EA044971B1 - COMBINATION OF MCL-1 INHIBITOR AND MIDOSTAURIN, ITS APPLICATIONS AND PHARMACEUTICAL COMPOSITIONS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a combination of Midostaurin, a multi-target tyrosine kinase inhibitor as defined below, and an Mcl-1 inhibitor. The invention also relates to said combination for use in the treatment of malignancy, in particular acute myeloid leukemia, and a pharmaceutical composition suitable for administering such a combination.

Prerequisites for creating the invention

Midostaurine is also called 4'-N-benzoylstaurosporine or PKC412. The chemical structure of Midostaurin is presented below:

N

Midostaurin, also referred to herein as Compound A, its synthesis, its uses for the treatment of cancer and its pharmaceutical compositions are described in US Patent No. 5,093,330, the contents of which are incorporated by reference. Midostaurin is specifically described in Example 18 of US Pat. No. 5,093,330. As a single agent for the treatment of solid tumors in phase 1 clinical trials, Midostaurin demonstrated low toxicity but limited efficacy (Propper et al., Journal of Clinical Oncology 2001, 19, 14851492). When added to the standard treatment regimen of cytarabine and daunorubicin induction and cytarabine consolidation, Midostaurin demonstrated statistically significant efficacy and improved overall survival in patients with FLT3 mutation-positive AML (Stone et al., New England Journal of Medicine 2017, 377, 454-464).

Midostaurin received US Food and Drug Administration (US FDA) approval in April 2017 as the active component of RYDAPT™ for the treatment of patients with newly diagnosed acute myeloid leukemia (AML) who have a FLT3 anchoring mutation, in combination with cytarabine induction and daunorubicin and consolidation of cytarabine.

Cytarabine and daunorubicin induction followed by cytarabine consolidation is the standard treatment regimen for AML. Cytarabine and daunorubicin are chemotherapy drugs that work by killing proliferating cells. Cytarabine is a DNA polymerase inhibitor and daunorubicin is an anthracycline that blocks replication. Administration of cytarabine or daunorubicin may cause nausea and vomiting and more serious side effects. Both drugs must be administered by injection and must be administered in specialized facilities so that patients can be closely monitored after administration.

The Mcl-1 inhibitor of the present invention is a compound of formula (I) as described herein. Said compounds of formula (I), their synthesis, their uses for the treatment of malignancy and their pharmaceutical compositions are described in WO 2015/097123, WO 2016/207216, WO 2016/207217, WO 2016/207225, WO 2016/207226, and WO 2017/125224, the contents of which are incorporated herein by reference.

Specifically, the Mcl-1 inhibitor is Compound B: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl4-[2-(4-methylpiperazin-1-yl)ethoxy] phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl) propanoic acid, or a pharmaceutically acceptable salt thereof, which is described in example 30 of WO 2015/097123, the contents of which are incorporated by reference.

In another embodiment, the Mcl-1 inhibitor is Compound C (S63845):

(2R)-2-{[(5 S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran -2yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl )propanoic acid, or a pharmaceutically acceptable salt thereof, as described in Example 185 of WO 2015/097123, the contents of which are incorporated by reference.

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 evasion of apoptosis is a hallmark of malignancy and that the effectiveness of many chemotherapeutic agents depends on activation of the intrinsic mitochondrial pathway. Three different subgroups of Bcl-2 family proteins control the intrinsic apoptotic pathway: (I) proapoptotic BH3 (Bcl-2 homology 3)-only proteins; (ii) pro-survival members such as Bcl-2 itself, Bcl-x1, Bcl-w, Mcl-1 and Bcl-2a1; and (III) the proapoptotic effector proteins BAX and BAK (Czabotar et al., Nature Reviews Molecular Cell Biology 2014, 15, 49-63). Overexpression of anti-apoptotic members of the Bcl-2 family is observed in various malignancies, particularly in hematologic malignancies such as mantle cell lymphoma (MCL), follicular lymphoma/diffuse large B-cell lymphoma (FL/DLBCL), and multiple myeloma (Adams and Cory, Oncogene 2007, 26, 13241337). Pharmacological inhibition of the anti-apoptotic proteins Bcl-2, Bcl-xl, Bcl-w and Mcl-1 using recently developed BH3-mimetic drugs such as ABT-199 (venetoclax), ABT-263 (navitoclax) and S63845 is a therapeutic strategy to induce apoptosis and causes tumor regression in malignancy (Zhang et al., Drug Resist. Update. 2007, 10, 207-217; Kotschy et al., Nature 2016, 538, 477-482). However, mechanisms of resistance to BH3 mimetics have been observed (Choudhary et al., Cell Death and Disease 2015, 6, e1593) and the use of combination therapies may improve efficacy and slow or even reverse the development of resistance.

Acute myeloid leukemia (AML) is a rapidly developing, fatal malignant blood disease that arises from clonal transformation of hematopoietic stem cells, resulting in paralysis of normal bone marrow function and death due to complications from absolute pancytopenia. AML is responsible for 25% of all leukemias in adults, and the highest incidence rates are observed in the United States of America, Australia and Europe (WHO. GLOBOCAN 2012. Estimated cancer incidence, mortality and prevalence worldwide in 2012. International Agency for Research on Cancer). Worldwide, approximately 88 thousand new cases are diagnosed annually. AML continues to maintain the lowest survival rate of all leukemias, with an estimated 5-year survival of only 24%. Although standard therapy for AML (cytarabine in combination with an anthracycline such as daunorubicin) was developed more than 4 decades ago, the introduction of successful targeted therapies for this disease remains an elusive goal. The concept of targeted therapy in AML is complicated by the fact that the disease develops as a multiclonal hierarchy, with the rapid growth of leukemic subclones as the main cause of drug resistance and disease relapse (Ding et al., Nature 2012, 481, 506-510).

Activating FLT3 mutations (mainly FLT3-ITD) are present in approximately 30% of AML patients at diagnosis, resulting in subsequent activation of the downstream signaling cascade, including the FLT3/JAK/STAT, MAPK, and PI3K signaling pathways.

Activation of FLT3 signaling suppresses apoptotic signals by inducing phosphorylation of the pro-apoptotic protein BAD and expression of anti-apoptotic Bcl2 family members. Inhibition of FLT3-ITD kinase activity induces apoptosis through down-regulation of BAD phosphorylation and Bcl-xL expression in FLT3-ITD cells (Minami et al., Blood 2003, 102, 2969-2975).

There remains a need for new treatments and therapies for the treatment of malignancy. Surprisingly, it has been found that Midostaurin, in combination with the Mcl-1 inhibitor of formula (I) as described herein, interacts in a synergistic manner to potently inhibit cell proliferation, particularly in AML, and more preferably in FLT3 mutant AML.

The essence of the invention

According to a first aspect of the invention, there is provided a novel combination of (a) midostaurin or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and (b) an Mcl-1 inhibitor of formula (I):

- 2 044971 where:

D represents a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

E represents a furyl, thienyl or pyrrolyl ring,

X ₁ , X ₃ , X ₄ and X ₅ independently of each other represent a carbon atom or a nitrogen atom,

X2 represents a C-R26 group or nitrogen atom,

denotes that the ring is aromatic,

Y represents a nitrogen atom or CR ₃ group,

Z represents a nitrogen atom or a C-R4 group,

R1 represents a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C2-C ₆ )alkenyl group, a linear or branched (C2-C ₆ )alkynyl group, a linear or branched (C ₁ - C ₆ )polyhaloalkyl group, hydroxy group, hydroxy(C ₁ -C ₆ )alkyl group, linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, cyano group, nitro group, -Cy ₈ , -αalkyl(Co-C ₆ )-NR ₁₁ R ₁₁ ', -O-alkyl(C ₁ -C ₆ )-NR ₁₁ R ₁₁ ',

-O-alkyl(C1-C6)-R12, -C(O)-ORn, -OC(O)-R _n , -C(O)-NR _n R _n ', -NR _n -C(O)- _Rn ',

-NR ₁₁ -C(O)-OR ₁₁ ', -αalkyl(C ₁ -C ₆ )-NR ₁₁ -C(O)-R ₁₁ ', -SO2-NR ₁₁ R ₁₁ ', or -SO ₂ -alkyl (C ₁ -C ₆ ),

