OA19180A - Combination of A BCL-2 inhibitor and A MCL1 inhibitor, uses and pharmaceutical compositions thereof. - Google Patents

Abstract

A combination comprising a BCL-2 inhibitor and a MCL1 inhibitor, and compositions and uses thereof.

Description

COMBINATION OF A BCL-2 INHIBITOR AND A MCL-1 INHIBITOR, USES AND PHARMACEUTICAL COMPOSITIONS THEREOF

FIELD OF THE INVENTION

The présent invention relates to a combination of a BCL-2 inhibitor and a MCL1 inhibitor.

The invention also relates to the use of said combination in the treatment of cancer, in particular leukaemia, lymphoma, multiple myeloma, neuroblastoma and lung cancer, and 5 more especially acute myeloid leukaemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma and small cell lung cancer. Also provided are pharmaceutical formulations suitable for the administration of such combinations.

BACKGROUND OF THE INVENTION

Apoptosis is a highly regulated cell death pathway that is initiated by various cytotoxic stimuli, including oncogenic stress and chemotherapeutic agents. It has been shown that évasion of apoptosis is a hallmark of cancer and that efficacy of many chemotherapeutic agents is dépendent upon the activation of the intrinsic mitochondrial pathway. Three distinct subgroups ofthe BCL-2 family proteins control the intrinsic apoptosis pathway:

(i) the pro-apoptotic BH3 (the BCL-2 homology 3)-only proteins; (ii) the pro-survival members such as BCL-2 itself, BCL-XL, Bcl-w, MCL1 and BCL-2al; and (iii) the proapoptotic effector proteins BAX and BAK (Czabotar et al, Nature Reviews Molecular cell biology 2014 Vol 15:49-63). Overexpression of the anti-apoptotic members of BCL-2 family is observed in many cancers, particularly in hematological malignancies such as mantle cell lymphoma (MCL), follicular lymphoma/diffuse large B-cell lymphoma (FL/D) and multiple myeloma (Adams and Cory Oncogene 2007 Vol 26:1324-1337). Pharmacological inhibition of the anti-apoptotic proteins BCL-2, BCL-XL and Bcl-w by the recently developed BH3-mimetics drugs such as ABT-199 and ABT-263 has emerged as a therapeutic strategy to induce apoptosis and cause tumor régression in cancer (Zhang et al, Drug Resist Updat 2007 Vol 10(6):207-17). Nevertheless, mechanisms of résistance to these drugs hâve been observed and investigated (Choudhary GS et al, Cell Death and Disease 2015 Vol 6, el593; doi:10.1038/cddis.2014.525).

Acute myeloid leukaemia (AML) is a rapidly fatal blood cancer arising from clonal transformation of hematopoietic stem cells resulting in paralysis of normal bone marrow function and deaths due to complications from profound pancytopenia. AML accounts for 25% of ail adult leukaemias, with the highest incidence rates occurring in the United States, Australia and Europe (WHO. GLOBOCAN 2012. Estimated cancer incidence, mortality and prevalence worldwide in 2012. International Agency for Research on Cancer). Globally, there are approximately 88,000 new cases diagnosed annually. AML continues to hâve the lowest survival rate of ail leukaemias, with expected 5-year survival of only 24%. Although the standard therapy for AML (cytarabine in combination with anthracyclines) was conceived over 4 décades ago, the introduction of successful targeted thérapies for this disease has remained an elusive goal. Furthermore, there remains a need for a chemotherapy-free treatment option for patients with AML. The concept of targeted therapy in AML has been hampered by the réalisation that this disease evolves as a multiclonal hierarchy, with rapid outgrowth of leukaemic sub-clones as a major cause of drug résistance and disease relapse (Ding L et al, Nature 2012 481:506-10). Recent clinical investigations hâve demonstrated the efficacy of BCL-2 inhbibitors in the treatment of AML (Konopleva M et al, American Society of Hematology 2014:118). Although these inhibitors are clinically active, it is likely that other BCL-2 family members will need to be targeted in order to enhance the overall efficacy in AML. In addition to BCL-2, MCL1 has also been identified as an important regulator of cell survival in AML (Glaser SP et al, Genes & development 2012 26:120-5).

Multiple myeloma (MM) is a rare and incurable disease that is characterized by the accumulation of clonal plasma cells in the bone marrow (BM) and accounts for 10% of ail haematological malignancies. In Europe, there are approximately 27,800 new cases each year. Due to the availability of new agents in recent years including bortezomib and lenalidomide, and autologous stem cell transplant (ASCT), the survival rate has improved. However, the response to these new agents is frequently not durable and it became an evidence that new treatments are needed, especially for relapsed/ refractory patients and patients with unfavorable prognostic (unfavorable cytogenetic profil). Recent investigations suggest a promising activity of BCL-2 inhibitors in a sub-group of multiple myeloma patients (Touzeau C, Dousset C, Le Gouill S, et al. Leukemia. 2014; 28(1):210212). MCL1 has also been identified as an important regulator of cell survival in multiple myeloma (Derenne S, Monia B, Dean NM, et al. Blood. 2002;l00(l).l 94-199, Zhang B, Gojo I, Fenton RG. Blood. 2002;99(6):l 885-1893).

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common type (25-35%) of NonHodgkin Lymphoma with 24 000 new patients/year. DLBCL is a heterogeneous disease with over a dozen subtypes, including double-hit/MYC translocation, Activated B-Cell (ABC) and Germinal Center B-cell (GCB). Modem immune chemotherapy (R-CHOP) cures approximately 60% of patients with DLBCL, but for the 40% remaining, there is little therapeutic option and the prognostic is poor. Thus, there is a high medical need to increase cure rates and clinical outcomes in high risk DLBCL such as ABC subtype (35% of DLBCL) that display constitutive activation ofthe prosurvival NF-κΒ pathway.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in infants and children, representing 8%-10% of ail childhood tumors stratified currently into low-, intermediate-, or high-risk. It accounts for approximately 15% of ail cancer-related deaths in the pédiatrie population. The incidence of NB is 10.2 cases per million children under 15 years of âge, and nearly 500 new cases are reported annually. The médian âge of diagnosis is 22 months. Outcomes in patients with NB hâve improved steadily over the last 30 years with 5-year survival rates rising from 52% to 74%. However, it is estimated that 50-60% of patients in the high-risk group expérience relapse, and as such, they hâve only seen a modest decrease in mortality. The médian time to relapse was 13.2 months, and 73% of those who relapsed were 18 months or older. Taken together, NB overall survival rates remain quite abysmal (~20% at 5 years) despite more aggressive thérapies (Colon and Chung, Adv Pediatr 2013 58:297-311). The mainstay of treatment consists of chemotherapy, surgical resection, and/or radiotherapy. However, many aggressive NB hâve developed résistance to chemotherapeutic agents, making the likelihood of relapse quite high (Pinto et al, J Clin Oncol 201533:3008-11). Standards of care for NB depending on risk stratification are frequently carboplatin, cisplatin cyclophosphamide, doxorubicin, etoposide, cytokines (GM-CSF and IL2), and vincristine. Relapse after initial response to chemotherapy is the major reason for treatment failure especially in high-risk NB.

Chemoresistance may dérivé from the activation of prosurvival BCL-2 proteins (e.g. BCL2 and MCL1 proteins). NB express high level of BCL-2 and MCL1 and low level of BCL19180

XL. Inhibition of BCL-2 sensitizes cell to death and induces NB tumor régression in vivo (Ham et al, Cancer Cell 29:159-172). Antagonisms of BCL-2 and MCL1 restore chemotherapy in high-riskNB (Lestini et al, Cancer Biol Ther 2009 8:1587-1595; Tanos et al, BMC Cancer 2016 16:97). Thus, there is strong rational to combine BCL-2 and MCL1 inhibitors in naïve or résistant patients.

The présent invention provides a novel combination of a BCL-2 inhibitor and a MCL1 inhibitor. The results show that with the development of potent small molécules targeting BCL-2 and MCL1, highly synergistic pro-apoptotic activity is revealed in primary human AML samples (Figure 2A and 17) as well as in AML (Figures 9, 13 and 14), multiple myeloma (Example 4), lymphoma (Figures 4 and 12), neuroblastoma (Figure 10), T-ALL, B-ALL cell lines (Figure 11) and in small cell lung cancer cell lines (Figures 15 (a)-(e)). We also show that combined BCL-2 and MCL1 targeting in vivo is efficacious at tolerated doses in AML and lymphoma xenograft models in mouse and rats (Figures 2, 5, 6, 7, 8 and 16), and dramatically increases time to relapse in AML (Figures 2B and 2C). Furthermore, in clonogenic assays, we demonstrate that BCL-2+MCL1 targeting is specifically toxic to leukemogenic cells, but not normal hematopoietic stem cells (Figure 3), in contrast to prior MCL1 gene targeting experiments in mice. Prior to the development of these potent and sélective inhibitors, the feasibility of targeting both BCL-2 and MCL1, remained uncertain. Previous lineage-specific délétion models indicated potential risk to cardiac (Wang X et al, Genes & development. 2013;27( 12): 1351 -1364; Thomas RL et al, Genes & development. 2013;27( 12): 1365-1377), granulocyte/hematopoietic (Opferman J et al, Science's STKE. 2005;307(5712): 1101; Dzhagalov 1 et al, Blood. 2007;109(4):1620-1626; Steimer DA et al, Blood. 2009;! 13(12):2805-2815), thymocyte (Dunkle A et al, Cell Death & Différentiation. 2010; 17(6):994-1002), neuronal (ArbourN et al, Journal of Neuroscience. 2008;28(24):6068-6078) and liver fonction (Hikita H et al, Hepatology. 2009;50(4):12171226 ; Vick B et al, Hepatology. 2009;49(2):627-636) resulting from long-term ablation of MCL1. Despite these concerns, weekly, twice weekly and even daily (during 5 consecutive days) intravenous delivery of a new potent short-acting pharmacological inhibitor of MCL1 has recently been shown to be well tolerated and active against a range of cancers in vivo, including AML (Kotschy A et al, Nature. 2016;538(7626):477-482; WO 2015/097123). The short half-life of MCL1 protein may permit sufficient time for its régénération in critical organs, thereby permitting physiological tolérance to MCLl inhibitors short-term exposure (Yang T et al, Journal of cellular physiology. 1996;166(3):523-536). Untii now, pulsatile inhibition of BCL-2 and MCLl mimicking a drug-like effect has not been possible using genetically engineered approaches. The studies using BCL-2 and MCLl inhibitors according to the présent invention provide proof-ofconcept démonstration that intermittent exposure to these drugs may be sufficient to trigger apoptosis and clinical response among highly sensitive diseases, such as AML, without concurrent toxicity to major organ Systems.

The synergistic effect of targeting both BCL-2 and MCLl in vitro and in vivo and the nontoxicity to normal marrow production when targeting both anti-apoptotic proteins hâve only been demonstrated through combination of potent small molécule inhibitors. These aspects were not anticipated by the results of gene targeting experiments, which would predict that MCLl délétion is poorly tolerated by hematopoietic stem cells.

SUMMARY OF THE INVENTION

The présent invention relates to a combination comprising (a) a BCL-2 inhibitor of formula wherein:

(D ♦ X and Y represent a carbon atom or a nitrogen atom, it being understood that they may not simultaneously represent two carbons atoms or two nitrogen atoms, ♦ Ai and A2, together with the atoms carrying them, form an optionally substituted, aromatic or non-aromatic heterocycle Het composed of 5, 6 or 7 ring members which may contain, in addition to the nitrogen represented by X or by Y, from one to 3 hetero atoms selected independently from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a group -C(O)-O-Alk wherein Alk is a linear or branched (Ci-Cô)alkyl group, or Ai and A2 independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)polyhaloalkyl, a linear or branched (Ci-Cô)alkyl group or a cycloalkyl, ♦ T represents a hydrogen atom, a linear or branched (Ci-Cô)alkyl group optionally substituted by from one to three halogen atoms, a group (Ci-CQalkyl-NRiIù, or a group (Ci-CQalkyl-ORô, ♦ Ri and R₂ independently of one another represent a hydrogen atom or a linear or branched (Ci-C6)alkyl group, or Ri and R2 form with the nitrogen atom carrying them a heterocycloalkyl, ♦ R3 represents a linear or branched (Cj-Côjalkyl group, a linear or branched (C2-C₆)alkenyl group, a linear or branched (C₂-C₆)alkynyl group, a cycloalkyl group, a (C₃-Cio)cycloalkyl-(Ci-C6)alkyl group wherein the alkyl moiety is linear or branched, a heterocycloalkyl group, an aryl group or a heteroaryl group, it being understood that one or more of the carbon atoms of the preceding groups, or of their possible substituents, may be deuterated, ♦ R4 represents an aryl group, a heteroaryl group, a cycloalkyl group or a linear or branched (Ch-Côjalkyl group, it being understood that one or more of the carbon atoms ofthe preceding groups, or of their possible substituents, may be deuterated, ♦ R5 represents a hydrogen or halogen atom, a linear or branched (Ci-C6)alkyl group, or a linear or branched (Ci-Cô)alkoxy group, ♦ R₆ represents a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, ♦ R_a, Rb, Rc and Rd, each independently of the others, represent R₇, a halogen atom, a linear or branched (Ci-C₆)alkoxy group, a hydroxy group, a linear or branched (Ci-Cô)polyhaloalkyl group, a trifluoromethoxy group, -NR7R7', nitro, R₇-CO-(Co-C₆)alkyl-, R₇-CO-NH-(Co-C₆)alkyl-, NR₇R₇'-CO-(Co-C₆)alkyl-,

NR₇R₇'-CO-(C₀-C₆)alkyl-O-, R₇-SO₂-NH-(C₀-C6)alkyl-,

R₇-NH-CO-NH-(Co-C₆)alkyl-, R₇-0-CO-NH-(Co-C₆)alkyl-, a heterocycloalkyl group, or the substituents of one of the pairs (R_a,Rb), (Rb,R_c) or (R_c,Rd) form together with the carbon atoms carrying them a ring composée! of from 5 to 7 ring members, which may contain from one to 2 hetero atoms selected from oxygen and sulphur, it also being understood that one or more carbon atoms of the ring defined hereinbefore may be deuterated or substituted by from one to 3 groups selected from halogen and linear or branched (Ci-Cô)alkyl, ♦ R₇ and R₇' independently of one another represent a hydrogen, a linear or branched (Ci-Cô)alkyl, a linear or branched (C₂-Cô)alkenyl, a linear or branched (C₂-C6)alkynyl, an aryl or a heteroaryl, or R₇ and R₇' together with nitrogen atom carrying them form a heterocycle composed of from 5 to 7 ring members, it being understood that when the compound of formula (I) contains a hydroxy group, the latter may be optionally converted into one of the following groups: -OPO(OM)(OM’), -OPO(OM)(OMi⁺), -OPO(O-Mi⁺)(Q-M2⁺), -OPO(O-)(O)M3²⁺,

-OPO(OM)(O[CH₂CH₂O]_nCH₃), or -OPO(O’Mi⁺)(O[CH2CH2O]nCH3), wherein M and M' independently of one another represent a hydrogen atom, a linear or branched (Ci-C6)alkyl group, a linear or branched (C2-Cô)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a cycloalkyl or a heterocycloalkyl, both composed of from 5 to 6 ring members, while Mi⁺ and M2⁺ independently of one another represent a pharmaceutically acceptable monovalent cation, M₃ ²⁺ represents a pharmaceutically acceptable divalent cation, and n is an integer from l to 5, it being understood that:

- aryl means a phenyl, naphthyl, biphenyl or indenyl group, heteroaryl means any mono- or bi-cyclic group composed of trom 5 to 10 ring members, having at least one aromatic moiety and containing from l to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary nitrogens),

- cycloalkyl means any mono- or bi-cyclic, non-aromatic, carbocyclic group containing from 3 to 10 ring members,

- heterocycloalkyl means any mono- or bi-cyclic, non-aromatic, condensed or spiro group composed of 3 to 10 ring members and containing from l to 3 hetero atoms selected from oxygen, sulphur, SO, SO₃ and nitrogen, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined and the groups alkyl, alkenyl, alkynyl and alkoxy to be substituted by from l to 3 groups selected from: linear or branched (Ci-Cô)alkyl optionally substituted by a hydroxyl, a morpholine, 3-3-difluoropiperidine or a 3-3-difluoropyrrolidine; (C₃-C6)spiro; linear or branched (Ci-Cô)alkoxy optionally substituted by a morpholine; (Ci-Cô)alkyl-S-; hydroxyl; oxo; V-oxide; nitro; cyano; -COOR'; -OCOR'; NR'R; linear or branched (Ci-C₆)polyhaloalkyl; trifluoromethoxy; (Ci-C₆)alkylsulphonyl; halogen; aryl optionally substituted by one or more halogens; heteroaryl; aryloxy; arylthio; cycloalkyl; heterocycloalkyl optionally substituted by one or more halogen atoms or alkyl groups, wherein R' and R independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group optionally substituted by a methoxy, it being possible for the Het group defined in formula (I) to be substituted by from one to three groups selected from linear or branched (Ci-Cô)alkyl, hydroxy, linear or branched (Ci-Cô)alkoxy, NRi'Ri and halogen, it being understood that Ri' and Ri are as defined for the groups R' and R mentioned hereinbefore, or its enantiomers, diastereoisomers, or addition salts thereof with a pharmaceutically acceptable acid or base, and (b) a MCLl inhibitor.

Said compounds of formula (I), their synthesis, their use in the treatment of cancer and pharmaceutical formulations thereof, are described in WO 2013/Π0890, WO 2015/011397, WO 2015/011399 and WO 2015/011400, the contents of which are incorporated by reference.

In certain embodiments, the MCLl inhibitor is selected from A-1210477 (Cell Death and Disease 2015 6, el590; doi:10.1038/cddis.2014.561) and the compounds described in WO 2015/097123, WO 2016/207216, WO 2016/207217, WO 2016/207225,

WO 2016/207226, or in WO 2016/033486, the contents of which are incorporated by reference.

