Patent Application
20100076013
The present invention relates to methods of treating hematological malignancies, including acute myeloid leukemia (AML), comprising the use of compounds that inhibit the binding of the Smac protein to IAPs (“IAP inhibitor”).
The present invention also relates to the use of IAP inhibitors for the preparation of a medicament to treat hematological malignancies, including AML.
AML is a hematologic malignancy characterized by a block in cellular differentiation and aberrant growth of myeloid precursor cells. Approximately 30% of AML patients, and a portion of acute lymphoblastic leukemia (ALL) patients, express a mutated form of the class III receptor tyrosine kinase, FLT3 (Fms-Like Tyrosine kinase-3; STK-1, human Stem Cell Tyrosine Kinase-1; or FLK-2, Fetal Liver Kinase-2). See Rosnet and Birnbaum (1993) and Stirewalt and Radich (2003). Constitutively activated FLT3 occurs most often as internal tandem duplications within the juxtamembrane domain [see Nakao et al. (1996)], and is observed in approximately 20-25% of AML patients, but in less than 5% of patients with myelodysplastic syndrome (MDS). See Nakao et al. (1996); Horiike et al. (1997); Kiyoi et al. (1998); Kondo et al. (1999); Kiyoi et al. (1999) and Rombouts et al. (2000). The transplantation of murine bone marrow cells infected with a retrovirus expressing a FLT3-ITD mutant has been shown to lead to the development of a rapidly lethal myeloproliferative disease in mice. See Kelly et al. (2002). Gain-of-function FLT3 occurs less often as point mutations in the activation loop (in approximately 7% of AML cases), and is often characterized by an asparagine (Asp) residue at position 835. See Yamamoto et al. (2001). Occurring less frequently are additional point mutations in the kinase domain, including N841I [see Jiang et al. (2004)] and Y842C [see Kindler et al. (2005)]. There is a need to develop small molecules for the treatment of acute leukemia patients.
It has been found that members of the IAP protein family play a role in mediating apoptosis and these proteins are a viable target in leukemia, as they have been found to be variably expressed in acute leukemias, and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival.
The present invention relates to a method of treating a warm-blooded animal, especially a human, having leukemia, especially AML, in particular, AML which is resistant to conventional chemotherapy, comprising administering to said animal a therapeutically effective amount of an IAP inhibitor; useful in AML treatment.
In another embodiment, the present invention relates to the use of IAP inhibitors in the preparation of a medicament for the treatment of hematological malignancies, including AML.
Mediators of apoptotic signaling represent an attractive target for therapeutic intervention. “Second mitochondria-derived activator of caspase” (“Smac”) mediates apoptosis occurring through the intrinsic apoptotic pathway [see Du et al. (2000)], and binds to and inhibits the IAP family of proteins. See Liu et al. (2000) and Wu et al. (2000). Smac is likely the functional equivalent of Drosophila Reaper, Hid and Grim [see Vucic et al. (1998); McCarthy and Dixit (1998) and Goyal et al. (2000)]; the mouse Smac ortholog is DIABLO. See Verhagen et al. (2000). Identified human IAPs (c-IAP-1, c-IAP-2, and X-chromosome-linked IAP, or XIAP) bind procaspase-9 and prevent its activation. See Deveraux et al. (1998). IAPs also directly bind and inhibit active caspases [see Deveraux et al. (1997); Roy et al. (1997) and Deveraux et al. (1998)]; the BIR (“baculovirus IAP repeat”) domain is responsible for the anti-apoptotic activity of IAPs. See Takahashi et al. (1998). Members of the IAP protein family play a role in mediating apoptosis.
