₂ for an additional 30-90 minutes. Fluorescence at 380,400 nm excitation/505 nm emission was read on a fluorometer and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the blank well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and

- 15 043826 averaging the CI values for effective doses (ED) 50, 75 and 90. As shown in FIG. 7, the addition of Compound I to olaparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.2.

Example 10 Compound I and niraparib in HCC-70 cells (BRCA1/2 wild type).

Synergistic inhibition of HCC-70 cell viability by combination of compound I with niraparib.

HCC-70 cells were seeded at a density of 2500 cells per well in 96-well flat-bottom plates in 1640-RPMI medium containing 10% FBS and penicillin/streptomycin and incubated for 24 h at 37°C, 5% CO ₂ . The medium was replaced with 1640-RPMI containing 10% FBS at constant ratios of either Compound I or niraparib as monotherapy or a combination of both drugs at four different concentrations (2x IC ₅₀ , 1x IC ₅₀ , 0.5x IC ₅₀ , 0.25 x IC ₅₀ ) and incubated at 37°C, 5% CO2 for 7 days. Cells were re-treated as described above on day 3 or 4. Triplicate wells were used for each concentration, and wells containing only 0.1% DMSO medium were used as a control. To measure cell viability, 100 µl of GF-AFC substrate diluted 1:100 in assay buffer (CellTiter Fluor Cell Viability Assay Kit (Promega)) was added to each well and incubated at 37°C, 5% CO2 in for an additional 30-90 minutes. Fluorescence at 380,400 nm excitation/505 nm emission was read on a fluorimeter and the cell titer was calculated as a percentage of DMSO-treated cells after background correction was made by subtracting the empty well signal. IC ₅₀ values for monotherapy were calculated using GraphPad Prism software. Quantification of synergy was performed by calculating additivity indices (CI) using CalcuSyn software (Biosoft) based on the Chou-Talalay algorithm (Chou and Talalay, 1984) and averaging the CI values for effective doses (ED) 50, 75, and 90. As shown. in fig. 8, the addition of Compound I to niraparib resulted in increased inhibition of cell viability compared to each monotherapy, with a mean CI of 0.2-0.4.

Example 11: Clinical trials.

Part 1 may be an open-label, non-randomized dose escalation of Compound I in combination with talazoparib in patients with TNBC without terminal BRCA1/2 mutations to evaluate safety, pharmacokinetics and activity. A standard 3+3 group design will be used. Groups of up to 6 patients will be included in the study at each dose level, and each patient will participate in only one group. Each cycle will last 28 days. Dose escalation will continue after all patients enrolled in the cohort have completed the 28-day DLT cycle 1 observation period. Toxicity will be assessed and reported according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), revision 5.0. A DLT is defined as a clinically significant adverse event or laboratory abnormality that is considered possible, probable, or definitely related to the study drug and that meets any of the following criteria.

Non-hematologic clinical toxicity grade 3 or higher, excluding grade 3 nausea or grade 3/4 vomiting or diarrhea, unless persisting for more than 72 hours despite maximal medical therapy. An increase of at least 2 grades in the severity of fatigue observed at baseline.

Anemia 4 degrees. Neutropenia grade 4 lasting more than 5 days. Febrile neutropenia grade 3 or higher (temperature >38.5°C). Grade 4 thrombocytopenia or grade 3 thrombocytopenia with clinically significant bleeding or any requirement for platelet transfusion. Any other grade 3 or 4 laboratory abnormalities requiring hospitalization.

An ALT value greater than 3 times the upper limit of the normal range, with an accompanying total bilirubin value greater than 2 times the upper limit of the normal range. Any toxicity that results in more than 25% missed doses during cycle 1 of treatment. Determination of Maximum Tolerated Dose: The MTD is defined as the highest dose level of Compound 1 in combination with talazoparib at which no more than 1 in 6 patients experience DLT during the first cycle of therapy.

Part 2. 2-step design by Simon.

Phase 1: Once the recommended dose of Compound I in combination with talazoparib has been determined in the dose escalation portion of the study, 17 patients will be enrolled in the Phase 1 study with a Simon 2-stage design to assess objective response (complete response (CR) ), partial response (PR) or stable disease (SD) for >4 cycles) according to RECIST 1.1. If >4 objective responses are observed, the study will advance to phase 2. The patient population in the phase 2 Simon study design is the same as the patient population in the dose escalation portion of the study.