The présent invention also relates to a combination comprising (a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II):

wherein:

♦ A represents a linear or branched (Ci-Côjalkyl group, a linear or branched (C2-Cô)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-C6)alkyl group, a linear or branched (Ci-Cô)polyhaloalkyl, a hydroxy group, a cyano, -NWioWio’, -Cy6 or an halogen atom, ♦ Wi, W₂, W₃, W₄ and W₅ independently of one another represent a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-Cô)alkenyl group, a linear or branched (C2-C6)alkynyl group, a linear or branched (Ci-C₆)polyhaloalkyl, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-Cô)alkyl group, a cyano, a nitro group, -alkyl(Co-C₆)-NW₈W₈’, -O-Cyi, -alkyl(Co-C₆)-Cyi, -alkenyl(C2-C₆)-Cyi, -alkynyl(C2-Cô)-Cyi, -O-alkyl(Ci-Cô)-W9, -C(O)-OW₈, -O-C(O)-W₈,

ΙΟ

-C/Oj-NWsWs’, -NW₈-C(O)-Ws’, -NW8-C(O)-OWs’,

-alkyl(Ci-C₆)-NW8-C(O)-W8’, -SO₂- NW₈W₈’, -SO₂-alkyl(Ci-C₆), or the substituents of one of the pairs (Wi, W₂), (W₂, W₃), (Wi, W₃), (W₄, W5) when grafted onto two adjacent carbon atoms, form together with the carbon atoms carrying them an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that resulting ring may be substituted by a group selected from a linear or branched (Ci-C₆)alkyl group, -NWioWio’, -alkyl(Co-Cô)-Cyi or an oxo, ♦ X’ represents a carbon or a nitrogen atom, ♦ Wô represents a hydrogen, a linear or branched (Ci-Cs)alkyi group, an aryl, an heteroaryl group, an arylalkyl(Ci-C6) group, an heteroarylalkyl(Ci-Cé) group, ♦ W7 represents a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cy₃, -alkyl(Ci-C₆)-Cy₃, -alkenyl(C₂-C₆)-Cy₃, -alkynyl(C₂-C₆)-Cy₃, -Cy₃-Cy₄, -alkynyl(C₂-C₆)-O-Cy₃, -Cy₃-alkyl(C₀-C₆)-0-alkyl(Co-C6)-Cy₄, an halogen atom, a cyano, -C(O)-Wn, -C(O)-NWi iWu’, ♦ Ws and Ws’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, or -alkyl(Co-Cô)-Cyi, or (Ws, Ws’) form together with the nitrogen atom carrying them an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, or a linear or branched (Ci-Côjalkyl group and it being understood that one or more of the carbon atoms of the possible substituents, may be deuterated, ♦ W9 represents -Cyi, -Cyi-alkyl(Co-C6)-Cy₂, -Cyi-alkyl(Co-Cô)-0-alkyl(Co-C6)-Cy₂,

-Cyi-alkyl(Co-C6)-NW8-alkyl(Co-C₆)-Cy₂, -Cyi-Cy₂-0-alkyl(Co-C₆)-Cy5,

-C(O)-NWsW₈’, -NW₈Ws’, -OWs,-NW₈-C(O)-W8’, -O-alkyl(Ci-C₆)-OW₈, -SO₂-Ws -C(O)-OW₈, -NH-C(O)-NH-Ws,

H

it being possible for the ammonium so defined to exist as a zwitterionic form or to hâve a monovalent anionic counterion, ♦ Wio, Wio’, Wh and Wn’ independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, ♦ W i2 represents a hydrogen or a hydroxy group, ♦ Wb represents a hydrogen atom or a linear or branched (Ci-Côjalkyl group, ♦ Wi4 represents a -O-P(O)(Q-)(O-) group, a -O-P(O)(O-)(OWi₆) group, _a -O-P(O)(OWi₆)(OWi6’) group, a -O-SO2-O’ group, a -O-SO2-OW16 group, -Cy₇, a -O-C(O)-W|₅ group, a -O-C(O)-OWi₅ group or a -O-C(O)-NWi5Wis’ group, ♦ W15 and W15’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a linear or branched amino(Ci-C6)alkyl group, ♦ Wi6 and Wiô’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or an arylalkyl(Ci-Cô) group, ♦ Cyi, Cy₂, Cy₃, Cy₄, Cy₅, Cy6 and Cy₇ independently of one another, represent a cycloalkyl group, a heterocycloalkyl group, an aryl or an heteroaryl group, ♦ n is an integer equal to 0 or l, it being understood that:

- aryl means a phenyl, naphthyl, biphenyl, indanyl or indenyl group,

- heteroaryl means any mono- or bi-cyclic group composed of from 5 to IO ring members, having at least one aromatic moiety and containing from l to 3 heteroatoms selected from oxygen, sulphur and nitrogen,

- cycloalkyl means any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to IO ring members, “heterocycloalkyl” means any mono- or bi-cyclic non-aromatic caibocyclic group containing from 3 to IO ring members, and containing from l to 3 heteroatoms selected from oxygen, sulphur and nitrogen, which may include fused, bridged 01 spiro ring Systems, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined and the alkyl, alkenyl, alkynyl, alkoxy, to be substituted by from l to 4 groups selected from linear or branched (Ci-Cô)alkyl which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy which may be substituted by a linear 5 or branched (Ci-C₆)alkoxy, a linear or branched (Ci-C₆)polyhaloalkyl, hydroxy, halogen, oxo, -NW’W”, -O-C(O)-W’, or -CO-NW’W”; linear or branched (C₂-C₆)alkenyl group; linear or branched (C₂-C₆)alkynyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; linear or branched (Ci-C₆)alkoxy which may be substituted by a group representing a linear or iO branched (Ci-C₆)alkoxy, a linear or branched (Ci-C₆)polyhaloalkyl, a linear or branched (C₂-C₆)alkynyl, -NW’W”, or hydroxy; (Ci-C₆)alkyl-S- which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; hydroxy; oxo; V-oxide; nitro; cyano; -C(O)-OW’; -O-C(O)-W’; -CO-NW’W”; -NW’W”; (C=NW’)-0W”; linear or branched (Ci-C₆)polyhaloalkyl; trifluoromethoxy; or 15 halogen; it being understood that W’ and W” independently of one another represent a hydrogen atom or a linear or branched (Ci-C₆)alkyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; and it being understood that one or more of the carbon atoms of the preceding possible substituents, may be deuterated, its enantiomers, diastereoisomers or atropisomers, or addition salts thereof with a pharmaceutically acceptable acid or base.

Said compounds of formula (II), their synthesis, their use in the treatment of cancer and pharmaceutical formulations thereof, are described in WO 2015/097123, the content of which is incorporated by reference.

In certain embodiments, the BCL-2 inhibitor is selected from the following compounds. 4_(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-l-en-l-yl]methyl}piperazin-l-yl)-N-[(3_nitr_O-4-{[(oxan-4-yl)methyl]amino}phenyl)sulfonyl]-2-[(lH-pyrrolo[2,3-ô]pyridm-5yl)oxy]benzamide (venetoclax or ABT-199); 4-(4-{[2-(4-chlorophenyl)-5,5 dimethylcyclohex-l-en-l-yl]methyl}piperazin-l-yl)-A-(4-{[(2R)-4-(morpholin-4-yl)-l(phenylsulfanyl)butan-2-yl]amino}-3-(trifluoromethanesulfonyl)benzenesulfonyl] benzamide (navitoclax or ABT-263); oblimersen (G3139); obatoclax (GXl5-070); HA14l; (±)-gossypol (BL-193); (-)-gossypol (AT-lOl); apogossypol; TW-37; antimycin A, ABT-737 (Oltersdorf T et al, Nature 2005 June 2;435(7042):677-8l) and compounds described in WO 2013/H0890, WO 2015/0H397, WO 2015/0H399 and WO 2015/011400, the contents of which are incorporated by reference.

According to a first aspect of the invention, there is provided a combination comprising:

(a) a BCL-2 inhibitor of formula (I) as described herein, and (b) a MCLl inhibitor of formula (II) as described herein.

In another embodiment, the invention provides a combination comprising:

(a) Compound l: W(4-hydroxyphenyl)-3-{6-[((35)-3-(4-morpholinylmethyl)-3,4-dihydro2(l/7)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}-/V-phenyl-5,6,7,8-tetrahydro-lindolizine carboxamide, or a pharmaceutically acceptable sait thereof, and (b) a MCLl inhibitor, for simultaneous, sequential or separate use.

In another embodiment, the invention provides a combination comprising:

(a) Compound 4: 5-(5-chloro-2-{[(35)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin2(lH)-yl]carbonyl}phenyl)-V-(5-cyano-l,2-dimethyl-l/f-pyrrol-3-yl)-A-(4hydroxyphenyl)-l,2-dimethyl-l/7-pyrrole-3-carboxamide, or a pharmaceutically acceptable sait thereof, and (b) a MCLl inhibitor, for simultaneous, sequential or separate use.

Alternatively, the invention provides a combination comprising:

(a) a BCL-2 inhibitor, and (b) Compound 2: (27?)-2-{[(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l- y|)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-J|pyrimidin-4-yl]oxy}-3-(2-{[l(2,2,2-trifluoroethyl)-l H-pyrazol-5-yl]methoxy}phenyl)propanoic acid, for simultaneous, sequential or separate use.

In another embodiment, the invention provides a combination comprising:

(a) a BCL-2 inhibitor, and (b) Compound 3: (27?)-2-{[(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l- yI)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-J]pyrimidin-4-yl]oxy}-3-(2-{[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, for simultaneous, sequential or separate use.

In another embodiment, the invention provides a combination as described herein, for use in the treatment of cancer.

In another embodiment, the invention provides the use of a combination as described herein, in the manufacture of a médicament for the treatment of cancer.

In another embodiment, the invention provides a médicament containing, separately or together, (a) a BCL-2 inhibitor of formula (I) and (b) a MCLl inhibitor, or (a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II), for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in effective amounts for the treatment of cancer.

In another embodiment, the invention provides a method of treating cancer, comprising administering a jointly therapeutically effective amount of:

(a) a BCL-2 inhibitor of formula (I) and (b) a MCLl inhibitor, or (a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II), to a subject in need thereof.

In another embodiment, the invention provides a method for sensitizing a patient who is (i) refractory to at least one chemotherapy treatment, or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering a jointly therapeutically effective amount of:

(a) a BCL-2 inhibitor of formula (I) and (b) a MCLl inhibitor, or (a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II), to said patient.

In a particular embodiment, the BCL-2 inhibitor is N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4morpholinylmethyl)-3,4-dihydro-2(l/7)-i^s°q^uinol‘ⁿy^l)^cart⁾onyl]-l,3-benzodioxol-5-yl}-Nphenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide hydrochloride (Compound l, HCl).

In a particular embodiment, the BCL-2 inhibitor is 5-(5-chloro-2-{[(3S)-3-(morpholin-4y lmethyl)-3,4-dihydroisoquinolin-2( 177)-yl]carbonyl} phenyl)-N-(5-cyano-1,2-dimethyll7/-pyrrol-3-yl)-N-(4-hydroxyphenyl)-l,2-dimethyl-H7-pyrrole-3-carboxamide hydrochloride (Compound 4, HCl).

In another embodiment, the BCL-2 inhibitor is ABT-199.

In another embodiment, the MCLl inhibitor is (2À)-2-{[(5S_o)-5-{3-chloro-2-methyl-4-[2(4-methylpiperazin-l -y l)ethoxy] phenyl }-6-(5-fluorofuran-2-yl)thieno[2,j-</]pyrimidin-4yl]oxy}-3-(2-{[l-(2,2,2-trifluoroethyl)-lH-pyrazol-5-yl]methoxy}phenyl)propanoic acid (Compound 2).

In another embodiment, the MCLl inhibitor is (2À)-2-{[(5S_a)-5-{3-chloro-2-methyl-4-[2(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-419180 yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid (Compound 3).

BRIEF DESCRIPTION OF THE FIGURES

Figure l. Expression of BCL-2 and MCL1 is prévalent in AML. 7 AML cell Unes and 13 primary AML samples with >70% blasts were immunoblotted for indicated proteins, showing that BCL-2 and MCLl proteins are dominantly expressed in contrast to BCL-XL, which was expressed in a lower proportion of samples.

Figure 2. Combined BCL-2/MCL1 targeting has synergistic activity in AML in vitro and in vivo. (A) 54 primary AML samples were incubated with a 6-log concentration range of Compound l (HCl sait), Compound 2 or a l:l concentration in RPMI/15% FCS for 48h and the LC50 determined (B) Four cohorts of NSG mice were engrafted with luciferase expressing MV4;ll cells. Tumour engraftment was verified on day IO (baseline) and then Compound l, HCl lOOmg/d orally on weekdays (expressed as the free base) or Compound 2 25mg/kg IV twice weekly administration commenced for 4 weeks. The impact of Compound 2 and the combination with Compound l was evidenced by reduced luciferase bulk on days 14 and 28 after starting therapy and increased overall survival (C).

Figure 3. Toxicity assessment of combined BCL-2/MCL1 targeting on normal CD34+ cells from normal donors or leukaemic blasts. Sorted normal CD34+ or leukaemic blasts were plated and treated with Compound l, HCl and Compound 2 at l:l ratio at the indicated concentrations. Combined Compound l + Compound 2 is toxic to leukaemic but not normal CD34+ progenitors.

Figure 4. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 in combination with Compound 1, HCl in DB cells (A) and Toledo cells (B). Values in the effect matrix range from 0 (no inhibition) to 100 (total inhibition). Values in the synergy matrix represent the extent of growth inhibition in excess of the theoretical additivity calculated based on the single agent activities of Compound 3 and

Compound l, HCl at the concentrations tested. Synergistic effects occurred across a broad range of single agent concentrations.

Figure 5. Anti-tumor effects of Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in lymphoma Karpass422 xenograft model in rats.

Figure 6. Body weight changes in animais treated with Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in lymphoma Karpass422 xenograft model in rats.

Figure 7. Anti-tumor effects of Compound 1, HCl, Compound 3 and the combination of Compound 1, HCl + Compound 3 in DLBCL Toledo xenograft model in mice.

Figure 8. Body weight changes in animais treated with Compound 1, HCI, Compound 3 and the combination of Compound 1, HCl + Compound 3 in DLBCL Toledo xenograft model in mice.

Figure 9. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in the AML cell line OCI-AML3 in two independent experiments.

Values in the effect matrix range from 0 (no inhibition) to 100 (total inhibition). Values in the synergy matrix represent the extent of growth inhibition in excess ot the theoretical additivity calculated based on the single agent activities of Compound 3 and Compound 1, HCl at the concentrations tested. Synergistic effects occurred across a broad range of single agent concentrations.

Figure 10. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in the NB cell line LAN-6 in two independent experiments (NI: upper panel; N2: lower panel). Values in the effect matrix range from 0 (no inhibition) to 100 (total inhibition). Values in the synergy matrix represent the extent of growth inhibition in excess of the theoretical additivity calculated based on the single agent activities of Compound 3 and Compound l, HCl at the concentrations tested.

Figure H. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in the B-ALL cell line NALM-6 in two independent experiments (NI: upper panel; N2: lower panel)

Figure 12. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Corn pound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in the MCL cell line Z-138.

Figure 13. Cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 (MCL1 inhibitor) in combination with ABT-199 (BCL-2 inhibitor) in AML cell line OCI-AML3 in two independent experiments (NI: upper panel; N2: lower panel).

Figure 14. Exemplary cell growth inhibition effect and synergy combination matrices for inhibition of cell growth (left) and Loewe excess inhibition (right) afforded by Compound 3 (MCL1 inhibitor) in combination with Compound 4, HCl (BCL-2 inhibitor) in AML cell lines (ML-2 cells in A and OCI-AML-3 in B).

Figures 15 (a)-(e). Dose matrices for inhibition (left), Loewe excess inhibition (middle) and growth inhibition afforded by Compound 3 (MCL1 inhibitor) in combination with Compound 1, HCl (BCL-2 inhibitor) in a panel of SCLC cell lines.

Figures 16 (a)-(b). Anti-tumor effects of Compound 1, HCl, ABT-199, Compound 3 and the combination of Compound 1, HCl or ABT-199 + Compound 3 in Patientderived primary AML mode! HAMLX5343 in mice.

Figure 17. Heat-map comparison of AML sensitivity (LCso) to BH3-mimetic monotherapy, or drug combinations (tested in 1:1 ratio), relative to chemotherapy (idarubicin) after 48h exposure. Cell viability of each primary AML samples after 48h in DMSO is shown.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore provides in Embodiment El, a combination comprising (a) a BCL-

wherein:

♦ X and Y represent a carbon atom or a nitrogen atom, it being understood that they may not simultaneously represent two carbons atoms or two nitrogen atoms, ♦ Ai and A2, together with the atoms carrying them, form an optionally substituted, aromatic or non-aromatic heterocycle Het composed of 5, 6 or 7 ring members which may contain, in addition to the nitrogen represented by X or by Y, from one to 3 hetero atoms selected independently from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a group -C(O)-O-Alk wherein Alk is a linear or branched (Ci-Cô)alkyl group, or A, and A₂ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)polyhaloalkyl, a linear or branched (Ci-Cô)alkyl group or a cycloalkyl, ♦ T represents a hydrogen atom, a linear or branched (Ci-Cô)alkyl group optionally substituted by from one to three halogen atoms, a group (Ci-C4)alkyl-NR|R₂, or a group (Ci-C4)alkyl-ORô, ♦ Ri and R₂ independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, or Ri and R₂ form with the nitrogen atom carrying them a heterocycloalkyl, ♦ R3 represents a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a cycloalkyl group, a (C3-Cio)cycloalkyl-(Ci-Cô)alkyl group wherein the alkyl moiety is linear or branched, a heterocycloalkyl group, an aryl group or a heteroaryl group, it being understood that one or more of the carbon atoms of the preceding groups, or of their possible substituents, may be deuterated, ♦ R₄ represents an aryl group, a heteroaryl group, a cycloalkyl group or a linear or branched (Ci-C₆)alkyl group, it being understood that one or more of the carbon atoms of the preceding groups, or of their possible substituents, may be deuterated, ♦ R5 represents a hydrogen or halogen atom, a linear or branched (Ci-Cô)alkyl group, or a linear or branched (Ci-Cô)alkoxy group, ♦ Rô represents a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, ♦ R_a, Rb, Rc and Ra, each independently of the others, represent R₇, a halogen atom, a linear or branched (Ci-C₆)alkoxy group, a hydroxy group, a linear or branched (Ci-C₆)polyhaloalkyl group, a trifluoromethoxy group, -NR7R7, nitro, R7-CO-(Co-C₆)alkyl-, R7-CO-NH-(Co-C₆)alky l-, N R₇R₇'-CO-(Co-C6)alkyl-,

NR7R7'-CO-(Co-C₆)alkyl-0-, R7-SQ₂-NH-(Co-C₆)alkyl-,

R₇-NH-CO-NH-(Co-C₆)alkyl-, R₇-0-CO-NH-(Co-C6)alkyl-, a heterocycloalkyl group, or the substituents of one of the pairs (R_a,Rb), (Rb,Rc) or (R_c,Rd) form together with the carbon atoms carrying them a ring composed of from 5 to 7 ring members, which may contain from one to 2 hetero atoms selected from oxygen and sulphur, it also being understood that one or more carbon atoms of the ring defined hereinbefore may be deuterated or substituted by from one to 3 groups selected from halogen and linear or branched (Ci-Cô)alkyl, ♦ R? and R₇' independently of one another represent a hydrogen, a linear or branched (Ci-Cô)alkyl, a linear or branched (Ca-Côjalkenyl, a linear or branched (C2-Cè)alkynyl, an aryl or a heteroaryl, or R7 and R7' together with nitrogen atom carrying them form a heterocycle composed of from 5 to 7 ring members, it being understood that when the compound of formula (I) contains a hydroxy group, the latter may be optionally converted into one of the following groups: -OPO(OM)(OM’), -OPO(OM)(O’Mi⁺), -OPO(O Mi⁺)(O’M2⁺), -OPO(O )(O )M₃ ²⁺,

-OPO(OM)(O[CH₂CH₂O]nCH3), or -OPO(O-Mi⁺)(O[CH2CH2O]nCH3), wherein M and M' independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-C6)alkenyl group, a linear or branched (C2-C6)alkynyl group, a cycloalkyl or a heterocycloalkyl, both composed of from 5 to 6 ring members, while Mi⁺ and M2⁺ independently of one another represent a pharmaceutically acceptable monovalent cation, M3²⁺ represents a pharmaceutically acceptable divalent cation, and n is an integer from 1 to 5, it being understood that:

- aryl means a phenyl, naphthyl, biphenyl or indenyl group,

- heteroaryl means any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary mtrogens), cycloalkyl means any mono- or bi-cyclic, non-aromatic, carbocyclic group containing from 3 to 10 ring members, heterocycloalkyl means any mono- or bi-cyclic, non-aromatic, condensed or spiro group composed of 3 to 10 ring members and containing from 1 to 3 hetero atoms selected from oxygen, sulphur, SO, SO2 and nitrogen, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined and the groups alkyl, alkenyl, alkynyl and alkoxy to be substituted by from 1 to 3 groups selected from: linear or branched (Ci-C₆)alkyl optionally substituted by a hydroxyl, a morpholine, 3-3-difluoropiperidine or a 3-3-difluoropyrrolidine; (Cs-Côjspiro; linear or branched (Ci-Cô)aikoxy optionally substituted by a morpholine; (Ci-Côjalkyl-S-; hydroxyl; oxo; V-oxide; nitro; cyano; -COOR'; -OCOR'; NR'R; linear or branched 5 (Ci-Cô)polyhaloalkyl; trifluoromethoxy; (Ci-C₆)alkylsulphonyl; halogen; aryl optionally substituted by one or more halogens; heteroaryl; aryloxy; arylthio; cycloalkyl; heterocycloalkyl optionally substituted by one or more halogen atoms or alkyl groups, wherein R' and R independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group optionally substituted by a methoxy, it being possible for the Het group defined in formula (I) to be substituted by from one to three groups selected from linear or branched (Ci-C6)alkyl, hydroxy, linear or branched (Ci-Cô)alkoxy, NRi'Ri and halogen, it being understood that Ri' and Ri are as defined for the groups R' and R mentioned hereinbefore, or its enantiomers, diastereoisomers, or addition salts thereof with a pharmaceutically 15 acceptable acid or base, and (b) a MCLl inhibitor, for simultaneous, sequential or separate use.