Examples of IAP inhibitors for use in the present invention include A compound according to formula (I):
wherein
Compounds within the scope of formula (I) and the process for their manufacture are disclosed in U.S. 60/835,000, which is hereby incorporated into the present application by reference. The preferred compounds are selected from the group consisting of:
In another embodiment, the IAP inhibitor is a compound of formula III:
or pharmaceutically acceptable salts thereof, wherein
R1 is H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl or C3-C10 cycloalkyl, which R1 may be unsubstituted or substituted;
R2 is H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C10 cycloalkyl which R2 may be unsubstituted or substituted;
R3 is H, CF3, C2F5, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CH2—Z or R2 and R3 taken together with the nitrogen atom to which they are attached form a heterocyclic ring, which alkyl, alkenyl, alkynyl or het ring may be unsubstituted or substituted;
Z is H, OH, F, Cl, CH3, CH2Cl, CH2F or CH2OH;
R4 is C0-10 alkyl, C3-C10 cycloalkyl, wherein the C0-10 alkyl, or cycloalkyl group is unsubstituted or substituted;
A is het, which may be substituted or unsubstituted;
D is C1-C7 alkylene or C2-C9 alkenylene, C(O), O, NR7, S(O)r, C(O)—C1-C10 alkyl, O—C1-C10 alkyl, S(O)r—C1-C10 alkyl, C(O)C0-C10 arylalkyl OC0-C10 arylalkyl, or S(O)r C0-C10 arylalkyl, which alkyl and aryl groups may be unsubstituted or substituted;
r is 0, 1, or 2;
A1 is a substituted aryl or unsubstituted or substituted het which substituents on aryl and het are halo, lower alkoxy, NR5R6, CN, NO2 or SR5;
each Q is independently H, C1-C10 alkyl, C1-C10 alkoxy, aryl C1-C10 alkoxy, OH, O—C1-C10-alkyl, (CH2)0-6—C3-C7 cycloalkyl, aryl, aryl C1-C10 alkyl, O—(CH2)0-6 aryl, (CH2)1-6het, het, O—(CH2)1-6-het, —OR11, C(O)R11, —C(O)N(R11)(R12), N(R11)(R12), SR11, S(O)R11, S(O)2R11, S(O)2—N(R11)(R12), or NR11—S(O)2—(R12), wherein alkyl, cycloalkyl and aryl are unsubstituted or substituted;
n is 0, 1, 2 or 3, 4, 5, 6 or 7;
het is a 5-7 membered monocyclic heterocyclic ring containing 1-4 heteroring atoms selected from N, O and S or an 8-12 membered fused ring system that includes one 5-7 membered monocyclic heterocyclic ring containing 1, 2, or 3 heteroring atoms selected from N, O and S, which het is unsubstituted or substituted;
R11 and R12 are independently H, C1-C10 alkyl, (CH2)0-6—C3-C7cycloalkyl, (CH2)0-6—(CH)0-1(aryl)1-2, C(O)—C1-C10alkyl, —C(O)—(CH2)1-6—C3-C7cycloalkyl, —C(O)—O—(CH2)0-6-aryl, —C(O)—(CH2)0-6—O-fluorenyl, C(O)—NH—(CH2)0-6-aryl, C(O)—(CH2)0-6-aryl, C(O)—(CH2)1-6-het, —C(S)—C1-C10alkyl, —C(S)—(CH2)1-6—C3-C7cycloalkyl, —C(S)—O—(CH2)0-6-aryl, —C(S)—(CH2)0-6—O-fluorenyl, C(S)—NH—(CH2)0-6-aryl, —C(S)—(CH2)0-6-aryl, C(S)—(CH2)1-6-het, C(O)R11, C(O)NR11R12, C(O)OR11, S(O)nR11, S(O)mNR11R12, m=1 or 2, C(S)R11, C(S)NR11R12, C(S)OR11, wherein alkyl, cycloalkyl and aryl are unsubstituted or substituted; or R11 and R12 are a substituent that facilitates transport of the molecule across a cell membrane; or R11 and R12 together with the nitrogen atom form het;
wherein the alkyl substituents of R11 and R12 may be unsubstituted or substituted by one or more substituents selected from C1-C10alkyl, halogen, OH, O—C1-C6alkyl, —S—C1-C6alkyl, CF3 or NR11R12;