R2, _R3 , R4 and _R5 independently of each other represent a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ ) alkyl group, a linear or branched (C ₂ -C ₆ ) alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, linear or branched (C ₁ -C ₆ )polyhaloalkyl, hydroxy group, hydroxy(C ₁ -C ₆ )alkyl group, linear or branched (C ₁ -C ₆ )alkoxy group, - S-(C ₁ -C ₆ )alkyl group, cyano group, nitro group, -alkyl(C ₀ -C ₆ )NR _n Rn', -O-Cy ₁ , -alkyl(C ₀ -C ₆ )-Cy ₁ , -alkenyl(C ₂ -C ₆ )-Cy ₁ , -alkynyl(C ₂ -C ₆ )-Cy ₁ , -O-alkyl(C ₁ -C ₆ )-NR ₁₁ R ₁₁ ', -O-alkyl( C1-C6)-R12, -C(O)-OR _n , -OC(O)-R _n , -C(O)-NR _n R _n ', -NR _n -C(O)-R _n ', -NR _n -C(O)-OR _n ', -alkyl(C ₁ -C ₆ )-NR ₁₁ -C(O)-R ₁₁ ', -SO ₂ -NR ₁₁ R ₁₁ ', or -SO ₂ - alkyl(C ₁ -C ₆ ), or substituent pairs (R ₁ , R2), (R2, R ₃ ), (R ₃ , R ₄ ), (R ₄ , R ₅ ) form, together with the carbon atoms to which they are attached, an aromatic or non-aromatic ring consisting of 5-7 ring members, which may contain from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is understood that the resulting ring can be substituted by 1-2 groups selected from halogen, linear or branched (C ₁ -C ₆ )alkyl, -alkyl(C ₀ -C ₆ )-NR ₁₁ R ₁₁ ', -NR ₁₃ R ₁₃ ', -alkyl(C ₀ -C ₆ )-Cy ₁ or oxo,

R6 and R7 independently represent a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, linear or branched (C ₁ -C ₆ )polyhaloalkyl, hydroxy group, linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, cyano group, nitro group , -alkyl(C ₀ -C ₆ )-NR ₁₁ R ₁₁ ',

-O-alkyl(C ₁ -C ₆ )-NR ₁₁ R ₁₁ ', -O-Cy ₁ , -alkyl(C ₀ -C ₆ )-Cy ₁ , -alkenyl(C ₂ -C ₆ )-Cy ₁ , -alkynyl(C ₂ -C ₆ )-Cy ₁ , -Oαalkyl(C1-C6)-R12, -C(O)-OR _u , -OC(O)-R _n , -C(O)-NR _n R _n ', -NR _n -C(O)-R _n ', -NR _n -C(O)-OR _n ', -alkyl(C1-C6)NR ₁₁ -C(O)-R ₁₁ ', -SO2 -NR ₁₁ R ₁₁ ', or -SO ₂ -alkyl(C ₁ -C ₆ ), or pair substituents (R ₆ , R ₇ ), when grafted onto two adjacent carbon atoms, form, together with the carbon atoms to which they attached, an aromatic or non-aromatic ring consisting of 5 to 7 ring members, which may contain from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is understood that the resulting ring may be substituted by a group selected from linear or branched (C ₁ -C ₆ )alkyl group, -NR ₁₃ R ₁₃ ', -alkyl(C ₀ -C ₆ )-Cy ₁ or oxo,

W represents -CH ₂ - group, -NH- group or oxygen atom,

R8 represents a hydrogen atom, a straight or branched (C ₁ -C ₈ )alkyl group, -CHR _a R _b group, an aryl group, a heteroaryl group, an arylalkyl(C ₁ -C ₆ ) group, or a heteroarylalkyl(C ₁ -C ₆ ) group,

R ₉ represents a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy ₂ , -alkyl(C ₁ -C ₆ )-Cy ₂ , -alkenyl (C ₂ -C ₆ )-Cy ₂ , -alkynyl (C ₂ -C ₆ )-Cy ₂ , -Cy ₂ -Cy ₃ , -alkynyl (C ₂ -C ₆ )-OCu ₂ , -Cu ₂ -alkyl(C ₀ -C ₆ )-O-alkyl(C ₀ -C ₆ )-Cu ₃ , halogen atom, cyano group, -C(O)-R ₁₄ , or -C(O)-NR ₁₄ R ₁₄ ',

R ₁₀ represents a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, arylalkyl (C ₁ -C ₆ ) group, cycloalkylalkyl(C ₁ -C ₆ ) group, linear or branched (C ₁ -C ₆ )polyhaloalkyl, or -alkyl(C ₁ -C ₆ )-O-Cy ₄ , or substituent pair (R ₉ , R ₁₀ ), when grafted onto two adjacent carbon atoms, form, together with the carbon atoms to which they are attached, an aromatic or non-aromatic ring consisting of 5-7 ring members, which may contain from 1 to 3 heteroatoms selected from oxygen , sulfur and nitrogen,

R11 and R11' independently of each other represent a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, or -alkyl(C ₀ -C ₆ )-Cy ₁ , or substituent pair (R11, R11' ) form, together with the nitrogen atom to which they are attached, an aromatic or non-aromatic ring consisting of 5-7 ring members, which may contain, in addition to the nitrogen atom, from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is intended that the nitrogen may be substituted by 1-2 groups representing a hydrogen atom, or a linear or branched (C ₁ -C ₆ )alkyl group, and it is intended that one or more of the carbon atoms of the possible substituents may be deuterated,

R ₁₂ represents -Cy ₅ , -Cy ₅ -alkyl(C ₀ -C ₆ )-O-αalkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ -alkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ alkyl(C ₀ -C ₆ )-NR ₁₁ -alkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ -Cy ₆ -O-alkyl(C ₀ -C ₆ )-Cy ₇ , - Su ₅ -alkyl(C ₀ -C ₆ )-O-alkyl(C ₀ -C ₆ )-Cu ₉ , -Cu ₅ -alkyl(C ₀ -C ₆ )-Cu ₉ , -NH-C(O)- NH-R ₁₁ , -Cy ₅ -αalkyl(C ₀ -C ₆ )-NR ₁₁ -alkyl(C ₀ -C ₆ )-Cy ₉ , -C(O)-NR ₁₁ R ₁₁ ', -NR11R11', - OR11, -NRn-C(O)-Rn', -O-alkyl(C1-C6)-OR11, -SO2-R11, -C(O)-OR _n ,

it seems possible for ammonium, defined in this way, to exist in a zwitterionic form or to have a monovalent anionic counterion,

R ₁₃ , R ₁₃ ', R ₁₄ and R ₁₄ ' independently of each other represent a hydrogen atom, or an optionally substituted linear or branched (C ₁ -C ₆ ) alkyl group,

Ra represents a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

Rb represents an -OC(O)-O-Rc group, an -OC(O)-NR _c Rc' group, or an -OP(O)(ORc) ₂ group,

R _c and R _c ' independently of each other represent a hydrogen atom, a linear or branched (C ₁ -C ₈ )alkyl group, a cycloalkyl group, a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group or ( C ₁ -C ₆ )alkoxycarbonyl (C ₁ -C ₆ )alkyl group, or substituents of the pair (R _c , R _c ') form, together with the nitrogen atom to which they are attached, a non-aromatic ring consisting of 5-7 ring members which may contain, in addition to the nitrogen atom, from 1 to 3 heteroatoms selected from oxygen and nitrogen, it is understood that this nitrogen may be replaced by a group representing a linear or branched (C ₁ -C ₆ )alkyl group, Cy ₁ , Cy ₂ , Cy ₃ , Cy ₄ , Cy ₅ , Cy ₆ , Cy ₇ , Cy ₈ and Cy ₁₀ independently of each other represent a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

Su ₉ is

or Cy ₉ represents a heteroaryl group which is substituted by a group selected from

-OP(O)(OR20)2; -OP(O)(O ^- M ⁺ )2; -(CH2)pO-(CHR18-CHR19-O) _q -R20;

hydroxy;

hydroxy(C ₁ -C ₆ )alkyl; -(CH ₂ ) _r -U-(CH ₂ ) _s -heterocycloalkyl; or -U-(CH ₂ ) _q -NR ₂₁ R ₂₁ ',

R ₁₅ represents a hydrogen atom; -(CH ₂ ) _p -O-(CHR ₁₈ -CHR ₁₉ -O) _q -R ₂₀ group; a linear or branched (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group; -U-(CH ₂ ) _q -NR ₂₁ R ₂₁ 'group; or -(CH ₂ ) _r -U-(CH ₂ ) _s heterocycloalkyl group,

R ₁₆ represents a hydrogen atom; hydroxy group; a hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r -and -(CH ₂ ) ₅ -heterocycloalkyl group; (CH ₂ ) _r -UVOP(O)(OR ₂₀ ) ₂ group; -O-P(O)((O ^- M ⁺ ) ₂ group; -(CH ₂ ) _p -O-(CHR ₁₈ -CHR ₁₉ -O) _q -R ₂₀ group; -(CH ₂ ) _p -OC( O)-NR ₂₂ R ₂₃ group; or -U-(CH ₂ ) _q -NR ₂₁ R ₂₁ ' group,

R ₁₇ represents a hydrogen atom; -(CH ₂ ) _p -O-(CHR ₁₈ -CHR ₁₉ -O) _q -R ₂₀ group;

-OP(O)(OR ₂₀ ) ₂ group; -O-P(O)(O ^- M ⁺ ) group ₂ ; hydroxy group; a hydroxy(C ₁ -C ₆ )alkyl group; -(CH ₂ ) _r -U-(CH ₂ ) _s -heterocycloalkyl group;