The invention also provides in embodiment E2 a combination comprising (a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II):

wherein:

♦ A represents a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-Cô)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-C6)alkyl group, a linear or branched (Ci-Cô)polyhaloalkyl, a hydroxy group, a cyano, -NWioWio’, -Cy6 or an halogen atom, ♦ Wi, W2, W3, W4 and W5 independently of one another represent a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched

I0 (C₂-C6)alkenyl group, a linear or branched (C₂-C6)alkynyl group, a linear or branched (Ci-Côjpolyhaloalkyl, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-C₆)alkyl group, a cyano, a nitro group, -alkyl(Co-C₆)-NW₈W₈’, -O-Cyi, -alkyl(Co-C₆)-Cyi, -alkenyl(C₂-C₆)-Cyi, -alkynyl(C2-C₆)-Cyi, -O-alkyl(Ci-C₆)-W9, -C(O)-OW₈, -O-C(O)-W₈,

-C(O)-NW₈W₈’, -NW₈-C(O)-W₈’, -NW₈-C(O)-OW₈’,

-alkyl(Ci-C₆)-NW₈-C(O)-W₈’, -SO2- NW₈W₈’, -SO₂-alkyl(Ci-C₆), or the substituents of one of the pairs (Wi, W2), (W2, W3), (Wi, W3), (W4, W5) when grafted onto two adjacent carbon atoms, form together with the carbon atoms carrying them an aromatic or non-aromatic ring composed of from 5 to 7 ring 20 members, which may contain from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that resulting ring may be substituted by a group selected from a linear or branched (Ci-C₆)alkyl group, -NW10W10’,

-alkyl(Co-C6)~Cyi or an oxo, ♦ X’ represents a carbon or a nitrogen atom, ♦ Wô represents a hydrogen, a linear or branched (Ci-Cs)alkyl group, an aryl, an heteroaryl group, an arylalkyl(Ci-Cô) group, an heteroarylalkyl(Ci-Cô) group, ♦ W7 represents a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-C6)alkenyl group, a linear or branched (C2-Cô)alkynyl group, -Cys, -alkyl(Ci-C₆)-Cy₃, -alkenyl(C2-C₆)-Cy₃, -alkynyl(C2-C₆)-Cy₃, -Cy₃-Cy₄, -alkynyl(C2-C₆)-O-Cy₃, -Cy₃-alkyl(Co-C₆)-0-alkyl(Co-C6)-Cy4, an halogen atom, a cyano, -C(O)-Wn, -C(O)-NWnWn’, ♦ Ws and Ws’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, or -alkyl(Co-C6)-Cyi, or (Ws, Ws’) form together with the nitrogen atom carrying them an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, or a linear or branched (Ci-C6)alkyl group and it being understood that one or more ofthe carbon atoms of the possible substituents, may be deuterated, ♦ W₉ represents -Cyi, -Cyi-alkyl(Co-Cô)-Cy2, -Cyi-alkyl(Co-C6)-0-alkyl(Co-Cô)-Cy2,

-Cyi-alkyl(Co-C6)-NW8-alkyl(Co-C₆)-Cy2, -Cyi-Cy2-0-alkyl(Co-C₆)-Cy₅,

-C(O)-NWsWs’, -NWsWs’, -OW8,-NWs-C(O)-Ws’, -O-alkyl(Ci-C₆)-OWs, -SO2-W8, -C(O)-OW₈, -NH-C(O)-NH-W₈,

it being possible for the ammonium so defined to exist as a zwitterionic form or to hâve a monovalent anionic counterion, ♦ W10, W10’, Wn and W11’ independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, ♦ W12 represents a hydrogen or a hydroxy group, ♦ Wi₃ represents a hydrogen atom or a linear or branched (Ci-Côjalkyl group, ♦ Wu represents a -O-P(O)(O')(O') group, a -O-P(O)(O‘)(OWi6) group, a -O-P(O)(OWiô)(OWi6’) group, a -O-SCh-O’ group, a -O-SO2-OW16 group, -Cy?, a -O-C(O)-Wi5 group, a -O-C(O)-OWis group or a -O-C(O)-NWi5Wi5’ group, ♦ W15 and W15’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Côjalkyl group or a linear or branched amino(Ci-C6)alkyl group, ♦ Wi6 and Wiô’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Côjalkyl group or an arylalkyl(Ci-Cô) group, ♦ Cyi, Cy2, Cya, Cy4, Cys, Cy6 and Cy? independently of one another, represent a cycloalkyl group, a heterocycloalkyl group, an aryl or an heteroaryl group, ♦ n is an integer equal to 0 or l, it being understood that:

- aryl means a phenyl, naphthyl, biphenyl, indanyl or indenyl group, heteroaryl means any mono- or bi-cyclic group composed of from 5 to IO ring members, having at least one aromatic moiety and containing from l to 3 heteroatoms selected from oxygen, sulphur and nitrogen, cycloalkyl means any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to IO ring members,

- “heterocycloalkyl” means any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to IO ring members, and containing from l to 3 heteroatoms selected from oxygen, sulphur and nitrogen, which may include fused, bridged or spiro ring Systems, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined and the alkyl, alkenyl, alkynyl, alkoxy, to be substituted by from l to 4 groups selected from linear or branched (Ci-Cô)alkyl which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy which may be substituted by a linear or branched (Ci-C₆)alkoxy, a linear or branched (Ci-C₆)polyhaloalkyl, hydroxy, halogen, oxo, -NW’W”, -O-C(O)-W’, or -CO-NWW”; linear or branched (C2-Cô)alkenyl group; linear or branched (C2-Cô)alkynyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; linear or branched (Ci-C₆)alkoxy which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy, a linear or branched (Ci-C6)polyhaloalkyl, a linear or branched (C₂-C6)alkynyl, -NW’W”, or hydroxy; (Ci-Cô)alkyl-S- which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; hydroxy; oxo; N-oxide; nitro; cyano; -C(O)-OW’; -O-C(O)-W’; -CO-NW’W”; -NW’W”; 5 (C=NW’)-OW”; linear or branched (Ci-C₆)polyhaloalkyl; trifluoromethoxy; or halogen; it being understood that W’ and W” independently of one another represent a hydrogen atom or a linear or branched (Ci-C6)alkyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; and it being understood that one or more of the carbon atoms of the preceding possible substituents, may be 10 deuterated, its enantiomers, diastereoisomers or atropisomers, or addition salts thereof with a pharmaceutically acceptable acid or base, for simultaneous, sequential or separate use.

Further enumerated embodiments (E) of the invention are described herein. It will be 15 recognized that features specifïed in each embodiment may be combined with other specifïed features to provide further embodiments ofthe présent invention.

E3. A combination according to El, wherein the MCLl inhibitor is a compound of formula (Π) as defined in E2.

E4. A combination according to any of El to E3, wherein the BCL-2 inhibitor is 7V-(420 hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(lH)-isoquinolinyl) carbonyl]-l,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide.

E5. A combination according to any of El to E3, wherein the BCL-2 inhibitor is 5-(5chloro-2-{[(3S)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoquinolin-2(lH)-yl]carbonyl} phenyl)-N-(5-cyano-l,2-dimethyl-lH-pyrrol-3-yl)-A-(4-hydroxyphenyl)-l,2-dimethyl-lÆ25 pyrrole-3-carboxamide.

E6. A combination according to E4, wherein A-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4morpholinylmethyl)-3,4-dihydro-2(l//)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}-7Vphenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide is in the form of the hydrochloride sait.

E7. A combination according to E5, wherein 5-(5-chloro-2-{[(3S)-3-(morpholin-4ylmethyl)-3,4-dihydroisoquinolin-2(l/7)-yl]carbonyl} phenyl)-A-(5-cyano-l,2-dimethyllH-pyrrol-3-yl)-A-(4-hydroxyphenyl)-l,2-dimethyl-lÆ-pyrrole-3-carboxamide is in the form of the hydrochloride sait.

E8. A combination according to E4 or E6, wherein the dose of 7V-(4-hydroxyphenyl)-3-{6[((3S)-3-(4-morpholinylmethyl)-3,4-dihydro-2(l/7)-isoquinolinyl)carbonyl]-l,3benzodioxol-5-yl}-A-phenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide during the combination treatment is from 50 mg to 1500 mg.

E9. A combination according to any of El to E8, wherein the BCL-2 inhibitor is administered once a week.

E10. A combination according to E6 or E8, wherein A-(4-hydroxyphenyl)-3-{6-[((3S)-3(4-morpholinylmethyl)-3,4-dihydro-2(l7/)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}A-phenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide is administered during the combination treatment once a day.

El l. A combination according to any of El to E3, wherein the BCL-2 inhibitor is ABT199.

El2. A combination according to any of El to El l, wherein the MCLl inhibitor is (2R)-2{[(5S_a)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(5fluorofuran-2-yl)thieno[2,3-<7]pyrimidin-4-yl]oxy}-3-(2-{[l-(2,2,2-trifluoroethyl)-lHpyrazol-5-yl]methoxy}phenyl)propanoic acid.

El3. A combination according to any of El to El l, wherein the MCLl inhibitor is (27()-2{[(5S₀)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-iZ]pyrimidin-4-yl]oxy} -3-(2-{[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy}phenyl)propanoic acid.

El4. A combination according to any of El to El3, wherein the BCL-2 inhibitor and the MCLl inhibitor are administered orally.

El5. A combination according to any of El to El3, wherein the BCL-2 inhibitor is administered orally and the MCLl inhibitor is administered intravenously.

El6. A combination according to any of El to El3, wherein the BCL-2 inhibitor and the MCLl inhibitor are administered intravenously.

El7. A combination according to any of El to El6, for use in the treatment of cancer.

El8. The combination for use according to El7, wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in amounts which are jointly therapeutically effective for the treatment of cancer.

El9. The combination for use according to El7, wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in amounts which are synergistically effective for the treatment of cancer.

E20. The combination for use according to El7, wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in synergistically effective amounts which enable a réduction of the dose required for each compound in the treatment of cancer, whilst providing an efficacious cancer treatment, with eventually a réduction in side effects.

E2L The combination for use according to any of El7 to E20, wherein the cancer is leukaemia.

E22. The combination for use according to E2l, wherein the cancer is acute myeloid leukaemia, T-ALL or B-ALL.

E23. The combination for use according to any of El7 to E20, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease.

E24. The combination for use according to any of El7 to E20, wherein the cancer is lymphoma.

E25. The combination for use according to any of E24, wherein the lymphoma is a nonHodgkin lymphoma.

E26. The combination for use according to any of E25, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma or mantle-cell lymphoma.

E27. The combination for use according to any of El7 to E20, wherein the cancer is multiple myeloma.

E28. The combination for use according to any of El7 to E20, wherein the cancer is neuroblastoma.

E29. The combination for use according to any of El7 to E20, wherein the cancer is small cell lung cancer.

E30. A combination according to any of El to El6, further comprising one or more excipients.

E3l. The use of a combination according to any of El to El6, in the manufacture of a médicament for the treatment of cancer.

E32. The use according to E3l, wherein the cancer is leukaemia.

E33. The use according to E32, wherein the cancer is acute myeloid leukaemia, T-ALL or B-ALL.

E34. The use according to E3l, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease.

E35. The use according to E31, wherein the cancer is lymphoma.

E36. The use according to E35, wherein the lymphoma is a non-Hodgkin lymphoma.

E37. The use according to E36, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma or mantle-cell lymphoma.

E38. The use according to E3l, wherein the cancer is multiple myeloma.

E39. The use according to E3l, wherein the cancer is neuroblastoma.

E40. The use according to E3l, wherein the cancer is small cell lung cancer.

E4l. A médicament containing, separately or together, (a) a BCL-2 inhibitor of formula (I) as defined in El, and (b) a MCLl inhibitor, for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in effective amounts for the treatment of cancer.

E42. A médicament containing, separately or together, (a) a BCL-2 inhibitor, and (b) a MCLl inhibitor of formula (II) as defined in E2, for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in effective amounts for the treatment of cancer.

E43. A method of treating cancer, comprising administering a jointly therapeutically effective amount of (a) a BCL-2 inhibitor of formula (I) as defined in El, and (b) a MCLl inhibitor, to a subject in need thereof.

E44. A method of treating cancer, comprising administering a jointly therapeutically effective amount of (a) a BCL-2 inhibitor, and (b) a MCLl inhibitor of formula (II) as defined in E2, to a subject in need thereof.

E45. A method for sensitizing a patient who is (i) refractory to at least one chemotherapy treatment, or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering a jointly therapeutically effective amount of (a) a BCL-2 inhibitor of formula (!) as defined in El, and (b) a MCLl inhibitor, to said patient.

E46. A method for sensitizing a patient who is (i) refractory to ai least one chemotherapy treatment, or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering a jointly therapeutically effective amount of (a) a BCL-2 inhibitor, and (b) a MCLl inhibitor of formula (II) as defined in E2, to said patient.

“Combination” refers to either a fixed dose combination in one unit dosage form (e.g., capsule, tablet, or sachet), non-fixed dose combination, or a kit of parts for the combined administration where a compound of the présent invention and one or more combination partners (e.g. another drug as explained below, also referred to as “therapeutic agent or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect.

The ternis “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessariiy administered by the same route of administration or at the same time.

The term “fixed dose combination” means that the active ingrédients, e.g. a compound of formula (I) and one or more combination partners, are both administered to a patient simultaneously in the form of a single entity or dosage.

The term “non-fixed dose combination” means that the active ingrédients, e.g. a compound ofthe présent invention and one or more combination partners, are both administered to a patient as separate entities either simultaneously or sequentially, with no spécifie time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingrédients.

“Cancer” means a class of disease in which a group of cells display uncontrolled growth. Cancer types include haematological cancer (lymphoma and leukaemia) and solid tumors including carcinoma, sarcoma, or blastoma. In particular “cancer” refers to leukaemia, lymphoma or multiple myeloma, and more especially to acute myeloid leukaemia.

The term “jointly therapeutically effective” means that the therapeutic agents may be given separately (in a chronologically staggered manner, especially a sequence-specific manner) in such time intervals that they prefer, in the warm-blooded animal, especially human, to be treated, still show a (preferably synergistic) interaction (joint therapeutic effect). Whether this is the case can, inter alia, be determined by following the blood levels, showing that both compounds are présent in the blood of the human to be treated at least during certain time intervals.

“Synergistically effective” or “synergy” means that the therapeutic effect observed following administration of two or more agents is greater than the sum of the therapeutic effects observed following the administration of each single agent.

As used herein, the term “treat”, “treating or treatment of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treat”, treating or treatment refers to alleviating or ameliorating at least one physical parameter including those which may not be discemible by the patient. In yet another embodiment, “treat”, treating or treatment refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.

As used herein, a subject is “in need of’ a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

In another aspect, provided is a method for sensitizing a human who is (i) refractory to at least one chemotherapy treatment, or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii), wherein the method comprises administering a BCL-2 inhibitor of formula (I) in combination with a MCLl inhibitor, as described herein, to the patient. A patient who is sensitized is a patient who is responsive to the treatment involving administration of a BCL-2 inhibitor of formula (I) in combination with a MCLl inhibitor, as described herein, or who has not developed résistance to such treatment.

“Médicament” means a pharmaceutical composition, or a combination of several pharmaceutical compositions, which contains one or more active ingrédients in the presence of one or more excipients.

‘AML’ means acute myeloid leukaemia.

‘T-ALL’ and ‘B-ALL’ means T-cell acute lymphoblastic leukemia and B-cell acute lymphoblastic leukemia.

‘free base’ refers to compound when not in sait form.

In the pharmaceutical compositions according to the invention, the proportion of active ingrédients by weight (weight of active ingrédients over the total weight of the composition) is from 5 to 50 %.

Among the pharmaceutical compositions according to the invention there will be more especially used those which are suitable for administration by the oral, parentéral and especially intravenous, per- or trans-cutaneous, nasal, rectal, perlingual, ocular or respiratory route, more specifically tablets, dragées, sublingual tablets, hard gelatin capsules, glossettes, capsules, lozenges, injectable préparations, aérosols, eye or nose drops, suppositories, creams, ointments, dermal gels etc.

The pharmaceutical compositions according to the invention comprise one or more excipients or carriers selected from diluents, lubricants, binders, disintegration agents, stabilisers, preservatives, absorbents, colourants, sweeteners, flavourings etc.

By way of non-limiting example there may be mentioned:

♦ as diluents·. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerol, ♦ as lubricants·. silica, talc, stearic acid and its magnésium and calcium salts, polyethylene glycol, ♦ as binders·. magnésium aluminium silicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, ♦ as désintégrants·. agar, alginic acid and its sodium sait, effervescent mixtures.

The compounds of the combination may be administered simultaneously or sequentially. The administration route is preferably the oral route, and the corresponding pharmaceutical compositions may allow the instantaneous or delayed release ofthe active ingrédients. The compounds ofthe combination may moreover be administered in the form oftwo separate pharmaceutical compositions, each containing one ofthe active ingrédients, or in the form of a single pharmaceutical composition, in which the active ingrédients are in admixture.

Preference is given to the pharmaceutical compositions being tablets.

Pharmaceutical composition of Compound 1 HCl sait film-coated tablet containing 50 mg and 100 mg of drug substance

	Amount (mg)	Function
Tablet	50 mg strength	100 mg strength
Compound 1 HCl sait	52,58	105,16	Drug Substance
équivalent in base to	50	100
Lactose monohydrate	178,51	357,02	Diluent
Maize starch	66,6	133,2	Disintegrant
Povidone	23,31	46,62	Binder
Magnésium stéarate	3,33	6,66	Lubricant
Silica, colloïdal anhydrous	0,67	1,34	Flow agent
Sodium starch glycolate (Type A)	10	20	Disintegrant
For an uncoated tablet with a mass of	335	670
Film-Coating
Glycerol	0,507	1,014	Plasticizing agent
hypromellose	8,419	16,838	Film-coating agent
Macrogol 6000	0,538	1,076	Smoothing agent
Magnésium stéarate	0,507	1,014	Lubricant
Titanium dioxide	1,621	3,242	Pigment
Intermediary Véhiculé
Water, purified	qs.	TY	Solvent
For a film-coated tablet with a mass of	346,6	693,2

PHARMACOLOGICAL DATA

MATERIAL AND METHOD FOR EXAMPLES 1-3:

Primary AML patient samples: Bone marrow or peripheral blood samples from patients with AML were collected after informed consent in accordance with guidelines approved by The Alfred Hospital Human research ethics committees. Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare, VIC, Aus) density-gradient centrifugation, followed by red cell déplétion in ammonium chloride (NH4CI) lysis buffer at 37°C for 10 minutes. Cells were then re-suspended in phosphate-buffered saline containing 2% Fêtai Bovine sérum (Sigma, NSW, Aus). Mononuclear cells were then suspended in RPMI-1640 (GIBCO VIC, Aus) medium containing penicillin and streptomycin (GIBCO) and heat inactivated fêtai bovine sérum 15% (Sigma).