substituted cycloalkyl substituents of R11 and R12 are substituted by one or more substituents selected from a C2-C10 alkene; C1-C6alkyl; halogen; OH; O—C1-C6alkyl; S—C1-C6alkyl, CF3; or NR11R12 and
substituted het or substituted aryl of R11 and R12 are substituted by one or more substituents selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, nitro, CN O—C(O)—C1-C4alkyl and C(O)—O—C1-C4-alkyl;
R5, R6 and R7 are independently hydrogen, lower alkyl, aryl, aryl lower alkyl, cycloalkyl, or cycloalkyl lower alkyl, and
wherein the substituents on R1, R2, R3, R4, Q, and A and A1 groups are independently halo, hydroxy, lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower alkoxy, aryl, aryl lower alkyl, amino, amino lower alkyl, diloweralkylamino, lower alkanoyl, amino lower alkoxy, nitro, cyano, cyano lower alkyl, carboxy, lower carbalkoxy, lower alkanoyl, aryloyl, lower arylalkanoyl, carbamoyl, N-mono- or N,N-dilower alkyl carbamoyl, lower alkyl carbamic acid ester, amidino, guanidine, ureido, mercapto, sulfo, lower alkylthio, sulfoamino, sulfonamide, benzosulfonamide, sulfonate, sulfanyl lower alkyl, aryl sulfonamide, halogen substituted aryl sulfonate, lower alkylsulfinyl, arylsulfinyl; aryl-lower alkylsulfinyl, lower alkylarylsulfinyl, lower alkylsulfonyl, arylsulfonyl, aryl-lower alkylsulfonyl, lower aryl alkyl lower alkylarylsulfonyl, halogen-lower alkylmercapto, halogen-lower alkylsulfonyl, phosphono (—P(═O)(OH)2), hydroxy-lower alkoxy phosphoryl or di-lower alkoxyphosphoryl, (R9)NC(O)—NR10R13, lower alkyl carbamic acid ester or carbamates or —NR8R14, wherein R8 and R14 can be the same or different and are independently H or lower alkyl, or R8 and R14 together with the N atom form a 3- to 8-membered heterocyclic ring containing a nitrogen heteroring atoms and may optionally contain one or two additional heteroring atoms selected from nitrogen, oxygen and sulfur, which heterocyclic ring may be unsubstituted or substituted with lower alkyl, halo, lower alkenyl, lower alkynyl, hydroxy, lower alkoxy, nitro, amino, lower alkyl, amino, diloweralkyl amino, cyano, carboxy, lower carbalkoxy, formyl, lower alkanoyl, oxo, carbarmoyl, N-lower or N,N-dilower alkyl carbamoyl, mercapto, or lower alkylthio, and
R9, R10, and R13 are independently hydrogen, lower alkyl, halogen substituted lower alkyl, aryl, aryl lower alkyl, halogen substituted aryl, halogen substituted aryl lower alkyl.
Some compounds which fall within compounds of formula III include: (S)—N—((S)-1-Cyclohexyl-2-{(S)-2-[4-(4-fluoro-benzoyl)-thiazol-2-yl]-pyrrolidin-1-yl}-2-oxo-ethyl)-2-methylamino-propionamide; (S)—N—[(S)-Cyclohexyl-(ethyl-{(S)-1-[5-(4-fluoro-benzoyl)-pyridin-3-yl]-propyl}carbamoyl)-methyl]-2-methylamino-propionamide; and (S)—N—((S)-1-Cyclohexyl-2-{(S)-2-[5-(4-fluoro-phenoxy)-pyridin-3-yl]-pyrrolidin-1-yl}-2-oxo-ethyl)-2-methylamino-propionamide; and pharmaceutically acceptable salts thereof.
These compounds of formula III are disclosed in PCT/US2007/074790 and U.S. Ser. No. 60/835,000; both herein incorporated by reference in their entirety.
Examples of other IAP inhibitors includes compounds disclosed in WO 05/097791 published on Oct. 20, 2005. A preferred compounds within the scope of formula (I) is N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide.