-U-(CH ₂ ) _q -NR ₂₁ R ₂₁ 'group; or aldonic acid,

M ⁺ is a pharmaceutically acceptable monovalent cation,

U represents a bond or oxygen atom,

V represents a -(CH ₂ ) _s - group or -C(O) - group,

Ri8 represents a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )αalkyl group,

R ₁₉ represents a hydrogen atom or a hydroxy(C ₁ -C ₆ )alkyl group,

R20 represents a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₂₁ and R ₂₁ ' independently of each other represent a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, or a hydroxy(C ₁ -C ₆ )αalkyl group, or the substituents of the pair (R21, R21') form , together with the nitrogen atom to which they are attached,

- 4 044971 aromatic or non-aromatic ring consisting of 5 to 7 ring members which may contain, in addition to the nitrogen atom, from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the resulting ring may be substituted by a group representing a hydrogen atom or a linear or branched (C ₁ -C ₆ ) alkyl group,

R22 is a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, -(CH ₂ ) _p -NR ₂₄ R ₂₄ ' group, or -(CH2)pO-(CHR1 ₈ -CHR1 ₉ -O ) _q -R20 group,

R ₂₃ represents a hydrogen atom or a (C ₁ -C ₆ )alkoxy(C ₁ -C ₆ )alkyl group, or the substituents of the pair (R ₂₂ , R ₂₃ ) form, together with the nitrogen atom to which they are attached, an aromatic or non-aromatic a ring consisting of 5 to 18 ring members which may contain, in addition to the nitrogen atom, from 1 to 5 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the resulting ring may be substituted by a group representing a hydrogen atom, linear or branched (C ₁ -C ₆ )alkyl group or heterocycloalkyl group,

R ₂₄ and R ₂₄ ' independently represent a hydrogen atom or a linear or branched (C ₁ -C ₆ ) alkyl group, or the substituent pairs (R ₂₄ , R ₂₄ ') form, together with the nitrogen atom to which they are attached , an aromatic or non-aromatic ring consisting of 5 to 7 ring members which may contain, in addition to the nitrogen atom, from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it being understood that the resulting ring may be substituted by a group representing a hydrogen atom or a linear or branched (C ₁ -C ₆ )alkyl group,

R ₂₅ represents a hydrogen atom, a hydroxy group, or a hydroxy(C ₁ -C ₆ )alkyl group,

R ₂₆ represents a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C6) alkyl group, or a cyano group,

R ₂₇ represents a hydrogen atom or a linear or branched (C ₁ -C ₆ ) alkyl group,

R ₂₈ represents -O-P(O)(O')(O') group, -OP(O)(O')(OR ₃₀ ) group, -OP(O)(0r ₃₀ )(OR ₃₀ ') group, -O-SO2-O ^- group, -O-SO ₂ -OR ₃₀ group, -Су ₁₀ , -OC(O)-R ₂₉ group, -OC(O)-OR ₂₉ group or -OC(O) -NR ₂₉ R ₂₉ 'group;

R ₂₉ and R ₂₉ ' independently of each other represent a hydrogen atom, a linear or branched (C ₁ -C ₆ )alkyl group or a linear or branched amino (C ₁ -C ₆ )alkyl group,

R ₃₀ and R ₃₀ ' independently of each other represent a hydrogen atom, a linear or branched (C ₁ -C ₆ ) alkyl group or an arylalkyl (C ₁ -C ₆ ) group, n is an integer equal to 0 or 1, p is an integer equal to 0, 1 or 2, q is an integer equal to 1, 2, 3 or 4, r and s are independently an integer equal to 0 or 1, aryl is intended to mean phenyl, naphthyl, biphenyl, indanyl or indenyl group, heteroaryl means any mono- or bicyclic group consisting of 5 to 10 ring members, having at least one aromatic component and containing from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, cycloalkyl means any mono- or bicyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, heterocycloalkyl means any mono- or bicyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, and containing from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen , which may include conjugated, bridged or spiro cyclic systems, it is possible for aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups as defined in this manner, and alkyl, alkenyl, alkynyl, alkoxy, to be substituted by 1 to 4 groups selected from the optionally substituted linear or branched (C ₁ -C ₆ )alkyl, optionally substituted linear or branched (C ₂ -C ₆ )alkenyl group, optionally substituted linear or branched (C ₂ -C ₆ )alkynyl group, optionally substituted linear or branched (C ₁ - C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, if necessary), nitro, cyano, -C(O)-OR',

-OC(O)-R', -C(O)-NRR, -NR'R, -(C=NR')-OR, linear or branched (C ₁ -C ₆ )polyhaloalkyl, trifluoromethoxy, or halogen, is implied that R' and R independently represent a hydrogen atom or an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, and it is intended that one or more carbon atoms of the preceding possible substituents may be deuterated, or enantiomers thereof , diastereoisomers, atropisomers, or addition salts thereof with a pharmaceutically acceptable acid or base, suitable for simultaneous, sequential or separate use.

In another aspect, the invention provides a combination as described herein for use in the treatment of cancer, in particular acute myeloid leukemia.

- 5 044971

In a further aspect, the invention provides a pharmaceutical composition comprising a combination as described herein and at least one pharmaceutically acceptable carrier.

Brief description of the figures

In fig. Figure 1 shows the effect on caspase 3.7 activity when combining the FLT3 inhibitor compound A (midostaurin), with the Mcl-1 inhibitor compound B, in the AML cell lines Molm13 and MV-4-11.

Fig. 2. Combined targeting of FLT3 and Mcl-1 is effective in FLT3-ITD AML primary samples. Mutation profiling of primary AML samples and sensitivity to 863845/Midostaurin combination treatment for 24 hours. Cell growth was measured using CellTiter-Glo. Additivity index (AI) was calculated using CalcuSyn software and represents the average PA at ED50, ED75, and ED90. PA<1 indicates a synergistic effect and PA>1 antagonism. Primary AML samples were treated with the following serial dilution doses of the combination: midostaurin, 3.1 nM to 800 nM; and S63845, from 0.31 nM to 80 nM. The ratio of midostaurin to S63845 was 10:1. The presence of a mutation was indicated by filled blue boxes for each sample.

Fig. 3. Midostaurin induces BIM accumulation, which contributes to the lethality of midostaurin/863845 (compound C).

A. Midostaurin increases the level of proapoptotic Bim, which may in turn bind and nullify remaining Mcl-1 activity. Elevated Bim persists with S63845 co-treatment, suggesting that Bim plays a functional role in midostaurin/863845-mediated lethality.

B. Western blot analysis of FLT3-mediated signaling pathways and expression of Bcl-2 family proteins in MV-4-11 cells treated for 6 hours with midostaurin, S63845 alone or in combination. Dephosphorylation of FLT3-ITD results in inactivation of STAT5 and MAPK and is accompanied by a significant down-regulation of MAPK-mediated phosphorylation of Mcl-1 at Thrl63, a site required for Mcl-1 stability. Consequently, midostaurin reduces Mcl-1 levels.

Fig. 4 and 5. Combined targeting of FLT3 and Mcl-1 facilitates apoptosis.

Caspase activation was measured using the Caspase-Glo® 3/7 assay after 6 h of drug treatment. The percentage of apoptotic cells was measured by flow cytometry using Annexin-V-APC/DAPI staining. AML primary cells were treated for 16 h.

Fig. 6. Anti-leukemic synergism of S63845 (Compound C) and FLT3 Tki.

Sensitivity of AML cell lines to S63845 (Compound C) and selected FLT3 inhibitors (FLT3 TKi) after exposure for 24 hours. Cell growth was measured using CellTiter-Glo. Additivity index (AI) values were calculated using CalcuSyn software, and mean PA values at ED50, ED75, and ED90 are presented. The ratios at which the drugs were combined are presented in parentheses.

Fig. 7 and 8. Antileukemic synergism of S63845 and FLT3 Tki.

Representative synergistic interactions between S63845 in combination with midostaurin on MV4-11 FLT3-ITD cells were determined using drug synergism in the BLISS extended size independence model. Cells were treated with nine 2-fold serial dilutions of each compound, either individually or in all possible combinations in a staggered manner. Cell growth was assessed after 24 h (Fig. 7). Synergistic interactions were assessed using the BLISS independence model. BLISS index values for each combination dose >0 represent synergism, while BLISS index values <0 represent antagonism (Fig. 8).

Fig. 9. BH3 profiling: midostaurin increases mitochondrial priming for apoptosis in FLT3-ITD cells exposed to S63845.

BH3 profiling analysis showed that midostaurin increases mitochondrial priming in MV4-11 FLT3-ITD to the Mcl-1 inhibitor S63845 and especially Bim peptide. Cells were subjected to BH3 profiling analysis 4 h after treatment with midostaurin (100 nM). Increased cytochrome C release is a quantitative indicator of the initiation of mitochondrial apoptosis.

Fig. 10, 11, 12, and 13. The combination of S63845 and midostaurin is effective in venetoclax-resistant AML cells with FLT3-ITD, but not in FLT3-WT.