Cell Unes, cell culture and generating luciferase reporter cell Unes: Cell lines MV4;11, OCI-AML3, HL-60, HEL, K562, KG-1 and EOL-1 were maintained at 37°C, 5% CO2 in RPMI-1640 (GIBCO) supplemented with 10% (v/v) fêtai bovine sérum (Sigma) and penicillin and streptomycin (GIBCO). MV4;I1 luciferase cell lines were generated by lentivral transductions.

Antibodies: Primary antibodies used for western blot analysis were MCLl, BCL-2, Bax, Bak, Bim, BCL-XL (generated in-house WEHI) and tubulin (T-9026,Sigma).

Cell Viability: Freshly purified mononuclear cells from AML patient samples were adjusted to a concentration of 2.5xl0⁵/ml and ΙΟΟμΕ of cells aliquoted per well into 96 well plates (Sigma). Cells were then treated with Compound 1, HCl, Compound 2, ABT199 (Active Biochem, NJ, USA) or idarubicin (Sigma), over a 6 log concentration range from InM to ΙΟμΜ for 48hr. For combinations assays, drugs were added at a 1:1 ratio from InM to lOuM and incubated at 37°C 5% CO₂. Cells were then stained with sytox blue nucleic acid stain (Invitrogen, VIC, Aus) and fluorescence measured by flow cytométrie analysis using the LSR-II Fortessa (Becton Dickinson, NSW, Aus). FACSDiva software was used for data collection, and FlowJo software for analysis. Blast cells were gated using forward and side scatter properties. Viable cells excluding sytox blue were determined at 6 concentrations for each drug and the 50% léthal concentration (LC50, in μΜ) determined.

LC50 détermination and synergy: Graphpad Prism was used to calculate the LC50 using non-linear régression. Synergy was determined by calculating the Combination Index (CI) based on the Chou Talalay method as described (Chou Cancer Res; 70(2) January 15, 2010).

Colony assays: Colony forming assays were performed on freshly purified and frozen mononuclear fractions from AML patients. Primary cells were cultured in duplicate in 35mm dishes (Griener-bio, Germany) at l x 10⁴ to l x 10⁵. Cells were plated in 0.6% agar (Difco NSW, Aus): AIMDM 2x (IMDM powder-Invitrogen), supplemented with NaHCOa, dextran, Pen/Strep, B mercaptoethanol and asparagine):Fetal Bovine Sérum (Sigma) at a 2: l : l ratio. For optimal growth conditions ail plates contained GM-CSF (lOOng per plate), IL-3(l00ng/plate R&D Systems, USA) SCF(l00ng/plate R&D Systems) and EPO (4U/plate) (Growth was for 2-3 weeks in the presence and absence of drug at 37°C at 5% CO2 in a high humidity incubator. After incubation plates were fixed with 2.5% glutaraldehyde in saline and scored using the GeiCount from Oxford Optronix (Abingdon, United Kingdom).

Western Blotting: Lysâtes were prepared in NP40 lysis buffer (10 mM Tris-HCI pH 7.4, 137 mM NaCl, 10% glycerol, l% NP40), supplemented with protease inhibitor cocktail (Roche, Dee Why, NSW, Australia). Protein samples were boiled in reducing loading dye before séparation on 4-12% Bis-Tris polyacrylamide gels (Invitrogen, Mulgrave, VIC, Australia), and transferred to Hybond C nitrocellulose membrane (GE, Rydalmere, NSW, Australia) for incubation with specified antibodies. Ail membrane-blocking steps and antibody dilutions were performed using 5% (v/v) skim milk in PBS containing 0.1% (v/v) Tween-20 phosphate-buffered saline (PBST) or Tris-buffered-saline, and washing steps performed with PBST or TBST. Western blots were visualized by enhanced chemiluminescence (GE).

In vivo expérimentation AML engraftment: Animal studies were performed under the institutional guidelines approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee, MV4;l l cells transduced with the luciferase reporter (pLUC2) were intravenously injected at l*lO⁵ cells into irradiated (lOORad) non-obese diabetic/severe combined immunodeficient (NOD/SCID/ IL2rynull) mice as previously described (Jin et al., Cell Stem Cell 2 July 2009, Volume 5, Issue l, Pages 31^-2). Engraftment was measured at day 7 by quantifying the percentage of hCD45+ cells in the PB by flow cytometry and by IVIS imaging of bioluminescent MV4;11 cells. At day 10, mice received daily oral gavage of Compound 1, HCl (200gL lOOmg/kg - dosage expressed as the free base) dissolved in PEG400 (Sigma), absolute éthanol (Sigma) and distilled H₂0 40:10:60 or Compound 2 (200pL 25mg/kg) twice weekly dissolved in 50% 2hydroxypropyl)-P-cyclodextrin (Sigma) and 50% 50mM HCl or the drug combination or vehicle, over 4 weeks. Blood counts were determined using a hematology analyzer (BioRad, Gladesville, NSW).

IVIS imaging: Bioluminescent imaging was performed using the Caliper IVIS Lumina III XR imaging system. Mice were anaesthetised with isofleurine and injected intraperitoneally with lOOgL of 125 mg/kg luciferin (Perkin Elmer, Springvale, VIC).

MATERIAL AND METHOD FOR EXAMPLE 4:

Cell lines: Human myeloma cell lines (HMCLs) were derived from primary myeloma cells cultured in RPMI 1640 medium supplemented with 5% fêtai calf sérum from and 3 ng/mL recombinant 1L-6 for 1L-6 dépendent cell lines. HMCLs are représentative of phenotypic and genomic heterogeneity and the variability in patient’s response to therapy.

MTT assay: Cell viability is measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) colorimétrie survival assay. Cells are incubated with compounds in 96-well plates containing a final volume of 100 μΙ/well time. (27?)-2-{[(5S_o)5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(5-fluorofuran-2yl)thieno[2,3-J]pyrimidin-4-yl]oxy}-3-(2-{[l-(2,2,2-trifluoroethyl)-l//-pyrazol-5 yl]methoxy}phenyl)propanoic acid (Compound 2) is used at 9 different concentrations accordingly to single agent sensitivity. ;V-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4morpholinylmethyl)-3,4-dihydro-2(l/7)-isoquinolinyI)carbonyl]-l,3-benzodioxol-5-yl}-7Vphenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide hydrochloride (Compound l, HCl) is used at a fixed dose - l μΜ. At the end of each treatment, cells are incubated with l mg/mL MTT (50 μΐ MTT solution 2.5 mg/ml for each well) at 37°C for 3 hours allowing the MTT to be metabolized. Lysis buffer (100 μΐ Lysis buffer: DMF (2:3) /SDS (l:3)) is added into each well to dissolve formazan cristals and after 18h of incubation, absorbance in viable cells is measured at 570 nm using a spectrophotometer.

As control, cells are incubated with medium alone and with medium containing 0.1% DMSO. As myeloma cell growth control, myeloma cell absorbance is recorded every day (D0, Dl, D2, D3 and D4).

Ail experiments are repeated 3 times, and each experimental condition is repeated at least in triplicate wells in each experiment.

The inhibition effect is calculated with the following formula:

Inhibition effect (%) = (l-Absorbance value of treated cells/Absorbance value of control cells)* 100

EXAMPLE 1: BCL-2 and MCL1 are the dominant pro-survival proteins expressed in AML

AML cell lines and 13 primary AML samples with >70% blasts were immunoblotted for proteins indicated in Figure 1.

As illustrated in Figure 1, a proteomic survey ofthe expression of BCL-2 family members in AML showed that, in addition to BCL-2, most primary AML samples and AML cell lines co-expressed the pro-survival protein MCL1. BCL-XL is less frequently expressed in AML.

EXAMPLE 2: Combined BCL-2 and MCL1 targeting displays synergistic killing in AML

AML patient samples were incubated with a 6-log concentration range of Compound l (HCl sait), Compound 2 or a l:l concentration in RPMI/15% FCS for 48h and the LC50 determined (Figure 2A).

Approximately 20% of primary AML samples were highly sensitive to either Compound l or Compound 2, with the léthal concentration of drug required to kiil 50% of primary AML blasts after 48 hours (LC50) in the low nanomolar range (LCso<lOnM) (Figure 2A). In contrast, when Compound l and Compound 2 were combined, the proportion of AML samples that were sensitive increased dramatically to 70%, indicating synergistic activity when BCL-2 and MCLl were simultaneously targeted (Figure 2A). Some results are displayed in Figure 17.

To verify the in vivo activity of this approach, luciferase expressing MV4;ll AML cells were engrafted into NSG mice and treated with Compound l (HCl sait) or Compound 2 alone, or in combination and tumour burden assessed after 14 and 2I days of therapy (Figure 2B). At the completion of 28 days of therapy, mice were followed for survival (Figure 2C). These experiments showed that the combination of Compound l and Compound 2 was highly effective in vivo, validating the impressive activity observed using primary AML cells in vitro.

The data presented in Figures 2A-2C herein show the synergistic combination activity between Compound l, HCl and Compound 2 in AML.

EXAMPLE 3: Combined BCL-2 and MCLl inhibition targets leukaemic, but not normal progenitor fonction

To assess the toxicity of BCL-2 inhibition combined with MCLl inhibition on normal human CD34+ cells or ficolled blasts from patients with AML, clonogenic potential was assessed after 2 weeks exposure to combined thérapies. Colonies were grown in agar supplemented with 10% FCS, IL3, SCF, GM-CSF and EPO over 14 days and colonies enumerated with an automated Gelcount® analyser. Assays for primary AML samples were performed in duplicate and averaged. Errors for CD34+ represent mean +/- SD of 2 independent normal donor samples. Results were normalised to the number of colonies counted in DMSO control. Indicated drug concentrations were plated on Dl. Notably, Compound 1 + Compound 2 suppressed AML colony forming activity without affecting the function of normal CD34+ colony growth.

Taken altogether, Examples 2 and 3 show that dual pharmacological inhibition of BCL-2 and MCL1 is a novel approach to treating AML without need for additional chemotherapy and with an acceptable therapeutic safety window.

EXAMPLE 4: In vitro évaluation of multiple myeloma cell survival in response to a MCL1 inhibitor as a single agent or in combination with a BCL-2 inhibitor

The sensitivity of 27 human multiple myeloma cell lines to Compound 1, Compound 2 or to Compound 2 in the presence of ΙμΜ of Compound 1 was analyzed by using MTT cell viability assay. 50% inhibitory concentrations (IC50, in nM) were determined.

The results are displayed in the following table:

Cell lines	Compound 1, HCl (IC50 nM)	Compound 2 (IC50 nM)	ICso of Compound 2 in the presence of 1 μΜ of Compound 1, HCI (nM)
AMO1	8610,3	0,5	0,2
ANBL6	1905,0	79,5	20,8
BCN	22217,0	1111,4	59,3
JIM3	>30000	56,3	25,9
JJN3	2692,0	15,6	2,4
KMM1	23926,3	57,8	8,6
KMS11	10486,7	44,1	3,9
KMS12BM	1393,7	44,1	0,03
L363	7581,3	7,6	3,4
LP1	9770,0	158,2	2,9
MM1S	21407,0	138,5	23,0
ΝΑΝΙ	6659,0	5,7	1,4

NAN3	8241,3	1,5	1,2
NAN6	4074,8	7,1	1,8
NAN8	9096,3	75,4	32,5
NAN9	23157,6	9,7	1,1
NC1-H929	15688,3	2,3	1,0
OPM2	6460,7	9,4	1,2
RPMI8226	3204,0	27,4	3,0
SBN	21273,7	221,1	14,6
U266	>30000	170,1	14,9
XG1	9779,7	5,9	0,2
XG11	7912,0	374,7	8,3
XG2	15297,7	6,4	2,7
XG3	7224,7	6,1	1,3
XG6	8544,3	19,2	0,5
XG7	18121,7	16,3	8,0

Strong synergistic activity was demonstrated when combining Compound l and Compound 2 in the majority of the cell lines as compared to the compounds alone.

EXAMPLE 5: In vitro effect on prolifération of combining a MCLl inhibitor with a 5 BCL-2 inhibitor in a panel of 17 Diffuse Large B-Cell Lymphoma (DLBCL) cell lines

Material and Method

Cell lines were sourced and maintained in the basic media supplemented with FCS (Fêtai Calf Sérum) as indicated in Table 1. in addition, ail media contained penicillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM). Unless otherwise mentioned, 10 culture media and suppléments were from Amimed/Bioconcept (Allschwil, Switzerland).

Cell lines were cultured at 37°C in a humidified atmosphère containing 5% CO₂ and expanded in T-75 flasks. In ail cases cells were thawed from frozen stocks, expanded through >1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter (Omni Life Science, Bremen, Germany) prior to plating 25 ul/well at the densities indicated in Table l into 384-well plates (Corning). Ail cell lines were determined to be free of mycoplasma contamination by PCR assay performed at Idexx Radil (Columbia, MO, USA) and misidentification ruled out by assessment of a panel of 48 Small Nucléotide Polymorphisms (SNPs) at Asuragen (Austin, TX, USA) or in-house.

Stock solutions of compounds were prepared at a concentration of 10 mM in DMSO (Sigma) and stored at -20°C. Where necessary to afford a full dose-response curve, the stock solutions were pre-diluted in DMSO to ΓΟΟΟ-fold the desired start concentration (see Table 2). On the day after cell seeding, eight 2.5-fold serial dilutions of each compound were dispensed, either individually or in ail possible permutations in a checkerboard fashion, directly into the cell assay plates using a non-contact 300D Digital Dispenser (TECAN, Mannedorf, Switzerland) as outlined in Figure 4. The final concentration of DMSO was 0.2% in ail wells.

Effects of the single agents as well as their checkerboard combinations on cell viability were assessed after 2 days of incubation at 37 °C/5% CO₂ by quantification of cellular ATP levels using CellTiterGlo (Promega, Madison, WI, USA) at 25 pL reagent/well and n=2 replicate plates per condition. Luminescence was quantified on a Ml000 multipurpose platereader (TECAN, Mannedorf, Switzerland). The number/viability of cells at time of compound addition was likewise assessed and used to assess the degree of the population doubling time of a particular cell line.

Single agent ICsos were calculated using standard four-parametric curve fitting. Potential synergistic interactions between compound combinations were assessed using the Excess Inhibition 2D matrix according to the Loewe additivity model and are reported as Synergy Score (Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666). Ail calculations were performed using the Combination Analysis Module in-house software. IC50 are defïned as the compound concentration at which the CTG signal is reduced to 50% of that measured for the vehicle (DMSO) control.

The interprétation of the Synergy Score is as follows:

SS - 0 Additive

SS >l —* Weak Synergy

SS >2 —> Synergy

Table 1. Identity and assay conditions for the 17 Diffuse Large B-Cell Lymphoma cell lines used in the combination experiments.

Cell line	Medium (source)	%FCS	Doubling time (hours)	Cell number seeded/well
DB	RPMI (ATCC)	10	31.7	500
DOHH-2	RPMI (DSMZ)	10	25.3	500
HT	RPMI (ATCC)	10	34.3	2000
JM1	Iscove's MEM*(ATCC)	10	22.8	500
KARPAS-422	RPMI (DSMZ)	10	26.5	500
NU-DHL-1	RPMI (DSMZ)	20	28.0	500
OCI-LY-19	MEM alpha (DSMZ)	20	25.8	500
Pfeiffer	RPMI (ATCC)	10	46.2	2000
RL	RPMI (ATCC)	10	28.9	500
SU-DHL-10	RPMI (DSMZ)	20	105.7	1000
SU-DHL-4	RPMI (DSMZ)	10	25.2	500
SU-DHL-5	RPMI (DSMZ)	20	25.9	500
SU-DHL-6	RPMI (DSMZ)	20	30.1	500
SU-DHL-8	RPMI (DSMZ)	20	23.6	500
Toledo	RPMI (ATCC)	10	49.6	2000
U-937	RPMI (ATCC)	10	28.7	500
WSU-DLCL2	RPMI (DSMZ)	10	26.1	500

*This medium was further complemented with 50 μΜ 2-mercaptoethanol. Doubling times were calculated based on the différence in ATP levels at the end compared to the beginning of compound incubation.

Table 2. Single agent ICso values for Compound 3 and Compound l, HCl, as well as the synergy scores for their combination are indicated. Interactions were deemed synergistic when scores > 2.0 where observed.

Cell Line	Compound 3	Compound 1, HCl	Combination
Start conc [uM]	Abs IC50 [uM]	Max Inh l%]	Start conc [uM]	Abs IC50 [uM]	Max Inh i%]	Synergy Score	Synergy Score Error
DB	1	0.0129	99.2	10	2.76	95.7	17.3	0.18
DOHH-2	0.1	0.00122	98.3	10	0.156	101.9	2.90	0.11
HT	1	0.00638	99.3	10	>10	37.2	2.35	0.06
JM1	1	0.0588	99.7	10	0.697	99.1	5.83	0.25
KARPAS- 422	1	0.00214	98.3	10	2.18	90.9	9.74	0.32
NU-DHL-1	1	0.0579	98.1	10	0.0515	102.1	4.97	0.12
OCI-LY-19	1	0.0604	98.5	10	0.0895	100.1	3.92	0.07
Pfeiffer	1	0.0426	82.7	10	4.50	92.0	3.44	0.19
RL	10	3.03	95.4	10	0.281	99.0	12.7	0.38
SU-DHL- 10	1	0.00384	98.3	10	>10	43.9	2.44	0.26
SU-DHL-4	1	0.0178	99.5	10	0.86	96.9	10.8	0.28
SU-DHL-5	0.1	0.00094	98.2	10	>10	49.6	1.45	0.14
SU-DHL-6	1	0.00213	99.1	10	0.614	101.0	4.57	0.21
SU-DHL-8	10	0.305	95.6	10	>10	28.7	9.88	0.20
Toledo	1	>1	45.8	10	0.137	101.9	11.1	0.51
U-937	1	0.00832	97.1	10	6.83	62.1	5.63	0.20
wsu- DLCL2	1	0.00792	99.0	10	1.02	99.3	8.67	0.13

“Start conc” means start concentration.

“Abs IC50” means absolute IC50.

“Max Inh” means maximum inhibition.

Results

The effect on prolifération of combining the MCLl inhibitor Compound 3 with the BCL-2 inhibitor Compound l, HCl was assessed in a panel of 17 Diffuse Large B-Cell Lymphoma (DLBCL) cell lines.

Compound 3 as single agent strongly inhibited the growth of the majority of the 17 DLBCL lines tested (Table l). Thus, 14 cell lines displayed ÎC50S below 100 nM, and an additional l cell lines displayed IC50S between 100 nM and l uM. Only 2 cell lines displayed an IC50 above l uM.