Additional IAP inhibitors include compounds disclosed in WO 04/005284, PCT/US2006/013984 and PCT/US2006/021850.
Other IAP inhibitor compounds for use in the present invention include those disclosed in WO 06/069063, WO 05/069888, US2006/0014700, WO 04/007529, US2006/0025347, WO 06/010118, WO 05/069894, WO 06/017295, WO 04/007529, and WO 05/094818.
In each case where citations of patent applications are given above, the subject matter relating to the compounds is hereby incorporated into the present application by reference. Comprised are likewise the pharmaceutically acceptable salts thereof, the corresponding racemates, diastereoisomers, enantiomers, tautomers, as well as the corresponding crystal modifications of above disclosed compounds where present, e.g., solvates, hydrates and polymorphs, which are disclosed therein. The compounds used as active ingredients in the combinations of the invention can be prepared and administered as described in the cited documents, respectively. Also within the scope of this invention is the combination of more than two separate active ingredients as set forth above, i.e., a pharmaceutical combination within the scope of this invention could include three active ingredients or more.
The terms “treatment” or “therapy” refer to the prophylactic or preferably therapeutic including, but not limited to, palliative, curing, symptom-alleviating, symptom-reducing, regulating and/or inhibiting treatment of said diseases, especially of the diseases mentioned below.
The term “AML”, as used herein, relates to an uncontrolled, quickly progressing growth of myeloid cells, e.g., granulocytes, as well as erythroid and megakaryotic cells and progenitors. In patients with AML the immature myeloid, erythroid or megakaryotic cells severely outnumber erythrocytes (red blood cells) leading to fatigue and bleeding, and also to increased susceptibility to infection. In children, as well as in adults, AML has a poor prognosis despite the use of aggressive chemotherapeutic protocols. Overall survival rates are 40-60%. Autologous bone marrow transplant preceded by myeloablative chemotherapy does not change the survival but an allogeneic bone marrow transplant preceded by aggressive chemotherapy might increase the survival rates up to 70%. Unfortunately, the availability of a matched sibling donor is limited. Therefore, new therapeutic strategies in AML treatment are necessary.
A warm-blooded animal (or patient) is preferably a mammal, especially a human.
The precise dosage of an IAP inhibitor compound to be employed depends upon several factors including the host, the nature and the severity of the condition being treated, the mode of administration. The IAP inhibitor compound can be administered by any route including orally, parenterally, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally. Preferably, the IAP inhibitor compound is administered orally, preferably at a daily dosage of 1-300 mg/kg body weight or, for most larger primates, a daily dosage of 50-5,000, preferably 500-3,000 mg. A preferred oral daily dosage is 1-75 mg/kg body weight or, for most larger primates, a daily dosage of 10-2,000 mg, administered as a single dose or divided into multiple doses, such as twice daily dosing.
Usually, a small dose is administered initially and the dosage is gradually increased until the optimal dosage for the host under treatment is determined. The upper limit of dosage is that imposed by side effects and can be determined by trial for the host being treated.
Dosage regimens must be titrated to the particular indication, the age, weight and general physical condition of the patient, and the response desired but generally doses will be from about 10 mg/day to about 500 mg/day as needed in single or multiple daily administration.
IAP inhibitor compounds may be combined with one or more pharmaceutically acceptable carriers and, optionally, one or more other conventional pharmaceutical adjuvants and administered enterally, e.g., orally, in the form of tablets, capsules, caplets, etc. or parenterally, e.g., intraperitoneally or intravenously, in the form of sterile injectable solutions or suspensions. The enteral and parenteral compositions may be prepared by conventional means.
N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide inhibits PKC412-sensitive and resistant mutant FLT3-expressing cells in vitro. N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide induces apoptosis, as measured via annexin-pi staining and caspase assays, was modestly observed with effective concentrations in the micromolar range.
N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide is effective against mutant FLT3 at doses that are physiologically achievable and well-tolerated in vivo.