BLISS independence model for synergistic interactions between S63845 in combination with Midostaurin in MOLM13 FLT3-ITD cells and the Venetoclax-resistant subline. Venetoclax-resistant cells were generated as described in Zhang et al. Blood, 2015, 126, 328 and maintained routinely in medium containing 1 µM Venetoclax. Venetoclax was washed out before experiments and cells were treated with nine 2-fold serial dilutions of S63845 and Midostaurin, either individually or in all possible staggered combinations. After 24 hours

- 6 044971 treatment, cell growth was measured using Cell TiterGlo. BLISS index values for each combination dose >0 represent synergism, while BLISS index values <0 represent antagonism.

Fig. 14. Preliminary in vivo study: AML FLT3-ITD xenograft NSG mice were transplanted with 10 ⁶ patient-derived AML cells anchoring FLT3-ITD mutations. Tumor burden was measured by the percentage of hCD45+ cells in the blood of mice using FACS. After engraftment, mice were treated with (I) vehicle, (II) S63845 40 mg/kg IV once weekly, (III) Midostaurin 75 mg/kg gavage daily, or (IV) a combination.

Fig. 15 and 16. Synergistic effect of midostaurin and S63845 in mouse Ba/F3 FLT3-ITD cells. Synergistic interactions between S63845 in combination with midostaurin in murine Ba/F3 FLT3-ITD cells were determined using drug synergy in the BLISS extended size independence model. Cells were treated with nine 2-fold serial dilutions of each compound, either individually or in all possible combinations in a staggered manner. Cell growth was assessed after 24 hours (Fig. 15). Synergistic interactions were assessed using the BLISS independence model. BLISS index values for each combination dose >0 represent synergism, while BLISS index values <0 represent antagonism (Fig. 16).

Fig. 17 and 18. Synergistic effect of midostaurin and S63845 in mouse Ba/F3 FLT3-D835Y cells.

Synergistic interactions between S63845 in combination with Midostaurin in mouse Ba/F3 FLT3-D835Y cells were determined using drug synergy in the BLISS extended size independence model. Cells were treated with nine 2-fold serial dilutions of each compound, either individually or in all possible combinations in a staggered manner. Cell growth was assessed after 24 hours (Fig. 17). Synergistic interactions were assessed using the BLISS independence model. BLISS index values for each dose of the combination > 0 represent synergism, while BLISS index values < 0 represent antagonism (Fig. 18).

Definitions

Combination refers to either a fixed-dose combination in one unit dosage form (e.g., a capsule, tablet or sachet), a non-fixed-dose combination, or a combination administration kit, wherein a compound of the present invention and one or more components of the combination (e.g., another drug, as explained below, also referred to as a therapeutic agent or coagent) may be administered independently at the same time or separately over time intervals, particularly if these time intervals provide an opportunity for the components of the combination to exhibit cooperative, e.g., synergistic Effect.

The terms co-administration or combination administration or the like as used herein encompass the administration of a selected component of the combination to a single subject in need thereof (eg, a patient) and include treatment regimens in which the agents are not necessarily administered by the same route. administration or at the same time.

The term fixed-dose combination means that the active components, eg, a compound of formula (I) and one or more components of the combination, are both administered to the patient simultaneously in unit or dosage form.

The term non-fixed dose combination means that the active components, for example, a compound of the present invention and one or more components of the combination, are both administered to a patient as separate components, either simultaneously or sequentially, without specific time restrictions, where such administration provides therapeutically effective levels of the two compounds in the patient's body. The latter is also used for cocktail therapy, for example, the administration of three or more active components.

Malignancy refers to a class of diseases in which a group of cells exhibit uncontrolled growth. Types of malignancies include hematologic malignancies (lymphoma and leukemia). In particular, malignancy refers to hematological malignancy, in particular acute myeloid leukemia.

AML stands for acute myeloid leukemia. In a preferred embodiment, AML is present in patients carrying the FLT3 mutation. In a further embodiment, the mutation is FLT3-ITD. In another embodiment, the mutation is FLT3TKD. Specifically, the FLT3-TKD mutation includes the FLT3-D835Y or FLT3-F691 mutation within the tyrosine kinase domain (TKD) of FLT3. Treated AML may be resistant to previous cancer therapy. Such prior therapy may include one or more compounds used to treat AML, for example, venetoclax, decitabine, daunorubicin, and cytarabine, particularly venetoclax. In one embodiment, the AML being treated is venetoclax-resistant AML, particularly venetoclax-resistant AML with a FLT3-ITD mutation.

The term co-therapeutically effective means that the therapeutic agents can be administered separately (in a chronological order, especially sequence-specific) at such time intervals that they can preferably, in a warm-blooded animal, especially a human, treat while still exhibiting (preferably synergistic) interaction (joint therapeutic effect). In any case, this can be determined, in particular, by determining subsequent blood levels indicating that both compounds are present in the blood of the person being treated, at least over certain time intervals.

Synergistically effective or synergism means that the therapeutic effect observed after administration of two or more agents is greater than the sum of the therapeutic effects observed after administration of each single agent.

As used herein, the term treat, cure, or therapy for any disease or disorder refers, in one embodiment, to alleviating the disease or disorder (ie, relieving or stopping or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, treat, treatment, or therapy refers to the attenuation or alleviation of at least one physical parameter, including those that may not be felt by the patient. In yet another embodiment, treat, treatment, or therapy refers to modulating a disease or disorder, either physically (eg, stabilization of a discernible symptom), physiologically (eg, stabilization of a physical parameter), or both.

As used herein, a subject requires treatment if such subject will benefit biologically, medically, or quality of life from such treatment.

Drug means a pharmaceutical composition, or a combination of several pharmaceutical compositions, which contains one or more active ingredients in the presence of one or more excipients.

Detailed Description of the Invention

Numbered embodiments of the invention are described below, where, for convenience, E1 is identical to the first aspect of the invention described herein above. Further numbered embodiments (E) of the invention are described herein. It should be appreciated that the features specified in each embodiment may be combined with other specified features to provide further specific embodiments of the present invention.

E2. The combination according to E1, comprising (a) midostaurin or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and (b) an Mcl-1 inhibitor of formula (I), wherein the Mcl-1 inhibitor formula (I) is formula (IA):

Where:

Z represents a nitrogen atom or a C-R4 group,

R1 represents a linear or branched (C ₁ -C ₆ ) alkyl group, a linear or branched (C ₂ -C6) alkenyl group, a linear or branched (C ₂ -C ₆ ) alkynyl group, a linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )alkyl group, linear or branched (C ₁ -C ₆ )polyhaloalkyl, hydroxy group, cyano, -NR11R11', -Cy ₈ or halogen atom,

R ₂ , R ₃ and R 4 independently represent a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ ) alkyl group, a linear or branched (C ₂ -C ₆ ) alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, linear or branched (C ₁ -C ₆ )polyhaloalkyl, hydroxy group, linear or branched (C ₁ -C ₆ )alkoxy group, -S-(C ₁ -C ₆ )αalkyl group, cyano group, nitro group, -alkyl(C ₀ -C ₆ )-NR ₁₁ R ₁₁ ', -O-Cy1, -alkyl(C ₀ -C ₆ )-Cy1, -alkenyl(C ₂ -C ₆ )-Cy ₁ , -alkyl(C ₂ -C ₆ )-Cy ₁ , -O-alkyl(C ₁ -C ₆ )-NR ₁₁ R ₁₁ ', -O-alkyl(C ₁ -C ₆ )-R ₁₂ , -C( O)-OR ₁₁ ,

- 8 044971

-OC(O)-R _n , -C(O)-NR _n R _n ', -NR _n -C(O)-R _n ', -NR _n -C(O)-OR _n ', -alkyl( C1-C6)-NR _n -C(O)-R _n ', -SO2-NR11R11', or -SO ₂ -alkyl(C ₁ -C ₆ ), or substituents of one of the pairs (R2, R ₃ ), ( R ₃ , R ₄ ) form, together with the carbon atoms to which they are attached, an aromatic or non-aromatic ring consisting of 5 to 7 ring members, which may contain from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is implied that the resulting ring may be substituted by a group selected from a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₁₃ R ₁₃ ', -alkyl(C ₀ -C ₆ )-Cy1 or oxo,