Compound l, HCl as single agent also inhibited the growth of the majority of the 17 DLBCL lines tested, although slightly less potent (Table 2). Thus, 2 cell lines displayed IC50S below 100 nM, and 6 cell lines displayed IC50S between 100 nM and l uM. Nine cell line displayed an IC50 above l uM (four of which above IO uM).

In combination, Compound 3 and Compound l, HCl treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2 - Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666) in 16 out of 17 DLBCL cell lines tested (Table 2). In 5 cell lines, the synergy effect was marked, with synergy scores between 5 and IO. In 4 cell lines, the synergy effect was exceptional, achieving synergy scores between IO and 17.3. Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of Compound 3 and Compound l that did not display an anti-proliferative effect on their own. For example, in DB cells, Compound 3 and Compound l at the second lowest concentration tested elicited a growth inhibition of only l and 2 %, respectively, while the respective combination of the two compounds afforded a growth inhibition of 96% (Figure 4A, left panel), thus being 91% above the additivity calculated based on the single agent activities (Figure 4A, right panel). As an additional example, in Toledo cells, in which Compound 3 was less potent and achieved only partial growth inhibition (46%) at the highest concentration tested, the combination with the second lowest concentrations of Compound l resulted in synergistic growth inhibition of 98% (Figure 4B, left panel), thus being 52% above the additivity calculated based on the single agent activities (Figure 4B, right panel).

Furthermore, it is noteworthy that the synergistic effects occurred across a broad range of single agent concentrations, which should prove bénéficiai in vivo with respect to flexibility concerning dosing levels and scheduling.

In summary, the combination of Compound 3 and Compound l afforded strong to exceptional synergistic growth inhibition in the majority of DLBCL cell lines tested.

EXAMPLE 6: In vivo efficacy in Karpas422 xenografts with combination of a MCLl inhibitor (Compound 3) and a BCL-2 inhibitor (Compound 1)

Material and Method

Tumour Cell Culture and Cell Inoculation

Karpas 422 human B-cell non-Hodgkin's lymphoma (NHL) cell line was established from the pleural effusion of a patient with chemotherapy-resistant NHL. The cells were obtained from the DSMZ cell bank and cultured in RPMI-1640 medium (BioConcept Ltd. Amimed,) supplemented with 10% FCS (BioConcept Ltd. Amimed), 2 mM L-glutamine (BioConcept Ltd. Amimed), 1 mM sodium pyruvate (BioConcept Ltd. Amimed) and 10 mM HEPES (Gibco) at 37°C in an atmosphère of 5% CO₂ in air. Cells were maintained between 0.5 and 1.5 x 106 cells/mL. To establish Karpas 422 xenografts cells were harvested and re-suspended in HBSS (Gibco) and mixed with Matrigel (BD Bioscience) (1:1 v/v) before injecting 200 pL containing 1x107 cells subcutaneously in the right flanks of animais which were anesthetized with isoflurane. Twenty four hours prior to cell inoculation ail animais were irradiated with 5Gy over 2 minutes using a x-irradiator.

Tumour Growth

Tumour growth was monitored regularly post cell inoculation and animais were randomised into treatment groups (n=5) when tumour volume reached appropriate volume. During the treatment period tumour volume was measured about twice a week using calipers. Tumour size, in mm³, was calculated from: (L x W2 x π/6). Where W = width and L = length of the tumour.

Treatment

Tumour bearing animais (rats) were enrolled into treatment groups (n=5) when their tumours reached an appropriate size to form a group with a mean tumour volume of about

450 mm³. The treatment groups were as outlined in Table 3. The vehicle for Compound l, HCl or Compound l, HCl was administered by oral (po) gavage l h before vehicle for Compound 3 or Compound 3 which was administered by 15 minutes iv infusion. For iv infusion animais were anesthetized with isoflurane/O? and the vehicle or Compound 3 administered via a cannula in the tail vein. Animais were weighed at dosing day(s) and dose was body weight adjusted, dosing volume was 10 ml/kg for both compounds.

Body weights

Animais were weighed at least 2 times per week and examined frequently for overt signs of any adverse effects.

Data analysis and statistical évaluation

Tumour data were analyzed statistically using GraphPad Prism 7.00 (GraphPad Software). If the variances in the data were normally distributed, the data were analyzed using oneway ANOVA with post hoc Dunnett’s test for comparison of treatment versus control group. The post hoc Tukey’s test was used for intragroup comparison. Otherwise, the Kruskal-Wallis ranked test post hoc Dunn’s was used. When applicable, results are presented as mean ± SEM.

As a measure of efficacy the %T/C value is calculated at the end of the experiment according to:

(Atumour volume^treated/Atumour volume^contro1)* 100

Tumour régression was calculated according to:

-(Atumour volume^treated/tumour volume^{treated atstart})* 100 wherein Atumour volumes represent the mean tumour volume on the évaluation day minus the mean tumour volume at the start of the experiment.

Table 3. Treatment groups for combination efficacy in Karpass422 xenograft bearing rats

Groups	Treatment	Dose (expressed as the free base)	Schedule	Number of rats
1	Vehicle for Compound 1, HCl (PEG400/EtOH/Phosal 50 PG (30/10/60)), po Ih before vehicle for Compound 3, 15 minutes iv infusion 10 ml/kg	10 ml/kg + 10 ml/kg	QW, po Ih before + QW, iv infusion	5
2	Vehicle for Compound 1, HCl + Compound 3	0 mg/kg + 20 mg/kg	QW, po Ih before + QW, iv infusion	5
3	Compound 1, HCl + Vehicle for Compound 3	150 mg/kg + 0 mg/kg	QW, po Ih before + QW, iv infusion	5
4	Compound 1, HCl + Compound 3	150 mg/kg + 20 mg/kg	QW, po Ih before + QW, iv infusion	5

Treatments were initiated when the average tumour volume was about 450 mm³.

Compound l, HCl was formulated in PEG400/EtOH/Phosal 50 PG (30/I0/60) and

Compound 3 was placed in solution.

QW means once-weekly.

Results

Combination treatment with Compound l free base at 150 mg/kg po Ih before Compound at 20 mg/kg iv infusion induces complété régression in ail Karpas422 tumours by day 30 from start of treatment (Figure 5). Ail animais in the treatment group hâve remained 10 tumour free after treatment was stopped on day 35 up to day 90. A positive combination effect is observed in the combination group compared with single agent activity. On day 34 the tumour response in the single agent Compound 3 and the combination group are significantly different from the vehicle group (p<0.05).The combination treatment is well tolerated based on body weight changes (Figure 6).

EXAMPLE 7: In vivo efficacy in DLBCL Toledo xenograft with combination of a MCL1 inhibitor (Compound 3) and a BCL-2 inhibitor (Compound 1, HCl)

Material and Method

Cell implantation

The xenograft model was established by direct subcutaneous (sc) implantation of 3 million Toledo cell suspension with 50% matrigel into the subcutaneous area of SCID/beige mice. Ail procedures were carried out using aseptie technique. The mice were anesthetized during the entire period of the procedure.

In general, a total of 6 animais per group were enrolled in efficacy study. For single-agent and combination studies, animais were dosed via oral gavage (po) for Compound 1 and intravenously (iv) via tail vein for Compound 3. Compound 1, HCl was formulated as solution in PEG300/EtOH/water (40/10/50), and Compound 3 was placed in solution. When tumors reached approximately 220 mm³ at day 26 post cell implantation, tumourbearing mice were randomized into treatment groups.

The design of the study including dose schedule for ail treatment groups are summarized in the table below. Animais were weighed at dosing day(s) and dose was body weight adjusted, dosing volume was 10 ml/kg. Tumour dimensions and body weights were collected at the time of randomization and twice weekly thereafter for the study duration. The following data was provided after each day of data collection: incidence of mortality, individual and group average body weights, and individual and group average tumour volume.

Groups	Treatment	Dose (expressed as the free base)	Schedule	Number of mice
1	Vehicle	PEG300/EtOH/water (40/10/50)	QW,po	6
2	Compound 1, HCl	100 mg/kg	QW,po	6
3	Compound 3	25 mg/kg	QW, iv	6
4	Compound 1, HCl + Compound 3	100 mg/kg 25 mg/kg	QW,po QW, iv	6

For the study in Toledo model, treatments were initiated on day 26 following cell implantation, when the average tumour volume was -218 to 228 mm³.

QW means once-weekly.

Body Weight (BW)

The % change in body weight was calculated as (BW_CUrrent - BWi_nitiai)/(BWi_nitiai) x 100. Data is presented as percent body weight change from the day of treatment initiation.

Tumour Volume andpercent mice remaining on the study

Percent treatment/control (T/C) values were calculated using the following formula:

% T/C = 100 x AT/AC if AT >0 % Régression = 100 x AT/To if AT <0 where:

T = mean tumour volume of the drug-treated group on the final day of the study;

AT = mean tumour volume of the drug-treated group on the final day of the study - mean tumour volume ofthe drug-treated group on initial day of dosing;

To = mean tumour volume of the drug-treated group on the day of cohort;

C = mean tumour volume of the control group on the final day of the study; and

AC = mean tumour volume of the control group on the final day of the study - mean tumour volume of the control group on initial day of dosing.

Percent mice remaining on the study ⁼ 6- number of mice reaching end point/6 100

Statistical Analysis

Ail data were expressed as mean ± standard error of the mean (SEM). Delta tumour volume and percent body weight changes were used for statistical analysis. Between group comparisons were carried out using the One way ANOVA followed by a post hoc Tukey test. For ail statistical évaluations, the level of significance was set at p < 0.05. Significance compared to the vehicle control group is reported unless otherwise stated.

Results

Treatment	T/C% at day 42
Vehicle	100
Compound 1, HCl	37
Compound 3	102
Compound 1, HCI + Compound 3	3

In Toledo model, Compound l free base at 100 mg/kg produced statistically significant anti-tumour effects with 37% T/C. Compound 3 at 25 mg/kg resulted in no anti-tumour effects with 102% T/C (Figure 7). Combination of Compound 1 + Compound 3 led to tumour stasis with 3% T/C, which is statistically significant compared to Vehicle, Compound 1 and Compound 3 treated tumors (p<0.05, by one-way ANOVA test).

Therefore, combined inhibition of BCL-2 and MCLl in DLBCL may provide a therapeutic benefit in the clinic. In addition, the mean body weight change for Toledo is shown in Figure 8. Treatment of mice with Compound 1, HCl and Compound 3 exhibit body weight gain (1.081% and 2.3%, respectively). The combination group showed slight body weight loss (-3.2%). No other signs of adverse events were observed in this study. Ail 6 animais survived throughout the study.

Taken altogether, Examples 2, 6 and 7 show that the combination of a MCLl inhibitor and a BCL-2 inhibitor is efficacious at tolerated doses in mice and rats bearing xenografts of acute myeloid leukemia and lymphoma human derived cell lines, suggesting that a suitable therapeutic window is achievable with this combination in these diseases.

EXAMPLE 8: In vitro effect on prolifération of combining a MCLl inhibitor with a BCL-2 inhibitor in a panel of 13 Acute Myeloid Leukemia (AML) cell Unes.

Material and Method

Cell Unes were sourced and maintained in the basic media supplemented with FBS (Fêtai Bovine Sérum) as indicated in Table 1. In addition, ail media contained penicillin (100 lü/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM).

Cell lines were cultured at 37°C in a humidified atmosphère containing 5% CO₂ and expanded in T-150 flasks. In ail cases cells were thawed from frozen stocks, expanded through >1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter prior to plating 150 ul/well at the densities indicated in Table 1 into 96well plates. AH cell lines were determined to be free of mycoplasma contamination inhouse.

Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C.

In order to analyse the activity of the compounds as single agents, cells were seeded and treated with nine 2-fold serial dilutions of each compound dispensed individually directly into the cell assay plates. Effects of the compounds on cell viability were assessed after 3 days of incubation at 37 °C/5% CO2 by quantification of cellular ATP levels using CelITiterGlo at 75 pL reagent/well. Ail the experiments were performed in triplicates. Luminescence was quantified on a multipurpose plate reader. Single agent IC50S were calculated using standard four-parametric curve fitting. IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the vehicle (DMSO) control.

In order to analyse the activity of the compounds in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound dispensed, either individually or in ail possible permutations in a checkerboard fashion, directly into the cell assay plates as indicated in Figure 9. Effects of the single agents as well as their checkerboard combinations on cell viability were assessed after 3 days of incubation at 37 °C/5% CO2 by quantification of cellular ATP levels using CelITiterGIo at 75 gL reagent/welL Two independent experiments, each one performed in duplicates, were performed. Luminescence was quantified on a multipurpose plate reader.

Potential synergistic interactions between compound combinations were assessed using the Excess Inhibition 2D matrix according to the Loewe additivity model and are reported as Synergy Score (Lehar et al, Nat Biotechnol. 2009 Juiy ; 27(7): 659-666). Ail calculations were performed using Clalice™ Bioinformatics Software.

The doubling time indicated in Table 3 is the mean of the doubling time obtained in the IO different passages (in T-150 flasks) performed from the thawing of the cells to their seeding in the 96-weel plates.

The interprétation of the Synergy Score is as follows:

SS ~ 0 —* Additive

SS >l Weak Synergy

SS >2 —> Synergy

Table 3. Identity and assay conditions for the 13 Acute Myeloid Leukemia (AML) cell lines used in the combination experiments.

Cell line	Medium (source)	%FBS	Doubling time (hours)	Cell number seeded/well
MV4;11	RPMI (ATCC)	10	31.0	56520
MOLM-13	RPMI (DSMZ)	10	32.4	56520
PL-21	RPMI (DSMZ)	10	32.4	56520
ML-2	RPMI (DSMZ)	10	31.6	56520
Nomo-1	RPMI (DSMZ)	10	43.5	56520
THP-1	RPMI (ATCC)	10	49.6	56520
HL-60	IMDM (ATCC)	20	34.8	56520
Kasumi-1	RPMI (ATCC)	20	59.4	56520
OCI-AML3	MEM alpha (DSMZ)	20	25.7	56520
EOL-1	RPMI (DSMZ)	10	37.6	113040
GDM-1	RPMI (ATCC)	10	31.6	56520
KG1	IMDM (ATCC)	20	45.7	56520
KGla	IMDM (ATCC)	20	36.5	56520

Table 4a. Single agent IC50 values for Compound 3, Compound l, HCl and ABT-199 in

AML cell lines are indicated. Compounds were incubated with the cells during 3 days.

Cell Line	Compound 3	Compound 1, HCl	ABT-199
Start conc [uM]	IC50 [uM]	Start conc [uM]	IC50 [uM]	Start conc [uM]	IC50 [uM]
MV4;11	0.01	0.001	0.1	0.03	n.d.	n.d.
MOLM-13	0.01	0.002	0.1	0.04	n.d.	n.d.
PL-21	0.10	0.065	30.0	2.78	15.0	3.300
ML-2	0.10	0.005	2.0	0.04	n.d.	n.d.
Nomo-1	0.05	0.013	30.0	7.45	15.0	5.000
THP-1	0.10	0.017	30.0	0.75	2.0	0.900
HL-60	0.10	0.025	30.0	1.42	15.0	2.100
Kasumi-1	2.00	0.033	30.0	0.77	n.d.	n.d.
OCLAML3	2.00	0.146	30.0	8.09	15.0	8.500
EOL-1	0.10	0.001	2.0	0.04	0.2	0.004
GDM-1	0.10	0.008	2.0	0.06	n.d.	n.d.
KG1	30.00	0.390	30.0	4.70	15.0	3.400
KGla	30.00	2.000	30.0	1.75	15.0	0.900

Table 4b. Single agent IC50 values for Compound 4, HCl in 5 AML cell lines are indicated. Compound was incubated with the cells during 3 days.

Cell Line	Compound 4, HCl
Start conc [uM]	IC50 [uM]
MV4;11	0.5	0.01
MOLM-13	0.5	0.012
ML-2	0.5	0.01
OCI-AML3	15	5.41
GDM-1	0.5	0.002

Table 5a. Synergy scores for Compound 3 and Compound l combination in 13 AML cell lines are indicated. Interactions were deemed synergistic when scores > 2.0 where observed. Start concentrations of compounds, mean of max inhibition and the standard déviation (sd) of the synergy scores are indicated. Compounds were incubated with the 5 cells during 3 days.

Cell Line	Compound 3	Compound 1, HCl	Combination (a)
Start cône [uM]	Mean of Max Inh (%]	Start conc [uM]	Mean of Max Inh [%l	Mean of SynergyScore	Synergy Score Error (sd)
MV4;11	0.1	100.0	0.3	99.5	4.3	0.7
MOLM-13	0.1	99.5	0.3	90.0	8.2	1.3
PL-21	0.3	91.5	5.0	75.0	17.9	2.7
ML-2	0.1	99.5	0.3	88.0	10.9	1.8
Nomo-1	0.3	97.0	5.0	31.0	11.8	1.0
THP-1	0.3	99.0	5.0	55.5	13.2 :	0.1
HL-60	0.3	97.5	5.0	61.5	12.9	1.7
Kasumi-1	0.3	84.5	2.0	52.5	10.5	0.5
OCI-AML3	2.0	100.0	5.0	53.5	19.8	0.2
EOL-1	0.1	100.0	1.0	100.0	5.8	0.8
GDM-1	0.1	99.0	0.3	87.0	11.1	1.4
KG1	2.0	80.5	5.0	53.0	14.6	1.7
KG la	2.0	28.5	5.0	73.0	13.0	0.9

Table 5b. Synergy scores for Compound 3 and ABT-199 combination in 8 AML cell lines are indicated. Interactions were deemed synergistic when scores > 2.0 where observed. Start concentrations of compounds, mean of max inhibition and the standard déviation (sd) of the synergy scores are indicated. Compounds were incubated with the cells during 3 days.

Cell Line	Compound 3	ABT-199	Combination (b)
Start cône [uM]	Mean of Max Inh i%]	Start conc [uMJ	Mean of Max Inh [%]	lîillÎBlîïMli Mean of SynergyScore .	Synergy Score Error (sd)
PL-21	0.3	89.0	2.0	41.5	19.7	2.7
Nonio-1	0.3	95.5	2.0	45.5	10.7	1.6
THP-1	0.3	97.0	0.3	47.0		0.7
HL-60	0.3	97.5	2.0	56.0	12.9	1.6
OCI-AML3	2.0	100.0	2.0	58.5	16.4	0.5
EOL-1	0.1	100.0	0.1	97.5	4.0	0.3
KG1	2.0	89.0	2.0	54.5	12.2	0.8
KG1a	2.0	57.5	2.0	73.0	17.6	0.1

Table 5c. Synergy scores for Compound 3 and Compound 4, HCl combination in 5 AML cell lines are indicated. Interactions were deemed synergistic when scores > 2.0 where observed. Start concentrations of compounds, mean of max inhibition and the standard déviation (sd) of the synergy scores are indicated. Compounds were incubated with the cells during 3 days.

Cell Line	Compound 3	Compound 4, HCl	Combination (c)
Start conc [uM]	Mean of Max Inh [%]	Start conc [uM]	Mean of Max Inh [%]	Mean of SynergyScore	Synergy Score Error (sd)
MV4;11	0.01	100.0	0.03	70	3.37	0.75
MOLM-13	0.1	100	0.1	99	3.84	0.02
ML-2	0.1	100	0.1	99	7.09	0.96
OCLAML3	2.0	100.0	5.0	53.5	16.53	1.62
GDM-1	0.1	100	0.1	99	7.03	0.52

Results

Combination (a). The effect on prolifération of combining the MCLl inhibitor Compound 3 with the BCL-2 inhibitor Compound l was assessed in a panel of 13 Acute Myeloid Leukemia (AML) cell lines.