The IL-3-dependent murine hematopoietic cell line Ba/F3 are transduced with either FLT3-ITD or FLT3-D835Y-containing MSCV retroviruses harboring a neomycin selectable marker, and selected for resistance to neomycin. See Kelly et al. (2002). FLT3-ITD transduced cells are selected for growth in G418 (1 mg/mL). PKC412-resistant Ba/F3 cell lines, which express FLT3-ITD harboring a mutation in the ATP-binding pocket (F691L, A627T, G697R, N676D), are developed as described previously. See Cools et al. (2004). The human AML-derived, FLT3-ITD-expressing cell line, MV4; 11 [see Quentmeier et al. (2003)], is provided by Dr. Scott Armstrong, Dana Farber Cancer Institute, Boston, Mass. The human AML-derived, FLT3-ITD-expressing cell line, MOLM-13, is modified to express luciferase and provided as MOLM13-luc+ by Dr. Andrew Kung, Dana Farber Cancer Institute, Boston, Mass. All cell lines are cultured with 5% CO2 at 37° C., at a concentration of 2×105 to 5×105 in RPMI (Mediatech, Inc., Herndon, Va.) with 10% fetal calf serum and supplemented with 1% glutamine. Parental Ba/F3 cells expressing wild-type FLT3 are similarly cultured with 15% WEHI-conditioned medium as a source of IL-3. All transfected cell lines are cultured in media supplemented with 1 mg/mL G418.
N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide is synthesized by Novartis Pharma AG, Basel, Switzerland, and dissolved in DMSO to make 10 mM stock solutions. Serial dilutions are then made, also in DMSO, to obtain final dilutions for cellular assays.
The trypan blue exclusion assay has been previously described [see Weisberg et al. (2002)], and is used to determine proliferation of cells cultured in the presence and absence of LBW242. Cell viability is reported as percentage of control (untreated) cells. Error bars represent the standard error of the mean for each data point. Apoptosis of drug-treated cells is measured using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, Ind.), as previously described. See Weisberg et al. (2002).
N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide displays activity at relatively high doses (≧1 μM) against the PKC412-sensitive lines FLT3-ITD-Ba/F3 and D835Y-Ba/F3 cells, as well as the PKC412-resistant line G697R-Ba/F3 in culture, refer to
There are no inhibitory effects of N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide on cell growth of wild-type FLT3-expressing Ba/F3 cells at concentrations ≦1 μM; however, concentrations of N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide >1 μM led to death of these cells (
For the FLT3-ITD-Ba/F3 and G697R-Ba/F3 lines, induction of apoptosis and caspase activity following 2 days and 3 days, respectively, of culturing in the presence of 1 μM N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide are found. Mutant FLT3-expressing cells are treated for 2 days in parallel with either 1 μM N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide in the presence and absence of WEHI (used as a source of IL-3). In contrast to PKC412-treated cells, which are fully rescued from the cytotoxic effects of PKC412 by WEHI, supplementation of culture media with WEHI did not rescue N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide-treated cells, showing that N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide, consistent with its proposed mechanisms of inhibition of IAP, does not selectively inhibit mutant FLT3, but interferes with viability.
To directly assess the in vivo anti-tumor efficacy of N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide, a mouse model of acute leukemia in which tumor burden is quantified by non-invasive imaging of luminescent tumor cells (
Mice are given vehicle alone, N-[1-cyclohexyl-2-oxo-2-(6-phenethyl-octahydro-pyrrolo[2,3-c]pyridin-1-yl-ethyl]-2-methylamino-propionamide (50 mg/kg) (
The EC50s were determined using CellTiter-Glo (Promega), a bioluminescent, cell viability assay measuring ATP levels in viable cells. Cells were plated in assay plates and incubated with a range of compound concentrations for 72 hours. Cells were lysed and ATP levels were determined using CellTiter-Glo reagent on a luminometer according to manufacturer's instructions. EC50 refers to the concentration of compound that inhibited 50% of cell growth.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/85486 | 11/26/2007 | WO | 00 | 5/27/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60867450 | Nov 2006 | US |