R6 and R7 independently represent a hydrogen atom, a halogen atom, a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, linear or branched (C ₁ -C6)πpolyhaloalkyl, hydroxy group, linear or branched (C ₁ -C6)alkoxy group, -S-(C ₁ -C6)αalkyl group, cyano group, nitro group, -alkyl (C ₀ -C ₆ )-NR ₁₁ R ₁₁ ', -O-Cy1, -alkyl(Co-C ₆ )-Cy1, -alkenyl(C ₂ -C ₆ )-Cy ₁ , -alkynyl(C ₂ -C ₆ )-Cy ₁ , -O-alkyl(C ₁ -C ₆ )-R ₁₂ , -C(O)-OR ₁₁ , -OC(O)-R ₁₁ , -C(O)-NR ₁₁ R ₁₁ ' , -NR _n -C(O)-R _n ', -NR _n -C(O)-OR _n ', -alkyl(C1-C6)-NR _n -C(O)-R _n ', -SO2- NR11R11', or -SO2-alkyl(C1-C6), or pair substituents (R ₆ , R ₇ ), when grafted onto two adjacent carbon atoms, form, together with the carbon atoms to which they are attached, an aromatic or non-aromatic ring, consisting of 5 to 7 ring members, which may contain from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is understood that the resulting ring may be substituted by a group selected from a linear or branched (C ₁ -C ₆ )alkyl group, -NR ₁₃ R ₁₃ ', -alkyl(C ₀ -C ₆ )-Cy1 or oxo,

R8 represents a hydrogen atom, a straight or branched (C ₁ -C ₈ )alkyl group, an aryl group, a heteroaryl group, an arylalkyl(C ₁ -C ₆ ) group, or a heteroarylalkyl(C ₁ -C ₆ ) group,

R ₉ represents a linear or branched (C ₁ -C ₆ )alkyl group, a linear or branched (C ₂ -C ₆ )alkenyl group, a linear or branched (C ₂ -C ₆ )alkynyl group, -Cy ₂ , -alkyl( C ₁ -C ₆ )-Cy ₂ , -alkenyl(C ₂ -C ₆ )-Cy ₂ , -alkynyl(C ₂ -C ₆ )-Cy ₂ , -Cy ₂ -Cy ₃ , -alkynyl(C ₂ -C ₆ )-O-Cy ₂ , -Cy ₂ -alkyl(C ₀ -C ₆ )-O-alkyl(C ₀ -C ₆ )-Cy ₃ , halogen atom, cyano group, -C(O)-R ₁₄ , or -C(O)-NR ₁₄ R ₁₄ ',

R11 and R11' independently of each other represent a hydrogen atom, an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, or -alkyl(C ₀ -C ₆ )-Cy1, or substituent pair (R11, R11') form, together with the nitrogen atom to which they are attached, an aromatic or non-aromatic ring consisting of 5 to 7 ring members which may contain, in addition to the nitrogen atom, from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, it is understood that the nitrogen may be replaced by a group representing a hydrogen atom, or a linear or branched (C ₁ -C ₆ )alkyl group and it is understood that one or more of the carbon atoms of the possible substituents may be deuterated,

R ₁₂ represents -Cy ₅ , -Cy ₅ -alkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ -alkyl(C ₀ -C ₆ )-O-alkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ -alkyl(C ₀ -C ₆ )-NR ₁₁ -alkyl(C ₀ -C ₆ )-Cy ₆ , -Cy ₅ -Cy ₆ -O-alkyl(C ₀ -C ₆ )-Cy ₇ , -C(O)-NR ₁₁ R ₁₁ ', -NR11R11', -OR11, -NR _n -C(O)-R _n ', -O-αalkyl(C1-C6)-OR11, -SO2-R11, - C(O)-OR _n , or -NH-C(O)-NH-R _n ,

R ₁₃ , R ₁₃ ', R ₁₄ and R ₁₄ ' independently of each other represent a hydrogen atom, or an optionally substituted linear or branched (C ₁ -C ₆ ) alkyl group,

R ₂₅ represents a hydrogen atom, a hydroxy group, or a hydroxy(C ₁ -C ₆ )alkyl group,

Cy1, Cy ₂ , Cy ₃ , Cy ₅ , Cy ₆ , Cy ₇ and Cy ₈ independently of each other represent a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, n is an integer equal to 0 or 1, implying What:

aryl means a phenyl, naphthyl, biphenyl, indanyl or indenyl group, heteroaryl means any mono- or bicyclic group consisting of 5-10 ring members, having at least one aromatic component and containing from 1 to 3 heteroatoms selected from oxygen, sulfur and nitrogen, cycloalkyl means any mono- or bicyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, heterocycloalkyl means any mono- or bicyclic non-aromatic carbocyclic group containing from 3 to 10 ring members and containing from 1 to 3 heteroatoms selected of oxygen, sulfur and nitrogen, which may include conjugated, bridged or spiro cyclic systems, it is possible for aryl, heteroaryl, cycloalkyl and heterocycloalkyl groups so defined, and alkyl, alkenyl, alkynyl, alkoxy, to be substituted by 1 to 4 groups , selected from an optionally substituted linear or branched (C ₁ -C ₆ )alkyl group, an optionally substituted linear or branched (C ₂ -C ₆ )alkenyl group, an optionally substituted linear or branched (C2-C6)alkynyl group, an optionally substituted linear or branched (C ₁ -C ₆ )alkoxy, optionally substituted (C ₁ -C ₆ )alkyl-S-, hydroxy, oxo (or N-oxide, if necessary), nitro, cyano, -C(O)-OR', - OC(O)-R', -C(O)-NR'R, -NR'R, -(C=NR')-OR, linear or branched (C ₁ -C ₆ )polyhaloalkyl, trifluoromethoxy, or halogen, R' and R are meant to independently represent a hydrogen atom or an optionally substituted straight or branched (C ₁ -C ₆ )alkyl group, and it is meant that one or more carbon atoms of the preceding possible substituents may be present riated, or their enantiomers, diastereoisomers, atropisomers, or their addition salts with a pharmaceutically acceptable acid or base, suitable for simultaneous, sequential or separate use.

E3. Combination according to E1 or E2, wherein the Mcl-1 inhibitor of formula (I) is formula (IB):

where R ₉ , Rn, Rn' and R ₁₂ have the meanings as described in E1 or E2 above.

E4. Combination according to E1, E2 or E3, wherein the Mcl-1 inhibitor of formula (I) is compound B: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4- [2-(4-methylpiperazin-1 -yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2 -methoxyphenyl)nirimidin-4-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof.

E5. Combination according to E1, E2 or E3, wherein the Mcl-1 inhibitor of formula (I) is Compound C: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4- [2-(4-methylpiperazin-1 -yl)ethoxy]phenyl}-6-(5fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[ 1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof.

E6. Combination according to E4, where Compound B is administered intravenously and Midostaurin is administered orally.

E7. The combination according to any E1 to E6, which further comprises at least one additional anticancer agent, for example cytarabine and/or daunorubicin.

E8. Combination according to any E1 to E7, in the form of a non-fixed dose combination.

E9. Combination according to any E1 to E7, in the form of a fixed dose combination.

E10. Combination in accordance with any E1 - E9, for medical use.

E11. A combination in accordance with any E1 to E9, for use in accordance with E10, wherein said use is for the treatment of a malignant neoplasm.

E12. A combination according to any E1 to E9, for use according to E11, wherein the malignancy is a hematological malignancy.

E13. A combination in accordance with any E1 - E9, for use in accordance with E12, where the malignancy is acute myeloid leukemia. In particular, acute myeloid leukemia is present in patients carrying the FLT3 mutation. More preferably, said mutation is FLT3-ITD.

E14. A combination according to any E4 or E6 to E9, for use according to any E10 to E13, wherein Midostaurin and Compound B are provided in amounts that are jointly therapeutically effective for treating the cancer.

E15. A combination according to any E4 or E6 to E9, for use according to any E10 to E13, wherein Midostaurin and Compound B are provided in amounts that are synergistically effective for the treatment of cancer.

E16. A combination according to any E4 or E6-E9, for use according to any E10 to E13, wherein Midostaurin and Compound B are provided in synergistically effective amounts that are capable of reducing the dose required of each compound for the treatment of cancer while providing effective treatment of malignant neoplasms, with a reduction in side effects.

E17. A pharmaceutical composition containing a combination in accordance with any E1-E9, and at least one pharmaceutically acceptable carrier.

E18. Use of a combination in accordance with any E1-E9 for the preparation of a medicinal product for the treatment of a malignant neoplasm.

E19. Use in accordance with E18, combinations in accordance with any E1-E9, where the malignancy is acute myeloid leukemia. In particular, acute myeloid leukemia is present in patients carrying the FLT3 mutation. More preferably, said muta-

- 10 044971 tion is FLT3-ITD or FLT3-TKD.

E20. A medicinal product containing, separately or together, (a) midostaurin or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and (b) compound B: (2R)-2-{[( 5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4 -yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof, for simultaneous, sequential or separate administration, and wherein Midostaurin and Compound B are provided in effective amounts for the treatment of cancer, particularly AML.

E21. A method of treating a malignant neoplasm, in particular AML, comprising co-administering a therapeutically effective amount of (a) midostaurin or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and (b) compound B: (2R) -2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3 -d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof.

E22. A method of sensitizing or treating a patient who (I) is refractory to at least one chemotherapy treatment, or (II) has relapsed after treatment with chemotherapy, or both (I) and (II), where the method includes administering a co-therapeutically effective an amount of (a) midostaurin, and (b) Mcl-1 inhibitor of formula (I), as defined herein, to the specified patient.