Compound 3 as single agent strongly inhibited the growth of the majority of the 13 AML lines tested (Table 4a). Thus, 10 cell lines displayed ICsos below 100 nM, and an additional 2 cell lines displayed IC50S between 100 nM and l uM. Only l cell lines displayed an IC₅o above l uM.

Compound l, HCl as single agent also inhibited the growth of the several AML lines tested, although slightly less potent (Table 4a). Thus, 5 cell lines displayed ICsos below 100 nM, and 2 cell lines displayed IC50S between 100 nM and l uM. Six cell lines displayed an ICso above l uM.

In combination, Compound 3 and Compound l, HCl treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2) in the entire 13 cell lines tested (Table 5a). In 2 cell lines, the synergy effect was marked, with synergy scores between 5 and 10. In 10 cell lines, the synergy effect was exceptional, achieving synergy scores between 10 and 19.8.

Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of Compound 3 and Compound l that did not hâve an anti-proliferative effect on their own. For example, in OCI-AML3 cells, Compound 3 and Compound l at the third lowest concentration tested elicited a growth inhibition of 5 and l%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 84% (Figure 9A, top left panel), thus being 79% above the additivity calculated based on the single agent activities (Figure 9A, top right panel).

Furthermore, it is noteworthy that the synergistic effects occurred across a broad range of single agent concentrations, which should prove bénéficiai in vivo with respect to flexibility concerning dosing levels and scheduling.

In summary, the combination of Compound 3 and Compound l afforded synergistic growth inhibition in ail the 13 AML cell fines tested. Importantly, exceptional synergistic growth inhibition was observed in the majority AML cell fines tested (10/13).

Combination (b). The effect on prolifération of combining the MCLl inhibitor Compound 3 with the BCL-2 inhibitor ABT-199 was assessed in a panel of 8 Acute Myeloid Leukemia (AML) cell fines.

Compound 3 as single agent strongly inhibited the growth of the majority of the 8 AML fines tested (Table 4a). Thus, 5 cell fines displayed IC50S below 100 nM, and an additional 2 cell fines displayed IC50S between 100 nM and l uM. Only l cell fines displayed an IC50 above l uM.

ABT-199 as single agent also inhibited the growth of AML fines, although with less potency (Table 4a). Thus, only one cell line displayed IC50S below 100 nM, and 2 cell fines displayed IC50s between 100 nM and l uM. Five cell fines displayed IC50 above l uM.

In combination, MCLl inhibitor and ABT-199 treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2) in the entire panel of 8 cell fines tested (Table 5b). In the majority of the cell fines, the synergy effect was exceptional, achieving synergy scores between 10 and 17.6. Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of MCLl inhibitor and ABT-199 that did not hâve an anti-proliferative effect on their own. For example, in OCI-AML3 cells, MCLl and ABT-199 at the third lowest concentration tested elicited a growth inhibition of 26% and 18%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 91% (Figure 13, top left panel).

Furthermore, it is noteworthy that the synergistic effects occurred across a broad range of single agent concentrations, which should prove bénéficiai in vivo with respect to flexibility concerning dosing levels and scheduling.

In summary, the combination of Compound 3 and ABT-199 afforded synergistic growth inhibition in ail the 8 AML cell lines tested. Importantly, exceptional synergistic growth inhibition was observed in the majority AML cell lines tested (7/8).

Combination (c). The effect on prolifération of combining the MCLl inhibitor Compound 3 with the BCL-2 inhibitor Compound 4 was assessed in a panel of 5 Acute Myeloid Leukemia (AML) cell lines.

Compound 3 as single agent strongly inhibited the growth ofthe 5 AML lines tested (Table 4b). Thus, ail cell lines displayed IC50S below 200 nM. Compound 4, HCl as single agent also inhibited the growth of the 4 out of 5 cell lines tested with IC50 below or equal to 40 nM, one cell line being résistant to Compound 4 with an IC50 of ΙΟμΜ. In combination, Compound 3 and Compound 4, HCl treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2) in the entire 5 cell lines tested (Table 5c). In 2 cell lines, the synergy effect was marked, with synergy scores between 5 and ΙΟ. In l cell line, the synergy effect was exceptional, achieving synergy score of 16.5. Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of Compound 4, HCl and Compound 3 that hâve no or low antiproliferative effect on their own. For example, in OCI-AML3 cells, Compound 4, HCl and Compound 3 at the third lowest concentration tested elicited a growth inhibition of l and 40%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 98% (Figure IA, left panel; représentative of two independent experiments), thus being 53% above the additivity calculated based on the single agent activities (Figure 14A, right panel). In ML-2, Compound 4, HCl and Compound 3 at the fîfth lowest concentration tested elicited a growth inhibition of 18 and 26%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 100% (Figure 14B, left panel; représentative of two independent experiments), thus being

51% above the additivity calculated based on the single agent activities (Figure 15, right panel)

In summary, the combination of Compound 4 and Compound 3 afforded synergistic growth inhibition in ail the 5 AML cell lines tested.

EXAMPLE 9: In vitro effect on prolifération of combining a MCL1 inhibitor with a

BCL-2 inhibitor in a panel of of 12 neuroblastoma (NB) cell lines

Materials and methods

Cell lines were sourced and maintained in the basic media supplemented with FBS as 10 indicated in Table 1. In addition, ail media contained penicillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM). Cell lines were cultured at 37°C in a humidified atmosphère containing 5% CO2 and expanded in T-150 flasks. In ail cases cells were thawed from frozen stocks, expanded through >1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter prior to plating 150 ul/well at 15 the densities indicated in Table 6 into 96-well plates. Ail cell lines were determined to be free of mycoplasma contamination in-house.

Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. In order to analyse the activity of the compounds as single agents, cells were seeded and treated with nine 3.16-fold serial dilutions of each compound dispensed 20 individually directly into the cell assay plates. Effects of the compounds on cell viability were assessed after 2 or 3 days of incubation (as indicated in Table 6) at 37 °C/5% CO2 by quantification of cellular ATP levels using CelITiterGlo at 150 pL reagent/well. Two independent experiments, each one performed in duplicates were performed. Ail the experiments were performed in triplicates. Luminescence was quantified on a multipurpose 25 platereader. Single agent IC50S were calculated using standard four-parametric curve fitting. IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the vehicle (DMSO) control.

Identical experiments were performed to assess potential synergistic interactions between compound combinations. Synergy Score were assessed using the Excess Inhibition 2D 30 matrix according to the Loewe additivity mode! (Lehar et al, Nat Biotechnol. 2009 July ;

27(7): 659-666). Ail calculations were performed using Chalice TM Bioinformatics Software.

The doubling time indicated in Table 6 is the mean of the doubling time obtained in the different passages (in T-150 flasks) performed from the thawing of the cells to their 5 seeding in the 96-weel plates.

The interprétation of the Synergy Score is as follows:

SS ~ 0 —► Additive

SS >l —> Weak Synergy

SS >2 —> Synergy

Table 6. Identity and assay conditions for the 12 neuroblastoma (NB) cell lines used in the combination experiments.

Cell line	Medium (source)	%FBS	Doubling time (hours)	Cell number seeded/well	Days of incubation with cpds
SK-N-AS	DMEM (ATCC)	10	33	9375	3
SK-N-BE	EMEM/Ham F12 (ATCC)	10	50	37500	3
SK-N-DZ	DMEM (ATCC)	10	42	37500	3
LAN-6	DMEM (DSMZ)	20	100	9375	3
NBL-S	Iscove's MDM (DSMZ)	10	46	18750	3
SIMA	RPMI (DSMZ)	10	60	18750	3
KELLY	RPMI (ECACC)	10	34	3750	2
IMR-32	EMEM (ATCC)	10	55	28125	2
SH-SY-5Y	1/2 EMEM no glutamin+ 1/2 Ham F12 + 2mM Glutamin + NEAA (ECACC)	15	35	3750	2
SK-N-SH	EMEM (ATCC)	10	65	3750	2
NB-1	RPMI (JCRB)	10	35	15000	2
SK-N-FI	DMEM (ATCC)	10	60	7500	2

Table 7. Single agent IC50 values for Compound 3 and Compound l, HCl, are indicated.

Compounds were incubated with the cells during 2 or 3 days.

Cell Line	Compound 3	Compound 1, HCl
IC50 [uM]	IC50 [uM]
SK-N-AS	0.26	> 1
SK-N-BE	>2	>2
SK-N-DZ	>2	>2
LAN-6	>2	>2
NBL-S	>2	>2
SIMA	>2	>2
NB1	0.123	>3
SK-N-SH	>3	>3
SH-SY5Y	>3	>3
Kelly	0.031	>3
SK-N-FI	>3	>3

Table 8. Synergy scores for combination with Compound 3 and Compound l, HCl are indicated. Interactions were deemed synergistic when scores > 2.0 where observed.

Compounds were incubated with the cells during 2 or 3 days.

Cell Line	Compound 3	Compound 1, HCI	Combination
Start conc |uM|	Mean of Max Inh |%]	Start conc |uM|	Max Inh [%]	Synergy Score	Synergy Score Error
SK-N-AS	2	84	1	9	2.78	0.46
SK-N-BE	2	27	2	27	10.72	0.78
SK-N-DZ	2	2.5	2	10	0.34	0.06
LAN-6	2	17.5	2	26	10.51	0.39
NBL-S	2	13	2	10	17.81	3.7
SIMA	2	0	2	48.5	2.41	0.75
NB1	3	99	3	11	10.72	4.33
SK-N-SH	3	40	3	15	4.07	0.23
SH-SY5Y	3	24	3	10	10.21	0.54
Kelly	3	99	3	27	9.62	0.48
SK-N-FI	3	33	3	6	4.35	0.91

Results

The effect on prolifération of combining the MCLl inhibitor Compound 3 with the BCL-2 inhibitor Compound l was assessed in a panel of 12 neuroblastoma cell lines. Three out of the 12 cell lines tested are sensitive to Compound 3 as single agent (Table 7). One cell lines displayed IC50S below 100 nM, and an additional 2 cell lines displayed ICsos between

100 nM and l uM.

Ail cell lines are résistant to Compound l, HCl as single agent with ail cell lines tested displaying an IC50 above ΙμΜ. In combination, Compound 3 and Compound l treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2 - Lehar et al, Nat IO Biotechnol. 2009 July ; 27(7): 659-666) in 11 out of 12 NB cell lines tested (Table 8). In 5 cell lines, the synergy effect was exceptional, achieving synergy scores between IO and 17.8L Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of Compound 3 and Compound l, HCl that did not hâve an anti-proliferative effect on their own. For example, 15 in LAN-6 cells, Compound 3 and Compound l, HCl at 630 nM elicited a growth inhibition of only 12% and 0%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 95% (Figure IO, upper left panel), thus being 76% above the additivity calculated based on the single agent activités (Figure IO, upper right panel).

In summary, the combination of Compound 3 and Compound l afforded strong to 20 exceptional synergistic growth inhibition in the majority of neuroblastoma cell lines tested.

EXAMPLE 10: In vitro effect on prolifération of combining a MCLl inhibitor with a BCL-2 inhibitor in a panel of 8 B-cell acute lymphoblastic leukaemia (B-ALL) and 10 T-cell acute lymphoblastic leukaemia (T-ALL) cell lines.

Materials and methods

Cell lines were sourced and maintained in the basic media supplemented with FBS as indicated in Table 1. In addition, ail media contained penicillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM). Cell lines were cultured at 37°C in a humidified atmosphère containing 5% CO2 and expanded in T-150 flasks. In ail cases cells were thawed from frozen stocks, expanded through >1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter prior to plating 150 ul/well at the densities indicated in Table 9 into 96-well plates. Ail cell Unes were determined to be free of mycoplasma contamination in-house.

Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. In order to analyse the activity of the compounds as single agents, cells were seeded and treated with nine 2-fold serial dilutions of each compound dispensed individually directly into the cell assay plates. Effects of the compounds on cell viability were assessed after 3 days of incubation at 37 °C/5% CO2 by quantification of cellular ATP levels using CellTiterGlo at 75 pL reagent/welL Ail the conditions were tested in triplicates. Luminescence was quantified on a multipurpose plate reader. Single agent IC50S were calculated using standard four-parametric curve fitting. IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the vehicle (DMSO) control.

In order to analyse the activity of the compounds in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound dispensed, either individually or in ail possible permutations in a checkerboard fashion, directly into the cell assay plates as indicated in Figure L Effects of the single agents as well as their checkerboard combinations on cell viability were assessed after 3 days of incubation at 37 °C/5% CO2 by quantification of cellular ATP levels using CellTiterGlo at 75 pL reagent/welL For B-ALL cell lines, two independent experiments, each one performed in duplicates, were performed. For T-ALL cell lines, one experiment performed in triplicate was performed. Luminescence was quantified on a multipurpose plate reader.

Potentïal synergistic interactions between compound combinations were assessed using the Excess Inhibition 2D matrix according to the Loewe additivity model and are reported as Synergy Score (Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666). Ail calculations were performed using Chalice TM Bioinformatics Software available in Horizon website.

The doubling time indicated in Table 9 is the mean of the doubling time obtained in the different passages (in T-150 flasks) performed from the thawing of the cells to their seeding in the 96-weel plates.

The interprétation of the Synergy Score is as follows:

SS ~ 0 —► Additive

SS >l —> Weak Synergy

SS >2 —► Synergy

Table 9. Identity and assay conditions for the 8 B-ALL and 10 T-ALL cell lines used in the combination experiments.

Cell line	Cancer type	Medium (source)	%FBS	Doubling time (hours)	Cell number seeded/well
TOM-1	B-ALL	RPMI (DSMZ)	20	70.0	112500
SUP-B15	B-ALL	McCoy (DSMZ)	20	35.0	112500
NALM-21	B-ALL	RPMI (DSMZ)	10	50.0	112500
NALM-6	B-ALL	RPMI (DSMZ)	10	27.0	56250
TANGUE	B-ALL	RPMI (DSMZ)	10	26.0	30000
Kasumi-2	B-ALL	RPMI (DSMZ)	10	52.0	112500
RS4;11	B-ALL	RPMI (ATCC)	10	42.0	90000
BALL-1	B-ALL	RPMI (DSMZ)	10	38.0	112500
BE-13	T-ALL	RPMI 1640 (DSMZ)	10	37.0	13875
MOLT-4	T-ALL	RPMI 1640 (ATCC)	10	24.0	28125
TALL-104	T-ALL	IMDM (ATCC)	20	68.0	13875
HPB-ALL	T-ALL	RPMI 1640 (DSMZ)	20	42.0	56250
DND-41	T-ALL	RPMI 1640 (DSMZ)	10	38.0	56250
CML-T1	T-ALL	RPMI 1640 (DSMZ)	10	32.0	112500
J45.01	T-ALL	RPMI 1640 (ATCC)	10	25.0	56250
CCRF- CEM	T-ALL	RPMI 1640 (ATCC)	10	24.0	56250
J.RT3 T3.5	T-ALL	RPMI 1640 (ATCC)	10	24.0	56250
Loucy	T-ALL	RPMI 1640 (ATCC)	10	61.0	112500

Table 10. Single agent IC50 values for Compound 3 and Compound 1, HCl in the 8 B-ALL and 10 T-ALL cell lines are indicated. Compounds were incubated with the cells during 3 days.

Cell Line	Cancer type	Treatment duration(h)	Compound 3	Compound 1, HCl
Start cone |uM|	IC50 [uM]	Start conc |uM|	IC50 [uM|
TOM-1	B-ALL	72	0.10	0.024	0.15	0.019
SUP-B15	B-ALL	72	2.00	0.078	0.90	0.025

NALM-21	B-ALL	72	0.10	0.012	0.50	0.095
NALM-6	B-ALL	72	2.00	0.120	30.00	3.630
TANGUE	B-ALL	72	30.00	6.540	30.00	17.000
Kasumi-2	B-ALL	72	2.00	0.030	2.00	0.209
RS4;11	B-ALL	72	0.90	0.079	9.00	0.020
BALL-1	B-ALL	72	0.25	0.063	0.10	0.019
BE-13	T-ALL	72	0.15	0.015	30.00	6.700
MOLT-4	T-ALL	72	2.00	0.026	30.00	3.290
TALL-104	T-ALL	72	2.00	0.044	30.00	15.900
HPB-ALL	T-ALL	72	2.00	0.660	30.00	4.500
DND-41	T-ALL	72	30.00	7.000	30.00	9.000
CML-T1	T-ALL	72	30.00	6.000	30.00	15.000
J45.01	T-ALL	48	0.60	0.029	30.00	9.000
CCRF-CEM	T-ALL	48	0.90	0.047	30.00	7.500
J.RT3 T3.5	T-ALL	48	1.88	0.063	30.00	10.000
Loucy	T-ALL	48	0.90	0.064	3.75	0.231

Table 11. Synergy scores for Compound 3 and Compound 1, HCl combination in 8 ΒΑΤΕ and 10 T-ALL cell lines are indicated. Interactions were deemed synergistic when scores > 2.0 where observed. Start concentrations of compounds, mean of max inhibition and the standard déviation (sd) of the synergy scores are indicated. Compounds were 5 incubated with the cells during 3 days.

Cell Line	Cancer type	Treatment duration (h)	Compound 3	Compound 1, HCl	Combination
Start conc [uM]	Mean of Max Inh [%]	Start conc [uM]	Mean of Max Inh [%l	Mean of Synergy Score	Synergy Score Error (sd)
TOM-1	B-ALL	72	0.3	98.5	0.1	90.5	4.1	0.4
SUP-B15	B-ALL	72	2.0	99.0	0.3	97.0	5.6	0.4
NALM-21	B-ALL	72	0.3	99.0	0.3	84.5	9.6	0.0
NALM-6	B-ALL	72	2.0	71.5	5.0	56.5	15.9	1.4
TANGUE	B-ALL	72	5.0	78.5	5.0	13.0	1.0	0.6
Kasumi-2	B-ALL	72	0.3	99.0	2.0	82.0	10.1	1.9
RS4;11	B-ALL	72	0.3	87.5	0.3	98.0	7.0	1.3
BALL-1	B-ALL	72	0.3	96.0	0.3	100.0	6.3	0.3
BE-13	T-ALL	72	1.0	95.0	5.0	0.0	8.8	0.4
MOLT-4	T-ALL	72	1.0	99.0	5.0	63.0	4.4	0.1
TALL-104	T-ALL	72	1.0	99.0	2.0	29.0	15.1	0.5

HPB-ALL	T-ALL	72	l.O	49.0	5.0	15.0	5.6	0.3
DND-41	T-ALL	72	2.0	17.0	5.0	24.0	10.3	0.3
CML-T1	T-ALL	72	2.0	22.3	5.0	17.0	3.3	0.2
J45.01	T-ALL	48	2.0	100.0	2.0	23.0	2.9	0.1
CCRF- CEM	T-ALL	48	2.0	92.0	2.0	55.0	4.1	1.0
J.RT3 T3.5	T-ALL	48	2.0	99.0	2.0	32.0	3.8	0.1
Loucy	T-ALL	48	2.0	100.0	2.0	77.0	11.3	0.6

Results

The effect on prolifération of combining the MCLl inhibitor with the BCL-2 inhibitor was assessed in a panel of 8 B-ALL and 10 T-ALL cell lines.

MCLl inhibitor as single agent strongly inhibited the growth of the majority of the ALL 5 cell lines tested (Table 10). Thus, 13 ALL cell lines displayed ICsos below 100 nM, and an additional 2 ALL cell lines displayed ICsos between 100 nM and l uM. Only 3 ALL cell lines displayed IC50 above l uM.