Stage 2: If at least 4 patients in stage 1 have an objective response (CR, PR or SD for >4 cycles) by RECIST 1.1, 20 patients will be enrolled in a stage 2 study with a 2-stage Simon design. Patients will receive the recommended daily dose of Compound I in combination with talazoparib. Patients may continue taking Compound I in combination with talazoparib until the

Claims (17)

genographic or clinical progression, unacceptable toxicity, need for off-protocol therapy, or patient exclusion from the study. Example 12 Induction of immune response and interferon gamma signaling in tumors in response to the combination of Compound I with enzalutamide in patients with mCRPC. Patients with mCRPC who had previously demonstrated disease progression on enzalutamide were administered QD doses of Compound I while continued on enzalutamide. Tumor biopsies were obtained at screening (in which the patient received enzalutamide alone) and after 8 weeks of enzalutamide and Compound I. Whole transcriptome (RNA-Seq) analysis was performed on two biopsies and alignment was performed using STAR software, and differential gene expression analysis was performed with Cufflinks using BaseSpace™ Sequence Hub default settings from December 2018 to August 2019. Additional independent analysis was performed using SALMON and BioConductor alignment software. Identification of differential gene expression signatures was performed using gene group abundance analysis (GSEA) using gene signatures from the Molecular Signature Database (Subramanian A Tamayo P, et al. (2005, PNAS 102, 1554515550); Liberzon A, et al. (2011 , Bionformatics 27, 1739-1740) Liberzon A et al (2015, Cell Systems 1, 417-425) As shown in Figure 12A, several immune-related signatures showed significant increases in expression levels in biopsies obtained during time of treatment.The relevant gene groups are indicated in the figure, and the genes included in each gene group can be downloaded from MSigDB.In Figure 12B, some of the genes found in these gene groups are graphed to show the degree of upregulation. The increased expression levels of groups of genes involved in the adaptive immune response, antigen presentation, and interferon-gamma signaling suggested that the combination of Compound I and enzalutamide induced an immunoreactive phenotype. Given that PARP inhibitors have demonstrated the ability to enhance responses to immune checkpoint inhibitors by enhancing the patient's immune response, this indicates that the combination of Compound I, a PARP inhibitor, and an immune checkpoint inhibitor may also enhance responses in the context of breast cancer. CLAIM 1. A method of treating triple negative breast cancer (TNBC), comprising administering to a subject in need thereof a bromodomain inhibitor of BET proteins selected from 1-benzyl-6-(3,5dimethylisoxazol-4-yl)-N-methyl-1H-imidazo [4,5-b]pyridin-2-amine (compound I), 1-benzyl-6-(3,5dimethylisoxazol-4-yl)-1H-imidazo[4,5-b]pyridin-2-amine and their pharmaceutically acceptable salts, with a second therapeutic agent that is a PARP inhibitor. 2. The method according to claim 1, wherein the bromodomain inhibitor of BET proteins is compound I. 3. The method according to claim 1 or 2, wherein the bromodomain inhibitor of BET proteins is the mesylate salt of Form I of Compound I. 4. The method of claim 1, further comprising administering an immune checkpoint inhibitor. 5. The method according to any one of claims 1 to 3, wherein the PARP inhibitor is selected from olaparib, talazoparib, rucaparib, veliparib, niraparib, pamiparib, CEP9722 and E7016. 6. The method according to any one of claims 1 to 3, wherein the PARP inhibitor is talazoparib. 7. The method according to any one of claims 1 to 6, wherein the subject has previously been treated with breast cancer therapy. 8. The method according to claim 7, wherein the breast cancer therapy is chemotherapy. 9. The method according to claim 7, wherein the therapy for breast cancer is immunotherapy. 10. The method of any one of claims 1 to 9, wherein the subject has previously experienced disease progression when treated with a PARP inhibitor. 11. Method according to any one of claims 1 to 10, where the subject is a human. 12. The method according to any one of claims 1 to 11, wherein the subject with breast cancer has one or both terminal mutations of BRCA1 and BRCA2. 13. The method according to any one of claims 1 to 11, wherein the subject with breast cancer does not carry terminal BRCA1 or BRCA2 mutations. 14. The method according to any one of claims 1 to 11, wherein the subject with breast cancer has somatic mutations of either BRCA1 or BRCA2. 15. The method according to any one of claims 1 to 11, wherein the subject with breast cancer has one or more somatic mutations of genes involved in homologous recombination (HR), selected from ATM, CHEK2, NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, CHK1, FANCD2, FANCA, FANCC, FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIP1. 16. The method according to any one of claims 1 to 11, wherein the subject with breast cancer has one or more terminal mutations of genes involved in homologous recombination (HR), selected from ATM, CHEK2, NBN, PALB2, ATR, RAD51, RAD54, DSS1, RPA1, CHK1, FANCD2, FANCA, FANCC, FANCM, BARD1, RAD51C, RAD51D, RIF1 and BRIP1. 17. The method according to any one of claims 1 to 11, wherein the subject has a tumor characterized by a surplus -