E23. compound B for use in combination therapy with midostaurin, for the treatment of malignant neoplasms, in particular AML.

E24. Midostaurin for use in combination therapy with compound B, for the treatment of malignant neoplasms, in particular AML.

E25. A combination for use, method or use as described in any of the embodiments herein for the treatment of AML resistant to prior therapy.

E26. A combination of use, method or use as described in any of the embodiments herein for the treatment of AML resistant to one or more compounds selected from venetoclax, decitabine, daunorubicin and cytarabine, in particular venetoclax.

E27. A combination of use, method or use as described in any of the embodiments herein for the treatment of venetoclax-resistant AML with a FLT3-ITD mutation.

E28. A combination of use, method or uses as described in any of the embodiments herein for the treatment of AML with FLT3-TKD mutation.

E29. A combination for use, method or use as described in embodiment E28, wherein said FLT3-TKD mutation includes FLT3-D835Y or FLT3-F691 mutation within the tyrosine kinase domain (TKD) of FLT3.

E30. A combination for use, method or use as described herein, wherein the malignancy is acute myeloid leukemia present in patients carrying the FLT3-ITD mutation in the presence of the FLT3-TKD mutation.

In another aspect, the invention provides a combination, use combination, method, composition, drug or use as described herein, particularly comprising Compound B or Compound C and midostaurin, wherein said cancer, particularly AML, is resistant to previous anticancer therapy. Such prior therapy may include one or more compounds used to treat a malignancy such as AML, such as venetoclax, decitabine, daunorubicin, and cytarabine, particularly venetoclax. In one embodiment, the AML being treated is venetoclax-resistant AML, particularly venetoclax-resistant AML with FLT3-ITD.

In another embodiment, the AML being treated is AML with the FLT3TKD mutation. Specifically, the FLT3-TKD mutation includes FLT3-D835Y or FLT3-F691 mutations within the tyrosine kinase domain (TKD) of FLT3. In a further embodiment, the malignancy is acute myeloid leukemia present in patients carrying the FLT3-ITD mutation in the presence of the FLT3-TKD mutation.

In particular, AML treated as described herein is resistant to treatment with a FLT3 inhibitor.

In another embodiment, there is provided a method of treating a malignancy, particularly AML, comprising co-administering a therapeutically effective amount of:

(a) midostaurin or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and

- 11 044971 (b) compounds B: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl }-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid , or a pharmaceutically acceptable salt thereof, or (c) compound C: (2R)-2-{[(5S _a )-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazine-1- yl)ethoxy]phenyl}-6(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[1-(2,2,2- trifluoroethyl)-1H-pyrazol-5yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof.

In a further embodiment, there is provided a method of treating cancer as described in any of the embodiments herein, comprising co-administering a therapeutically effective amount of midostaurin and Compound B or Compound C.

In the pharmaceutical compositions according to the invention, the proportion of active ingredients by weight (weight of active ingredients in relation to the total weight of the composition) is 5-50%.

More specifically used as pharmaceutical compositions according to the invention are those which are suitable for administration by oral, parenteral and in particular intravenous, trans- or transdermal, nasal, rectal, sublingual, ocular or respiratory routes, more specifically tablets, tablets, sublingual tablets, hard gelatin capsules, sublingual tablets, capsules, lozenges, injectables, aerosols, eye or nasal drops, suppositories, creams, ointments, skin gels, etc.

In one embodiment, a compound of formula (I), in particular compound B, is administered intravenously, for example using a formulation as described in WO 2018/078064.

Pharmaceutical compositions in accordance with the invention may contain one or more excipients or carriers selected from diluents, lubricants, binders, disintegrants, stabilizers, preservatives, absorbents, coloring agents, sweeteners, flavoring agents, etc.

By way of non-limiting example:

as diluents: lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerin, as lubricants: silicon dioxide, talc, stearic acid and its magnesium and calcium salts, polyethylene glycol, as binders: magnesium aluminum silicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, as disintegrators: agar, alginic acid and its sodium salt, effervescent mixtures.

The combination compounds may be administered simultaneously, separately, or sequentially. Appropriate pharmaceutical compositions may provide the possibility of immediate or delayed release of the active components. The combination compounds may further be administered in the form of two separate pharmaceutical compositions, each containing one of the active ingredients, or in the form of a single pharmaceutical composition in which the active ingredients are present in a mixture.

A suitable dosage regimen will vary depending on the sex, age and body weight of the patient, the route of administration, the nature of the malignancy and any associated treatments and ranges from 25 mg to 1500 mg of Mcl-1 inhibitor per week, more preferably from 50 mg to 1400 mg per week.

The dosage of midostaurin for the treatment of AML that is positive for the FLT3 mutation is 50 mg orally, twice daily with meals. Midostaurin is provided to the patient in combination with standard induction of cytarabine and daunorubicin and consolidation of cytarabine. This dose may be adapted, if necessary, during combination treatment with Compound B.

In one embodiment, oral administration of Midostaurin is via a solid form. The dosage form of midostaurin is a soft gelatin capsule containing 25 mg of the drug.

Example 1. In vitro effect on caspase 3.7 activity when combining the FLT3 inhibitor compound A (midostaurin) with the Mcl-1 inhibitor compound B, in the AML cell lines Molml3 and MV-4-11.

We evaluated Mcl-1 and FLT3 inhibitors as monotherapy and in combination to determine their ability to induce apoptosis (ie, Caspase 3.7 activation) in leukemic cells. We used two AML cell lines with the FLT3-ITD mutation (Molml3 and MV-4-11). We tested the ability of compounds A and B to activate caspase 3.7 in combinations and as monotherapy. Compound A as monotherapy induces caspase activity with a maximum increase of 33% in Molm13 and 24% in MV-4-11. Compound B also induced caspase activity with a maximum increase of 87% a Molm13 and 89% a MV-4-11. When both compounds were combined, a synergistic induction of caspase 3.7 activity was observed. Synergy was observed in both cell lines with a synergy score of 3.1 in both. Strong synergism was particularly evident at the lowest doses of Compound B. The results are shown in FIG. 1.

Methods.

Both compounds were dissolved in 100% DMSO (Sigma, cat. no. D2650) at concentrations

- 12 044971 mM and stored at -20°C until use. The Molm13 cell line used in this study was obtained from Leibniz-Institut DSMZ (ACC 554) and MV-4-11 from the American Type Cell Collection (ATCC® CRL-9591). Cell lines were cultured at 37°C in a 5% _CO2 incubator, Molm13 in RPMI (Lonza, 12-702F) and MV-4-11 in IMDM (Hyclone, SH30228.01) medium supplemented with 10% fetal bovine serum (Seradigm, 1500-500). Cells were inoculated twice a week and the medium was replaced every 2-3 days.

Caspase 3.7 activity was measured using the Caspase-Glo® 3/7 kit from Promega (G8092). 9000 cells/well were transferred into black clear-bottom 384-well plates (Greiner, no. 781091) in triplicate with 30 μl/well growth medium. 10 µL/well of the 4X compound mixture was added to each well of a 384-well plate for 6 hours of treatment. 30 µL/well of Caspase-Glo® 3/7 reagents was then added to each well as a final step and luminescence was recorded on an Envision plate reader. (Perkin Elmer). The measured luminescence is proportional to the amount of caspase activity present.

To objectively assess the activity of caspase 3.7 under the influence of the combination, as well as to identify the synergistic effect at all possible concentrations, studies were carried out with a dose matrix. To do this, all possible combinations of serial dilutions of compound A and compound B were used.

The dose matrix of compound A and compound B consisted of the following: both compounds were subjected to a 1:3 serial dilution of 11 doses with the highest dose of 3 μM and reduced to no compound control. In combination studies, the agents were administered simultaneously. Luminescent signal values for treatment with each agent alone and in combination were compared with the corresponding values for untreated controls. The percentage of caspase 3.7 activity was calculated using the following calculation: (1-control/compound-treated)x100. Percent caspase 3.7 activity values for all wells were calculated using Chalice software (CombinatoRx, Cambridge MA) and Chalice Analyzer as described in Lehar et al., Nature Biotechnology 2009, 27(7), 659-66. In this study, caspase 3.7 activity data were analyzed instead of cell growth inhibition data with similar mathematical protocols. Caspase 3.7 activity compared to control is presented in the panel labeled Percent Caspase 3.7 Activity and the amount of activity greater than the expected amount in the panel labeled Excess Löwe Activity). Concentrations of compound A are presented in the bottom row from left to right, and increasing concentrations of compound B for the leftmost column from top to bottom. All remaining points on the grids represent results from the combination of two inhibitors, which correspond to the concentrations of the individual agents indicated on the two axes. Caspase 3.7 activity data analysis was performed using Chalice Analyzer. Excess activity was calculated using the Löwe synergy model, which determines the effect on caspase 3.7 activity to be greater than that expected if the two drugs acted in a dose-additive manner. Positive numbers represent areas of increasing synergy. Interpretations of synergy scores were: SS ~ 0 = Dose Additive, SS >1 = Weak Synergism, and SS >2 = Synergism.