BCL-2 inhibitor as single agent also inhibited the growth of several ALL cell lines tested, although it was less potent (Table IO). Thus, 5 cell lines displayed ICsos below IOO nM, IO and 2 cell lines displayed ICsos between IOO nM and l uM. Eleven ALL cell lines displayed an IC50 above l uM.

In combination, MCLl inhibitor and BCL-2 inhibitor treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2 - Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666) in the entire 17/18 ALL cell lines tested (Table H). In 6 cell lines, the synergy effect was marked, with synergy scores between 5 and IO. ln 5 cell lines, the synergy effect was exceptional, achieving synergy scores between IO and 15.9. Importantly, the synergy was not dépendent on single agent anti-proliferative effects, and in fact was particularly strong at concentrations of MCLl inhibitor and BCL-2 inhibitor that did not hâve an anti-proliferative effect on their own. For example, in NALM-6 cells, MCLl inhibitor and BCL-2 inhibitor at the fourth lowest concentration tested elicited a growth inhibition of 6 and 8%, respectively, while the respective combination of the two compounds afforded a growth inhibition of 61% (Figure 11, top left panel).

Furthermore, it is noteworthy that the synergistic effects occurred across a broad range of single agent concentrations, which should prove bénéficiai in vivo with respect to 25 flexibility concerning dosing levels and scheduling.

In summary, the combination of MCLl inhibitor and BCL-2 inhibitor afforded synergistic growth inhibition in the majority (17/18) of ALL cell lines tested. Importantly, exceptional synergistic growth inhibition was observed in 5/18 ALL cell lines tested.

EXAMPLE 11: 7n vitro effect on prolifération of combining a MCLl inhibitor with a BCL-2 inhibitor in a panel of 5 Mantle Cell Lymphoma (MCL) cell lines.

Materials and methods

Cell lines were sourced and maintained in the basic media supplemented with FBS as indicated in Table 12. In addition, ail media contained penicillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM).

Cell lines were cultured at 37°C in a humidified atmosphère containing 5% CO2 and expanded in T-150 flasks. In ail cases cells were thawed from frozen stocks, expanded through >1 passage using appropriate dilutions, counted and assessed for viability using a CASY cell counter prior to plating 150 ul/well at the densities indicated in Table 12 into 96-well plates. Ail cell lines were determined to be free of mycoplasma contamination inhouse.

Stock solutions of compounds were prepared at a concentration of 5 mM in DMSO and stored at -20°C. In order to analyse the activity of the compounds as single agents or in combination, cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound dispensed, either individually or in ail possible permutations in a checkerboard fashion, directly into the cell assay plates. Effects of the single agents as well as their checkerboard combinations on cell viability were assessed after 2 days of incubation at 37 °C/5% CO₂ by quantification of cellular ATP levels using CelITiterGlo at 150 pL reagent/well. Ail the conditions were tested in triplicates. Luminescence was quantified on a multipurpose plate reader.

Potential synergistic interactions between compound combinations were assessed using the Excess Inhibition 2D matrix according to the Loewe additivity model and are reported as Synergy Score (Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659—666). Ail calculations were performed using Chalice™ Bioinformatics Software available in Horizon website.

Single agent IC50S were calculated using standard four-parametric curve fitting. IC50 is defïned as the compound concentration at which the CTG signai is reduced to 50% of that measured for the vehicle (DMSO) control.

The doubling time indicated in Table 12 is the mean of the doubling time obtained in the 5 different passages (in T-150 flasks) performed from the thawing of the cells to their seeding in the 96-weel plates.

Synergy Score

SS ~ 0 —* Additive

SS >1 —* Weak Synergy

SS >2 —> Synergy

Table 12. Identity and assay conditions for the 5 Mantle Cell Lymphoma cell lines used in the combination experiments.

Cell line	Medium	%FBS	Source	Doubling time (hours)	Cell number seeded/well
Z-138	RPMI	10	ATCC	22.5	37500
Jeko	RPMI	20	ATCC	26.0	27000
Mino	RPMI	15	ATCC	31.1	56250
JVM-2	RPMI	10	ATCC	76.0	56250
REC-1	RPMI	10	ATCC	36.0	56250

Table 13. Single agent IC50 values for Compound 3 and Compound 1, HCl in the 5 Mantle

Cell Lymphoma cell lines are indicated. Compounds were incubated with the cells during 2 15 days.

Cell Line	Compound 3	Compound 1, HCl
Start conc [uM|	IC50 |uM|	Start conc [uM]	IC50 [uM]
Z-138	2	0.448	5	>5
Jeko	2	0.023	5	> 5
Mino	2	0.008	2	0.091
JVM-2	2	>2	5	>5
REC-1	2	0.077	2	0.703

Table 14. Synergy scores for Compound 3 and Compound 1, HCl combination in 5 Mantle Cell Lymphoma cell lines are indicated. Interactions were deemed synergistic when scores > 2.0 where observed. Start concentrations of compounds, max inhibition and the synergy scores are indicated. Compounds were incubated with the cells during 2 days.

Cell Line	Compound 3	Compound 1, HCl	Combination
Start cône [uM]	Max Inh [%]	Start conc [uM]	Max Inh [%]	Synergy Score
Z-138	2.0	96.0	5.0	25.0	11.1
Jeko	2.0	100.0	5.0	35.0	9.7
Mino	2.0	100.0	2.0	91.0	5.7
JVM-2	2.0	19.0	5.0	38.0	3.4
REC-1	2.0	99.0	2.0	78.0	5.1

Results

The effect on prolifération of combining the MCL1 inhibitor with the BCL-2 inhibitor was assessed in a panel of 5 Mantle Cell Lymphoma cell lines.

As single agents, MCL1 inhibitors displayed superior activity as compared with BCL-2 inhibitor. Thus, 3 cell lines displayed IC50S below 100 nM for MCL1 inhibitor while only one cell line displayed ICsos below 100 nM for BCL-2 inhibitor (Table 13).

In combination, MCL1 inhibitor and BCL-2 inhibitor treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2 - Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666) in ail cell lines tested (Table 14), as examplified in Figure 12. Importantly, in 4/5 cell lines, the synergy effect was marked, with synergy scores above 5.

EXAMPLE 12: In vitro effect on prolifération of combining a MCL1 inhibitor with a BCL-2 inhibitor in a panel of 5 Small Cell Lung Cancer (SCLC) cell lines.

Ail cell lines were obtained from ATCC. Culture media containing RPMI 1640 (Invitrogen) supplemented with 10% FBS (HyClone) was used for COR-L95, NCI-H146, NCI-H211, SHP-77, SW1271, NCI-H1339, NCI-H1963, and NCI-H889. Culture media containing Waymouth’s MB 752/1 (Invitrogen) with 10% FBS was used for DMS-273. Culture media containing DMEM/F12 (Invitrogen) containing 5% FBS, and supplemented with 0.005 mg/ml insulin, 0.01 mg/ml transferrin, and 30 nM sodium selenite solution (Invitrogen), 10 nM hydrocortisone (Sigma), IO nM beta-estradiol (Sigma), and 2 mM L-glutamine (HyClone) was used for NCI-Hl 105.

Cell lines were cultured in 37°C and 5% CO2 incubator and expanded in T-75 flasks. In ail cases cells were thawed from frozen stocks, expanded through >l passage using l:3 dilutions, counted and assessed for viability using a ViCell counter (Beckman-Coulter), prior to plating in 384-well. To split and expand cell lines, cells were dislodged from flasks using 0.25% Trypsin-EDTA (GIBCO). Ail cell lines were determined to be free of mycoplasma contamination as determined by a PCR détection methodology performed at Idexx Radil (Columbia, MO, USA) and correctly identified by détection of a panel of SNPs.

Cell prolifération was measured in 72hr CellTïter-GIo™ (CTG) assays (Promega G7571) and ail results shown are the resuit of at least triplicate measurements. For CellTiter-Glo™ assays, cells were dispensed into tissue culture treated 384-well plates (Corning 3707) with a final volume of 35 gL of medium and at density of 5000 cells per well. 24 hrs after plating, 5 gL of each compound dilution sériés were transferred to plates containing the cells, resulting in compound concentration ranges from 0-10 uM and a final DMSO (Sigma D8418) concentration of 0.16%. Plates were incubated for 72 hrs and the effects of compounds on cell prolifération was determined using the CellTiter-Glo™ Luminescent Cell Viability Assay (Promega G7571) and a Envision plate reader (Perkin Elmer).

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to détermine the number of viable cells in culture based on quantitation of the ATP présent, which signais the presence of metabolically active cells. The method is described in detail in the Technical Bulletin, TB288 Promega. Briefly, cells were plated in Opaque-walled multiwell plates in culture medium as described above. Control wells containing medium without cells were also prepared to obtain a value for background luminescence. 15 uL of CellTiter-Glo® Reagent was then added and contents mixed for 10 minutes on an orbital shaker to induce cell lysis. Next, luminescence was recorded using the plate reader.

The percent growth inhibition and excess inhibition were analysed using the Chalice software (CombinatoRx, Cambridge MA). The percentage of growth inhibition relative to

DMSO is displayed in the panel labelled inhibition, and the amount of inhibition in excess of the expected amount in the panel labelled ADD Excess Inhibition (Figures 15 (a)-(e)). Concentrations of Compound l, HCl are shown along the bottom row from left to right and increasing concentrations of Compound 3 along the leftmost column from bottom to top. Ail remaining points in the grids display results from a combination of the two inhibitors that correspond to the single agent concentrations denoted on the two axes. Data analysis of cell prolifération was performed using Chalice Analyser as described in Lehar et al, Nat Biotechnol. 2009 July ; 27(7): 659-666. Excess inhibition was calculated using the Loewe synergy model which measures the effect on growth relative to what would be expected if two drugs behave in a dose additive manner. Positive numbers represent areas of increasing synergy.

Synergy Score

SS ~ 0 —► Dose Additive

SS >2 —» Synergy

SS > l —> Weak Synergy

Results

In combination, Compound l and Compound 3 treatment caused synergistic growth inhibition (i.e. Synergy Scores above 2) in 8/10 small cell lung cancer cell lines. Importantly, in 6 cell lines, the synergy effect was marked, with synergy scores above 6.

EXAMPLE 13: In vivo efficacy in Patient-derived primary AML model HAMLX5343 with combination of a MCLl inhibitor (Compound 3) and a BCL-2 inhibitor (Compound 1, HCl or ABT-199)

Materials and Methods

Materials

Animais

NOD scid gamma (NSG) female mice weighing 17-27 grams (Jackson Laboratories) were allowed to acclimate with access to food and water ad libitum for 3 days prior to manipulation.

Primary tumor models

Patient-derived primary AML model HAMLX5343 carrying KRAS mutation and wild type FLT3 were obtained from Dana Farber Cancer Institute.

Test compounds, formulations

Compound l, HCl was formulated in 5% Ethanol, 20% Dexolve-7 as a solution for intravenous administration or formulated in PEG300/EtOH/water (40/10/50) for oral administration. ABT-199 was formulated in PEG300/EtOH/water (40/10/50) for oral administration. Ail of them are stable for at least one week at 4°C. Compound 3 was formulated in Liposomal formulation as a solution for intravenous formulation, which is stable for three weeks at 4°C. Vehicle and compound dosing solutions were prepared as needed. Ail animais were dosed at I0 mL/kg with Compound l (expressed as the free base) or ABT-199, or 5 mL/kg with Compound 3.

Methods

Study design

Eight treatment groups were used in study 7844HAMLX5343-XEF as summarized in Table 15. Ail treatments were initiated when the average tumor burden (%CD-45 positive cells) was between 8% and 15%.

In this study, Compound l was administered by oral gavage (po) or intravenous administration at 50 mg/kg once a week, ABT-199 was administered at 25mg/kg by oral gavage (po) once a week, either as a single agent or in combination with Compound 3 at 12.5mg/kg once a week, respectively, for 18 days.

Both Compound 1 (expressed as the free base) and ABT-199 were administered at 10 mL/kg. Compound 3 was administered at 5 mL/kg. The dose was body weight adjusted. Bodyweights were recorded twice/week and tumor burden was recorded once/week.

Table 15. Doses* and dose schedules for 7844HAMLX5343-XEF

Treatment groups	Number of animais	Dosing regimen
Vehicle (10 mL/kg)	4	QW
Compound 1 (50mg/kg po)	4	QW
Compound 1 (50mg/kg iv)	4	QW

Treatment groups	Number of animais	Dosing regimen
ABT-199 (25mg/kg po)	4	QW
Compound 3 (12.5mg/kg iv)	4	QW
Compound 1 + Compound 3 (po/iv)	4	QW + QW
Compound 1 + Compound 3 (iv/iv)	4	QW + QW
ABT-199 + Compound 3 (po/iv)	4	QW + QW

* Doses are expressed as the free base

Primary AML model

For this experiment, 32 mice were implanted with primary AML line HAMLX5343. Mice were injected intravenously with 2.0 million leukemia cells. When the tumor burden was between 8%-l5%, animais were randomized into eight groups of four mice each for vehicle, Compound l (po), Compound l (iv), ABT-199, Compound 3, or combination treatment. After 18 days of treatment, the study was terminated when the tumor burden reached 99%. Tumor burden was measured by FACS analysis.

Animal monitoring

Animal well-being and behavior, including grooming and ambulation were monitored twice daily. General health of mice was monitored and mortality recorded daily. Any moribund animais were sacrificed.

Tumor measurement

Mice were bled via tail snip once per week. Blood was split into an IgG control well and a CD33/CD45 well of a 96-well plate. Blood was lysed with 200μ! RBC lysis buffer twice at RT, then washed once with FACS buffer (5% FBS in PBS). Samples were then incubated for 10-30 minutes at 4C in ΙΟΟμΙ blocking buffer (5% mouse Fc Block + 5% human Fc Block + 90% FACS buffer). 20μ! IgG control mix (2.5μ1 Mouse igGl K isotype control-PE + 2.5μΙ Mouse igGl K isotype control-APC + 15μ1 FACS buffer) were added to the IgG control wells and 20ul CD33/CD45 mix (2.5μ1 Mouse anti-human CD33-PE + 2.5μ1 Mouse anti-human CD45-APC + 15μ! FACS buffer). Samples were incubated for 30-60 minutes at 4C then washed twice prior to analysis. Samples were run on Canto with

FACSDiva software. Analysis was performed with FloJo software. The percent of CD45positive, live, single cells was reported as the tumor burden.

Data analysis

Percent treatment/control (T/C) values were calculated using the following formula:

%T/C = 100 X AT/AC if AT >0 %Regression = 100 x AT/Tinitiai if AT <0 where:

T = mean tumor burden of the drug-treated group on the final day of the study;

AT = mean tumor burden of the drug-treated group on the final day of the study - mean tumor burden of the drug-treated group on initial day of dosing;

Tinitiai = mean tumor burden of the drug-treated group on initial day of dosing;

C = mean tumor burden of the control group on the final day of the study; and

AC = mean tumor burden of the control group on the final day of the study — mean tumor burden of the control group on initial day of dosing.

Ail data were expressed as Mean ± SEM. Delta tumor burden and body weight were used for statistical analysis. Between-groups comparisons for final measurements were performed using ANOVA with Tukey’s test. Statistical analysis was carried out using GraphPad Prism.

Statistical analysis

Ail data were expressed as mean ± standard error of the mean (SEM). Delta tumor volume and body weight were used for statistical analysis. Between-group comparisons were carried out using the Kruskal-Wallis ANOVA followed by a post hoc Dunn’s test or Tukey’s test. For ail statistical évaluations, the level of significance was set at p < 0.05. Significance compared to the vehicle control group is reported unless otherwise stated. The standard protocols used in pharmacology studies are not pre-powered to demonstrate statistically significant superiority of a combination over the respective single agent treatment. The statistical power is often limited by potent single agent response and/or model variability. The p-values for combination vs single agent treatments are, however, provided.

Results

Synergistic anti-tumor effect of combined MCLl and BCL-2 inhibition

In the 7844HAMLX5343-XEF study, Compound l, ABT-199 or Compound 3 alone did not show anti-tumor activity in the HAMLX5343 model carrying the KRAS mutation, when administered at 50mg/kg (oral or iv), 25mg/kg (oral) or 12.5mg/kg (zv) once a week, respectively (%T/C of 98, 92, 98 or 99%, respectively, p>0.05).

When orally administered, Compound 1 at 50mg/kg or ABT-199 at 25mg/kg in combination with Compound 3 (12.5mg/kg iv) once a week resulted in tumor stasis (%T/C of 3% or 6%, respectively, p<0.05) in this model.

On the other hand, the combination of intravenously administered Compound 1 with Compound 3 induced near complété tumor régression (%Regression of 100%), which is 15 significantly different from either single agent (p<0.05) or Compound 1/Compound 3 po/zv combination. The mean tumor burden for each treatment group is plotted against time for the 18 day treatment period, as shown in Figure 1. The change in tumor burden, %T/C or %Regression is presented in Table 16 and in Figures 16 (a)-(b).

Table 16. Summary of anti-tumor effect in 7844HAMLX5343-XEF study

Treatment	T/C %	Régression %
Vehicle	100
Compound 1 SOmpk po	98
Compound 1 50mpk iv	92
ABT-199 25mpkpo	98
Compound 3 12.5mpk iv	99
Compound 1 + Compound 3 (po/iv)	3*
Compound 1 + Compound 3 (iv/iv)		100**

ABT-199 + Compound 3 (po/iv)

6*

* p< 0.05 versus Vehicle and single agents (ANOVA, Tukey’s test) ** p < 0.05 versuspo/iv combination (ANOVA, Tukey’s test)

Conclusion

AML is an aggressive and heterogeneous hématologie malignancy, caused by the transformation of hematopoietic progenitor cells due to acquisition of genetic alterations (Patel et al, New England Journal of Medicine 2012 366:1079-1089). The 5-year survival rate of AML has been low due to lack of effective thérapies. Evasion of apoptosis is a hallmark of cancer (Hanahan et al Cell 2000 100:57-70). One of the primary means by which cancer cells évadé apoptosis is by up-regulating the pro-survival BCL-2 family proteins such as BCL-2, BCL-xL and MCL1.

MCL1 gene is of the most commonly amplified gene in cancer patients (Beroukhim et al, Nature 2010 463:899-905). Moreover, both BCL-2 and MCL1 are highly expressed in AML. Therefore, the combination of Compound 1 (BCL-2Î) and Compound 3 (MCL1) may provide synergy by enhancing pro-apoptotic signais as a general mechanism against AML.

We show here that BCL-2 inhibitor Compound 1 or ABT-199 in combination with Compound 3 (MCL1 inhibitor) has a dramatic synergistic effect in treating AML in an AML xenograft model with KRAS mutation (wt FLT3). The iv/iv Compound 1/Compound 3 combination is superior to the po/iv combination treatment at the same dose level. The results indicate that the combination of and MCL1 inhibitors would be an effective therapy for AML.