Example 2 The combination of the Mcl-1 inhibitor Compound C and Midostaurin is synergistic in FLT3-ITD mutated AML cells, including those resistant to the Bcl-2 inhibitor Venetoclax and in primary AML cells.

Materials and methods.

Fig. 2. Combined targeting of FLT3 and Mcl-1 is effective in FLT3-ITD AML primary samples.

Cell viability assay and additivity index (AA).

Primary AML cells were obtained from peripheral blood samples collected from patients at the MD Anderson Cancer Center with newly diagnosed or recurrent AML and high (>40%) blast cell counts. After Ficoll purification, AML blasts (8x10 /well) were plated in 96-well plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma). Midostaurin and S63845 were prepared as 10 mM stock solutions in DMSO and maintained at −80°C until analyses. Each drug was diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Control cells received 100 μl of medium containing DMSO (a volume of DMSO corresponding to the sum of the volumes of Midostaurin and S63845 stock solutions used to prepare 4x solutions). Cells were incubated with drugs used alone or in combination for 24 hours. Cell viability was measured using a CellTiter-Glo luminescence assay (Promega) according to the manufacturer's instructions. Briefly, cells were mixed gently by pipetting and 35 μl of cell suspension was transferred into white opaque 96-well plates. Thereafter, 80 μl of CellTiter-Glo reagent diluted 1:3 in PBS was added to each well, and the cells were incubated for 30 minutes in the dark on a plate shaker. Luminescence was recorded using a Tekan plate reader (Infinite m200 pro). Analysis of the combined effects of drugs was carried out using the additivity index (AA) calculated using software

CalcuSyn (BioSoft, Ferguson, MO, USA). PA <1, 1 and >1 indicate synergism, additive effect and antagonism, respectively.

Fig. 3A/3B. Midostaurin induces BIM accumulation, which contributes to the lethality of Mudocmaypun/S63845 (compound C).

Western blotting.

Cells (3 x 10 ⁶ /well) were seeded in a 6-well plate in 5 ml of complete RPMI medium and exposed to Midostaurin and S63845, administered alone or in combination. Control cells received DMSO. Cells were incubated for 6 h and then harvested, washed twice in PBS and lysed in RIPA buffer (ThermoFisher Scientific) supplemented with 1x protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). The lysates were kept on ice for 20 min with vortex mixing every 5 min, then sonicated briefly and centrifuged at 13,500 rpm. within 15 minutes. The supernatant was collected and protein content was measured by BSA assay. Cell lysates (30–50 μg of protein per well) were separated by electrophoresis on 4%–20% gradient preformed sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Bio-Rad). Membranes were first incubated in Odyssey blocking buffer (Li-Cor, Lincon, Nebraska, USA) for 1 hour to block nonspecific protein binding, then with primary antibody overnight at 4°C, washed with TBST three times, and incubated with IRDye-conjugated secondary antibody (1:15,000 dilution) for 1 hour at room temperature. Membranes were scanned using the Odyssay Imaging System (Li-Cor).

Fig. 4. Combined targeting of FLT3 and Mcl-1 facilitates apoptosis.

Caspase 3/7 activity assay to detect apoptosis.

AML cells or AML primary blast cells (3 x 10 ⁶ /well) were seeded in a 6-well plate in 5 ml of complete RPMI medium and exposed to midostaurin and S63845, administered alone or in combination. Control cells received DMSO. Cells were incubated for 6 h and then 100 μl of the cell suspension was transferred to a white opaque 96-well plate, mixed with 100 μl of Caspase-Glo3/7 reagent (Promega) and incubated in the dark for 30 minutes on a plate shaker at room temperature. Luminescence was recorded using a Tekan plate reader (Infinite m200 pro). Changes in caspase-3/7 activity after drug treatment were normalized to DMSO separately. The remaining cells were used for Western blotting.

Fig. 5. Combined targeting of FLT3 and Mcl-1 facilitates apoptosis.

Annexin-V/DAPI assay.

AML blast cells (8x105/well) were seeded in 96-well round-bottom plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma). Midostaurin and S63845 were diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Control cells received 100 μl of medium containing DMSO (the volume of DMSO corresponded to the sum of the volumes of Midostaurin and S63845 stock solutions used to prepare 4x solutions). The cells were incubated with drugs administered alone or in combination for 24 hours. The next day, the cell plates were centrifuged at 1500 rpm. within 5 minutes. Thereafter, cells were washed once in PBS and resuspended in 100 μl Annexin-V binding buffer containing 0.3 μl Annexin-V-APC (BD Bioscience) and 5 μl DAPI (2 μg/ml). Cells were stained in the dark for 20 min, supplemented with an additional 100 μl of Annexin-V binding buffer, and analyzed using a BD LRSII flow cytometer (Becton Dickinson). Annexin-V-APC positive cells were identified using FlowJo software (LLC, Ashland, Oregon, USA).

Fig. 6. Anti-leukemic synergism of S63845 (compound C) and FLT3 TKi.

Cell viability assay.

MV4-11 FLT3-ITD ^+/+ and MOLM13 FLT3-ITD ^+/ AML cells (8x105/well) were seeded in 96-well plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma ). Midostaurin and S63845 were diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Control cells received 100 μl of medium containing DMSO (the volume of DMSO corresponded to the sum of the volumes of midostaurin and S63845 stock solutions used to prepare 4x solutions). Cells were incubated with drugs administered alone or in combination for 24 hours. Cell viability was measured using a CellTiter-Glo luminescent assay (Promega) according to the manufacturer's instructions. Briefly, cells were mixed gently by pipetting and 35 μl of cell suspension was transferred into white opaque 96-well plates. Thereafter, 80 μl of CellTiter-Glo reagent diluted 1:3 in PBS was added to each well, and the cells were incubated for 30 minutes in the dark on a plate shaker. Luminescence was recorded using a Tekan plate reader (Infinite

- 14 044971 m200 pro). Combined drug effects were analyzed using the additivity index (AD) calculated using CalcuSyn software (BioSoft, Ferguson, MO, USA). PA <1, 1, and >1 indicated synergism, additive effect, and antagonism, respectively.

Fig. 7 and 8. Antileukemic synergism of S63845 and FLT3 TKi.

Cell viability analysis and BLISS index.

MV4-11 FLT3-ITD ^+/+ cells (8x105/well) were seeded in 96-well plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma). Midostaurin and S63845 were diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Cells were treated for 24 h with nine 2-fold serial dilutions of each compound, either individually or in all possible staggered combinations. Cell growth was measured, and viability was measured using a CellTiter-Glo luminescence assay (Promega) according to the manufacturer's instructions. Briefly, cells were mixed gently by pipetting and 35 μl of cell suspension was transferred into white opaque 96-well plates. Thereafter, 80 μl of CellTiter-Glo reagent diluted 1:3 in PBS was added to each well, and the cells were incubated for 30 minutes in the dark on a plate shaker. Luminescence was recorded using a Tekan plate reader (Infinite m200 pro). Cell growth was expressed as % of cells treated with DMSO. Synergistic interactions were assessed using the BLISS independence model. BLISS index values for each combination dose > 0 represent synergism, while BLISS index values < 0 represent antagonism.

Fig. 9. Midostaurin enhances mitochondrial priming for apoptosis in FLT3-ITD cells exposed to S63845.

Dynamic VN3 profiling.

BH3 profiling was carried out in black 384-well plates according to the method of A. Letai (https://letailab.dana-farber.org/bh3-profiling.html). Briefly, cells (3 x 10 ⁶ /well) were seeded in a 6-well plate in 5 ml of complete RPMI medium and exposed to Midostaurin and S63845, administered alone or in combination. Control cells received DMSO. Cells were incubated for 6 h, collected, washed twice in PBS and resuspended in MEB buffer (150 mM mannitol, 10 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate) at a final density of 1x10 ⁶ /ml. Cells (15 μl) were added to a 384-well plate containing peptides/BH3 mimetics and 15 μl of 0.002% digitonin in MEB buffer. Cells were incubated for 1 hour at 25°C and then fixed in 10 μl of 4% formaldehyde in PBS for 10 minutes. To stop fixation, 10 μl of N2 buffer (1.7 M Tris, 1.25 M Glycine pH 9.1) was added and the cells were left at room temperature for 5 minutes. After this, the cells were mixed with 10 μl of 10x CytoC staining buffer (10% BSA, 2% Tween20 in PBS) containing an antibody to cytochrome C (Clone 6H2.B4, fluorescently labeled, BioLegend) at a dilution of 1:40 and stained for nights at 4°C. Cytochrome C release was measured using flow cytometry.

Fig. 10, 11, 12, and 13. The combination of S63845 and midostaurin is effective in venetoclax-resistant AML cells with FLT3-ITD, but not in FLT3-WT. Cell viability analysis and BLISS index in venetoclax-resistant AML cells.