Claims (44)

1. A combination comprising:

(a) a BCL-2 inhibitor of formula (I):

r₃

wherein:

5 ♦ X and Y represent a carbon atom or a nitrogen atom, it being understood that they may not simultaneously represent two carbons atoms or two nitrogen atoms, ♦ Ai and A2, together with the atoms carrying them, form an optionally substituted, aromatic or non-aromatic heterocycle Het composed of 5, 6 or 7 ring members which may contain, in addition to the nitrogen represented by X or by Y, from one to 3 hetero atoms selected

10 independently from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, a linear or branched (Ci-Côjalkyl group or a group -C(O)-O-Alk wherein Alk is a linear or branched (CiCô)alkyl group, or Ai and A2 independently of one another represent a hydrogen atom, a linear or branched 15 (Ci-Cô)polyhaloalkyl, a linear or branched (Ci-Côjalkyl group or a cycloalkyl, ♦ T represents a hydrogen atom, a linear or branched (Ci-Cô)alkyl group optionally substituted by from one to three halogen atoms, a group (Ci-CQalkyl-NRiïG, or a group (Ci-C₄)alkyl-OR6, ♦ Ri and R₂ independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, or Ri and R₂ form with the nitrogen atom carrying them a heterocycloalkyl, ♦ R₃ represents a linear or branched (Ci-Ce)alkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, acycloalkyl group, a (C₃-Cio)cycloalkyl(Ci-Cô)alkyl group wherein the alkyl moiety is linear or branched, a heterocycloalkyl group, an aryl group or a heteroaryl group, it being understood that one or more of the carbon atoms of the preceding groups, or of their possible substituents, may be deuterated, ♦ R₄ represents an aryl group, a heteroaryl group, a cycloalkyl group or a linear or branched (Ci-Cô)alkyl group, it being understood that one or more of the carbon atoms of the preceding groups, or of their possible substituents, may be deuterated, ♦ Rs represents a hydrogen or halogen atom, a linear or branched (Ci-Cô)alkyl group, or a linear or branched (Ci-Côjalkoxy group, ♦ Rô represents a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, ♦ R_a, Rb, Rc and Ra, each independently of the others, represent R7, a halogen atom, a linear or branched (Ci-C6)alkoxy group, a hydroxy group, a linear or branched (Ci-C6)polyhaloalkyl group, a trifluoromethoxy group, -NR7R7', nitro, R₇-CO-(Co-C₆)alkyl-, R₇-CO-NH-(C₀-C₆)alkyl-, NR₇R7’-CO-(Co-C6)alkyl-, NR₇R7'-CO-(Co-C₆)alkyl-0-, R₇-S0₂-NH-(Co-C₆)alkyl-,

R₇-NH-CO-NH-(Co-C₆)alkyl-, R₇-0-CO-NH-(Co-C₆)alkyl-, a heterocycloalkyl group, or the substituents of one of the pairs (R_a,Rb), (Rb,Rc) or (Rc,Ra) form together with the carbon atoms carrying them a ring composed of from 5 to 7 ring members, which may contain from one to 2 hetero atoms selected from oxygen and sulphur, it also being understood that one or more carbon atoms of the ring defined hereinbefore may be deuterated or substituted by from one to 3 groups selected from halogen and linear or branched (Ci-Cô)alkyl, ♦ R7 and R7' independently of one another represent a hydrogen, a linear or branched (CiCô)alkyl, a linear or branched (C₂-Cô)alkenyl, a linear or branched (C₂-C6)alkynyl, an aryl or a heteroaryl, or R7 and R7' together with nitrogen atom carrying them form a heterocycle composed of from 5 to 7 ring members, it being understood that when the compound of formula (I) contains a hydroxy group, the latter may be optionally converted into one of the following groups: -0P0(0M)(0M’), -0P0(0M)(0‘

ΜΓ), -OPO(O-Mi⁺)(OM2⁺), -OPO(O)(O)M3²⁺,

-OPO(OM)(O[CH₂CH₂O]_nCH₃), or -OPO(OW)(O[CH₂CH₂O]_nCH₃), wherein M and M' independently of one another represent a hydrogen atom, a linear or branched (Ci-Côjalkyl group,

5 a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a cycloalkyl or a heterocycloalkyl, both composed of from 5 to 6 ring members, while Mi⁺ and M2⁺ independently of one another represent a pharmaceutically acceptable monovalent cation, M3²⁺ represents a pharmaceutically acceptable divalent cation, and n is an integer from 1 to 5, it being understood that:

10 - aryl means a phenyl, naphthyl, biphenyl or indenyl group, heteroaryl means any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quatemary nitrogens), cycloalkyl means any mono- or bi-cyclic, non-aromatic, carbocyclic group containing 15 from 3 to 10 ring members, heterocycloalkyl means any mono- or bi-cyclic, non-aromatic, condensed or spiro group composed of 3 to 10 ring members and containing from 1 to 3 hetero atoms selected from oxygen, sulphur, SO, SO₂ and nitrogen, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined and 20 the groups alkyl, alkenyl, alkynyl and alkoxy to be substituted by from 1 to 3 groups selected from:

linear or branched (Ci-Côjalkyl optionally substituted by a hydroxyl, a morpholine, 3-3difluoropiperidine or a 3-3-difluoropyrrolidine; (C₃-C₆)spiro; linear or branched (Ci-C₆)alkoxy optionally substituted by a morpholine; (Ci-Côjalkyl-S-; hydroxyl; oxo; A-oxide; nitro; cyano; COOR'; -OCOR'; NR'R; linear or branched (Ci-C₆)polyhaloalkyl; trifluoromethoxy; (Cj25 C₆)alkylsulphonyl; halogen; aryl optionally substituted by one or more halogens; heteroaryl;

aryloxy; arylthio; cycloalkyl; heterocycloalkyl optionally substituted by one or more halogen atoms or alkyl groups, wherein R' and R independently of one another represent a hydrogen atom or a linear or branched (Ci-Cô)alkyl group optionally substituted by a methoxy, it being possible for the Het group defined in formula (I) to be substituted by from one to three groups selected from linear or branched (Ci-Cô)alkyl, hydroxy, linear or branched (Ci-Côjalkoxy, NRi'Ri and halogen, it being understood that Rf and Ri are as defined for the groups R' and R mentioned hereinbefore, or its enantiomers, diastereoisomers, or addition salts thereof with a pharmaceutically acceptable acid or base, and (b) a MCLl inhibitor.

2. A combination comprising:

(a) a BCL-2 inhibitor and (b) a MCLl inhibitor of formula (II):

(Π) wherein:

♦ A represents a linear or branched (Ci-C6)alkyl group, a linear or branched (C₂-C₆)alkenyl group, a linear or branched (C₂-C₆)alkynyl group, a linear or branched (CiCô)alkoxy group, -S-(Ci-C6)alkyl group, a linear or branched (Ci-C6)polyhaloalkyl, a hydroxy group, a cyano, -NWioWio’, -Cy6 or an halogen atom, ♦ Wi, W₂, W3, W4 and W5 independently of one another represent a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (CiCô)polyhaloalkyl, a hydroxy group, a linear or branched

-S-(Ci-Cô)alkyl group, a cyano, a nitro group,

-O-Cyi, -alkyl(Co-C6)-Cyi, -alkenyl(C2-C6)-Cyi,

-O-alkyl(Ci-C₆)-W₉, -C(0)-0W₈, -0-C(0)-W₈,

-NW₈-C(O)-W₈’, -NW₈-C(O)-OW₈’, (Ci-Cô)alkoxy group, -alkyl(C₀-C₆)-NW₈W₈’, -alkynyl(C₂-C₆)-Cyi, -C(O)-NW₈W₈’,

-alkyl(Ci-C₆)-NW₈-C(O)-W₈’, -SO₂- NW₈W₈’, -SO₂-alkyl(Ci-C₆), or the substituents of one of the pairs (Wi, W₂), (W₂, W₃), (Wi, W₃), (W₄, W5) when grafted onto two adjacent carbon atoms, form together with the carbon atoms carrying them an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that resuiting ring may be substituted by a group selected from a linear or branched (CiCôjalkyl group, -N W10W10’,

-alkyl(Co-Cô)-Cyi or an oxo, ♦ X’ represents a carbon or a nitrogen atom, ♦ Wô represents a hydrogen, a linear or branched (Ci-C₈)alkyl group, an aryl, an heteroaryl group, an arylalkyl(Ci-C6) group, an heteroarylalkyl(Ci-C6) group, ♦ W7 represents a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C₆)alkenyl group, a linear or branched (C₂-C₆)alkynyl group, -Cys, -alkyl(Ci-C₆)-Cys, -alkenyl(C₂-C₆)-Cys, -alkynyl(C₂-C₆)-Cy3, -Cy₃-Cy₄, -alkynyl(C₂-C₆)-O-Cy₃, -Cy₃-alkyl(Co-C₆)-0-alkyl(Co-C6)-Cy4, an halogen atom, a cyano, -C(0)-Wn, -C(0)-NWnWn’, ♦ W₈ and W₈’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Côjalkyl group, or -alkyl(Co-C6)-Cyi, or (W₈, W₈’) form together with the nitrogen atom carrying them an aromatic or nonaromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from one to 3 heteroatoms selected from oxygen, sulphur and nitrogen, it being understood that the nitrogen in question may be substituted by a group representing a hydrogen atom, or a linear or branched (Ci-Côjalkyl group and it being understood that one or more of the carbon atoms of the possible substituents, may be deuterated, ♦ W9 represents -Cyi, -Cyi-alkyl(Co-C6)-Cy₂, -Cyi-alkyl(Co-C6)-0-alkyl(Co-C6)-Cy₂,

-Cyi-alkyl(Co-C₆)-NW₈-alkyl(Co-C₆)-Cy₂, -Cyi-Cy₂-0-alkyl(Co-C₆)-Cy₅,

-C(O)-NW₈W₈’, -NW₈W₈’, -OW₈,-NW₈-C(O)-W₈’, -O-alkyl(Ci-C₆)-OW₈,

-SÛ2-W₈, -C(O)-OW₈, -NH-C(O)-NH-W₈,

being possible for the ammonium so defïned to exist as a zwitterionic form or to hâve a monovalent anionic counterion, ♦ Wio, Wio’, Wn and Wn’ independently of one another represent a hydrogen atom or an optionally substituted linear or branched (Ci-C6)alkyl group, ♦ W12 represents a hydrogen or a hydroxy group, ♦ W13 represents a hydrogen atom or a linear or branched (Ci-Côjalkyl group, ♦ Wu represents a -O-P(O)(O)(O) group, a -O-P(O)(O')(OWi6) group, a -O-P(O)(OWi₆)(OWi6’) group, a -O-SCL-O’ group, a -O-SO2-OW16 group, -Cy₇, a -O-C(O)-Wi5 group, a -O-C(O)-OWi5 group or a -O-QOj-NWisWis’ group, ♦ W15 and W15’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a linear or branched amino(Ci-C6)alkyl group, ♦ Wi6 and Wiô’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or an arylalkyl(Ci-Cô) group, ♦ Cyi, Cy₂, Cy₃, Cy₄, Cy₅, Cy₆ and Cy₇ independently of one another, represent a cycloalkyl group, a heterocycloalkyl group, an aryl or an heteroaryl group, ♦ n is an integer equal to 0 or 1, it being understood that:

- aryl means a phenyl, naphthyl, biphenyl, indanyl or indenyl group,

- heteroaryl means any mono- or bi-cyclic group composed of from 5 to 10 ring members, having at least one aromatic moiety and containing from 1 to 3 heteroatoms selected from oxygen, sulphur and nitrogen,

- cycloalkyl means any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, “heterocycloalkyl” means any mono- or bi-cyclic non-aromatic carbocyclic group * containing from 3 to 10 ring members, and containing from 1 to 3 heteroatoms selected from oxygen, sulphur and nitrogen, which may include fused, bridged or spiro ring

Systems, it being possible for the aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined 5 and the alkyl, alkenyl, alkynyl, alkoxy, to be substituted by from 1 to 4 groups selected from linear or branched (Ci-Cô)alkyl which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy which may be substituted by a linear or branched (Ci-Cô)alkoxy, a linear or branched (Ci-Cô)polyhaloalkyl, hydroxy, halogen, oxo, -NW’W”, -O-C(O)-W’, or CO-NW’W”; linear or branched

10 (C2-C6)alkenyl group; linear or branched (Cz-Côjalkynyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; linear or branched (Ci-Cô)alkoxy which may be substituted by a group representing a linear or branched (Ci-Ce)alkoxy, a linear or branched (Ci-Cejpolyhaloalkyl, a linear or branched (C2-C6)alkynyl, -NW’W”, or hydroxy; (Cj-Côjalkyl-S- which may be substituted by a group representing a linear or branched (Ci15 Cé)alkoxy; hydroxy; oxo; A-oxide; nitro; cyano; -C(O)-OW’; -O-C(O)-W’; -CO-NW’W”; NW’W”; -(C=NW’)-0W”; linear or branched (Ci-C6)polyhaloalkyl; trifluoromethoxy; or halogen; it being understood that W’ and W” independently of one another represent a hydrogen atom or a linear or branched (Ci-Ce) alkyl group which may be substituted by a group representing a linear or branched (Ci-Cô)alkoxy; and it being understood that one or 20 more ofthe carbon atoms of the preceding possible substituents, may be deuterated, their enantiomers, diastereoisomers and atropisomers, and addition salts thereof with a pharmaceutically acceptable acid or base.

3. A combination according to claim 1, wherein the MCLl inhibitor is a compound of formula (II) as defined in claim 2.

25

4. A combination according to any of claims 1 to 3, wherein the BCL-2 inhibitor is A-(4hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinyhnethyl)-3,4-dihydro-2(lJ7)-isoquinolinyl) carbonyl]-!,3-benzodioxol-5-yl}-A-phenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide.

5. A combination according to any of claims l to 3, wherein the BCL-2 inhibitor is 5-(5-chloro-

2-{[(35)-3-(morpholin-4-ylmethyl)-3,4-dihydroisoqumohn-2(l/7)-yl]carbonyl} phenyl)-A-(5cyano-1,2-dimethyl- l/f-pyrrol-3 -yl)-7V-(4-hydroxyphenyl)-1,2-dimethyl-1 Æ-pyrrole-3 carboxamide.

6. A combination according to claim 4, wherein 7V-(4-hydroxyphenyl)-3-{6-[((35)-3-(4morpholinylmethyl)-3,4-dihydro-2(lZ/)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}-jVphenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide is in the form of the hydrochloride sait.

7. A combination according to claim 5, wherein 5-(5-chloro-2-{[(35)-3-(morpholin-4-ylmethyl)3,4-dihydroisoquinolin-2(l//)-yl] carbonyl} phenyl)-2V-(5-cyano-l,2-dimeÎhyl-l//-pyrrol-3-yl)-7V(4-hydroxyphenyl)-l,2-dimethyl-17/-pyrrole-3-carboxamide is in the form of the hydrochloride sait.

8. A combination according to any of claims 1 to 3, wherein the BCL-2 inhibitor is ABT-199.

9. A combination according to any of claims 1 to 8, wherein the MCLl inhibitor is (27?)-2-{[(5S_a)5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(5-fluorofuran-2yl)thieno[2,3-J]pyrimidin-4-yl]oxy}-3-(2-{[ 1 -(2,2,2-trifluoroethyl)-l/Z-pyrazol-5- y 1] methoxy } phenyl)propanoic acid.

10. A combination according to any of claims 1 to 8, wherein the MCLl inhibitor is (22?)-2{[(55'_fl)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-i/]pyrimidin-4-yl]oxy} -3-(2- {[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy} phenyl)propanoic acid.

11. A combination according to any of claims 1 to 10, for use in the treatment of cancer.

12. The combination for use according to claim 11, wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in amounts which are jointly therapeutically effective for the treatment of cancer.

13. The combination for use according to claim 11, wherein the BCL-2 inhibitor and the MCL1 inhibitor are provided in amounts which are synergistically effective for the treatment of cancer.

14. The combination for use according to claim 11, wherein the BCL-2 inhibitor and the MCL 1 inhibitor are provided in synergistically effective amounts which enable a réduction of the dose required for each compound in the treatment of cancer, whilst providing an efficacious cancer treatment, with eventually a réduction in side effects.

15. The combination for use according to claim 11, wherein:

(i) the BLC-2 inhibitor is /V-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinylmethyl)-3,4dihydro-2(177)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl} -7V-phenyl-5,6,7,8tetrahydro-1 -indolizine carboxamide, (ii) the dose of N-(4-hydroxyphenyl)-3-{6-[((3S)-3-(4-morpholinymethyl)-3,4-dihydro2(lH)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}-N-phenyl-5,6,7,8-tetrahydro-lindolizine carboxamide during the combination treatment is from 50 mg to 1500 mg.

16. The combination for use according to claim 11, wherein the BCL-2 inhibitor is for administration once a week.

17. The combination for use according to claim 15, whereinN-(4-hydroxyphenyl)-3-{6-[((3S)-

3-(4-morpholinylmethyl)-3,4-dihydro-2(lH)-isoquinolinyl)carbonyl]-l,3-benzodioxol-5-yl}-Nphenyl-5,6,7,8-tetrahydro-l-indolizine carboxamide is for administration during the combination treatment once a day.

18. The combination for use according to any of daims 11 to 17, wherein the BCL-2 inhibitor and the MCLl inhibitor are for administration orally.

19. The combination for use according to any of daims 11 to 17, wherein the BCL-2 inhibitor is for administration orally and the MCLl inhibitor is for administration intravenously.

20. The combination for use according to any of daims 11 to 17, wherein the BCL-2 inhibitor and the MCLl inhibitor are for administration intravenously.

21. The combination for use according to any of daims 11 to 20, wherein the cancer is leukaemia.

22. The combination for use according to claim 21, wherein the leukaemia is acute myeloid leukaemia, T-ALL or B-ALL.

23. The combination for use according to any of claims 11 to 20, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease.

24. The combination for use according to any of claims 11 to 20, wherein the cancer is lymphoma.

25. The combination for use according to claim 24, wherein the lymphoma is a non-Hodgkin lymphoma.

26. The combination for use according to claim 25, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma or mantle-cell lymphoma.

27. The combination for use according to any of claims 11 to 20, wherein the cancer is multiple myeloma.

28. The combination for use according to any of claims 11 to 20, wherein the cancer is neuroblastoma.

29. The combination for use according to any of claims 11 to 20, wherein the cancer is small cell lung cancer.

30. A combination according to any of claims 1 to 10, further comprising one or more excipients.

31. The use of a combination according to any of claims 1 to 10, in the manufacture of a médicament for the treatment of cancer.

32. The use according to claim 31, wherein the cancer is leukaemia.

33. The use according to claim 32, wherein the leukaemia is acute myeloid leukaemia, T-ALL or B-ALL.

34. The use according to claim 31, wherein the cancer is myelodysplastic syndrome or myeloproliferative disease.

35. The use according to claim 31, wherein the cancer is lymphoma.

36. The use according to claim 35, wherein the lymphoma is a non-Hodgkin lymphoma.

5

37. The use according to claim 36, wherein the non-Hodgkin lymphoma is diffuse large B-cell lymphoma or mantle-cell lymphoma.

38. The use according to claim 31, wherein the cancer is multiple myeloma.

39. The use according to claim 31, wherein the cancer is neuroblastoma.

40. The use according to claim 31, wherein the cancer is small cell lung cancer.

10

41. A médicament containing, separately or together, (a) a BCL-2 inhibitor of formula (I) as defined in claim 1, and (b) a MCLl inhibitor, for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in effective amounts for the treatment of cancer.

15

42. A médicament containing, separately or together, (a) a BCL-2 inhibitor, and (b) a MCLl inhibitor of formula (II) as defined in claim 2, for simultaneous, sequential or separate administration, and wherein the BCL-2 inhibitor and the MCLl inhibitor are provided in effective amounts for the treatment of cancer.

20

43. A combination according to any of claims 1 to 10 for use in a method for sensitizing a patient who is (i) refractory to at least one chemotherapy treatment, or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii).

44. Use of a combination according to any of claims 1 to 10 in the manufacture of a médicament for sensitizing a patient who is (i) refractory to at least one chemotherapy treatment, 25 or (ii) in relapse after treatment with chemotherapy, or both (i) and (ii).