Venetoclax-resistant cell lines (MOLM13 FLT3-ITD ^+/- and OCI-AML3 FLT3-WT) were generated by exposing cells to successively increasing concentrations of venetoclax. Cells were routinely maintained in complete RPMI medium containing 1 μM venetoclax. 24 hours before testing the effect of the combination of midostaurin and S63845 on cell viability, cells were cultured in RPMI medium without venetoclax. Venetoclax-resistant cells and their parental counterparts were then seeded into 96-well plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma). Midostaurin and S63845 were diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Cells were treated for 24 h with nine 2-fold serial dilutions of each compound, either individually or in all possible staggered combinations. Cell growth was measured, and viability was measured using a CellTiter-Glo luminescence assay (Promega) according to the manufacturer's instructions. Briefly, cells were mixed gently by pipetting and 35 μl of cell suspension was transferred into white opaque 96-well plates. Thereafter, 80 μl of CellTiter-Glo reagent diluted 1:3 in PBS was added to each well, and the cells were incubated for 30 minutes in the dark on a plate shaker. Luminescence was recorded using a Tekan plate reader (Infinite m200 pro). Cell growth was expressed as % of cells treated with DMSO. Synergistic interactions were assessed using the BLISS independence model. BLISS index values for each combination dose >0 represent synergism, while BLISS index values <0 represent antagonism.

Fig. 14. Preliminary in vivo study: AML FLT3-ITD xenograft.

AML-PDX model.

For the AML-PDX model, female NSG mice (6 weeks old, The Jackson Laboratory) were irradiated

-

Claims (21)

200 cGy and AML-PDX (1x10 ⁶ cells/100 μl) was injected i.v. Peripheral blood was collected once a week by retroorbital sinus sampling and processed to alter hCD45 cells by flow cytometry to confirm that leukemia had developed. When leukemia inoculation reached 2-10%, mice were divided into treatment groups with vehicle, midostaurin (75 mg/kg, once daily by gavage), S63845 (40 mg/kg IV once weekly) or combinations. Peripheral blood was collected biweekly by retroorbital sinus sampling and processed to measure leukemic load (hCD45 cells) by flow cytometry. The experiments presented in Fig. 2-14 indicate that the combination of the Mcl-1 inhibitor Compound C and midostaurin is synergistic in FLT3-ITD mutated AML cells, including those cells that are resistant to the Bcl-2 inhibitor venetoclax and in primary AML cells. Increased Bim levels and increased mitochondrial priming in response to hBIM peptide following midostaurin exposure suggest that Bim may play a functional role in midostaurin/S63845-mediated lethality. Example 3. Analysis of cell viability and BLISS index for Ba/F3 FLT3-ITD and FLT3-D835Y cells. Mouse Ba/F3 FLT3-ITD and FLT3-D835Y cells (8x105/well) were seeded in 96-well plates in 100 μl of complete RPMI medium containing 10% FBS (Sigma) and 1x Pen/Strep (Sigma). Midostaurin and S63845 were diluted in complete RPMI medium and administered as a 4x concentrated solution prepared in 50 μl of medium. Control cells received 100 μl of medium containing DMSO (the volume of DMSO corresponded to the sum of the volumes of Midostaurin and S63845 stock solutions used to prepare 4x solutions). Cells were incubated with drugs administered alone or in combination for 24 hours. Cell viability was measured using a CellTiter-Glo luminescent assay (Promega) according to the manufacturer's instructions. Briefly, cells were mixed gently by pipetting and 35 μl of cell suspension was transferred into white opaque 96-well plates. Thereafter, 80 μl of CellTiter-Glo reagent diluted 1:3 in PBS was added to each well, and the cells were incubated for 30 minutes in the dark on a plate shaker. Luminescence was recorded using a Tekan plate reader (Infinite m200 pro). Combined drug effects were analyzed using the additivity index (AD) calculated using CalcuSyn software (BioSoft, Ferguson, MO, USA). PA <1, 1, and >1 indicated synergism, additive effect, and antagonism, respectively. Results. In fig. 15 and 16 show synergistic interactions between S63845 and midostaurin in murine Ba/F3 FLT3-ITD cells determined by drug synergy in the BLISS extended size independence model. Cells were treated with nine 2-fold serial dilutions of each compound, either individually or in all possible combinations in a staggered manner. Cell growth was assessed after 24 h (heat map, left panel). BLISS index values for each combination dose > 0 represent synergism, while BLISS index values < 0 represent antagonism. In fig. 17 and 18 show synergistic interactions between S63845 and midostaurin in murine Ba/F3 FLT3-D835Y cells determined by drug synergy in the BLISS extended size independence model. Cells were treated with nine 2-fold serial dilutions of each compound, either individually or in all possible staggered combinations. Cell growth was assessed after 24 h (heat map, left panel). BLISS index values for each combination dose >0 represent synergism, while BLISS index values <0 represent antagonism. The combination of S63845 and Midostaurin exhibits strong synergy in murine Ba/F3 cells expressing either FLT3-ITD or FLT3-D835Y point mutations within the tyrosine kinase domain (TKD) of FLT3. This latter observation suggests that the 863845/midostaurin combination can provide significant efficacy in FLT3-ITD mutants even in the presence of FLT3 TKD mutations, which has been clinically shown to confer resistance to FLT3 inhibitor treatment. CLAIM 1. Pharmaceutical combination of (a) midostaurin, or a pharmaceutically acceptable salt thereof, or a complex thereof, or a co-crystal thereof, or a solvate thereof, including a hydrate, and (b) an Mcl-1 inhibitor, which is Compound B: (2R)-2 -{[(5Sa)-5-{3-chloro-2-methyl-4[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d ]pyrimidin-4-yl]oxy}-3-(2-{[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, or compound C: (2R)-2-{[(5S _a )-5- - 16 044971 {3-chloro-2-methyl-4-[2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d] pyrimidin4-yl]oxy} -3-(2-{[1 -(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy} phenyl)propanoic acid, or their enantiomers, diastereoisomers, atropisomers, or their addition salts with a pharmaceutically acceptable acid or base, for simultaneous, sequential or separate use. 2. The pharmaceutical combination according to claim 1, wherein the Mcl-1 inhibitor of formula (I) is compound B: (2R)-2-{[(5S _α )-5-{3-chloro-2-methyl-4-[ 2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-( 2-methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof. 3. Pharmaceutical combination according to claim 1, wherein the Mcl-1 inhibitor of formula (I) is compound C: (2R)-2-{[(5S _α )-5-{3-chloro-2-methyl-4-[ 2-(4-methylpiperazin-1-yl)ethoxy]phenyl}-6-(5-fluorofuran-2-yl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{ [1-(2,2,2-trifluoroethyl)-1H-pyrazol-5-yl]methoxy}phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof. 4. The pharmaceutical combination according to claim 2, wherein Compound B is administered intravenously and midostaurin is administered orally. 5. Pharmaceutical combination according to one of claims 1 to 4, which further contains at least one additional anticancer agent, for example cytarabine and/or daunorubicin. 6. Pharmaceutical combination according to one of claims 1-5, in the form of a combination with a non-fixed dose. 7. Use of a pharmaceutical combination according to one of claims 1 to 6, for use in medicine. 8. Use of a pharmaceutical combination according to one of claims 1-6, for the treatment of malignant neoplasms. 9. Use according to claim 8, wherein the malignancy is acute myeloid leukemia. 10. Use according to claim 9, wherein the malignancy is acute myeloid leukemia present in patients carrying the FLT3 mutation. 11. Use according to claim 10, wherein the malignancy is acute myeloid leukemia present in patients carrying the FLT3-ITD mutation. 12. Use according to claim 10, wherein the malignancy is acute myeloid leukemia present in patients carrying the FLT3-TKD mutation. 13. The use of claim 12, wherein said FLT3-TKD mutation includes FLT3-D835Y or FLT3-F691 mutations within the tyrosine kinase domain (TKD) of FLT3. 14. Use according to one of claims 8-13, where the malignant neoplasm is resistant to previous therapy. 15. Use according to claim 14, wherein the malignancy is acute myeloid leukemia resistant to one or more compounds selected from venetoclax, decitabine, daunorubicin and cytarabine. 16. Use according to claim 15, wherein the malignancy is acute myeloid leukemia resistant to venetoclax. 17. Use according to claim 11, wherein said cancer is acute myeloid leukemia present in patients carrying the FLT3-ITD mutation, and said cancer is resistant to venetoclax. 18. Use according to one of claims 7 to 17, wherein midostaurin and compound B are provided in amounts that are synergistically effective for the treatment of malignancy. 19. A pharmaceutical composition containing the pharmaceutical combination according to one of claims 16, and at least one pharmaceutically acceptable carrier. 20. Use of a pharmaceutical combination according to one of claims 1 to 6 for the preparation of a medicinal product for the treatment of a malignant neoplasm. 21. Use according to claim 10, wherein the malignancy is acute myeloid leukemia present in patients carrying the FLT3-ITD mutation in the presence of the FLT3-TKD mutation. -