Patent Grant
10351554
This application features pyrazol-3-one compounds that activate a pro-apoptotic function of BAX. Also featured are methods of using such compounds, e.g., for the treatment or prevention of diseases, disorders, and conditions associated with deregulated apoptosis of cells (e.g., diseased or damaged cells; e.g., insufficient apoptosis of diseased or damaged cells or reduced apoptosis of diseased or damaged cells). Examples of such diseases, disorders, and conditions include, but are not limited to, those associated with blockade(s) of cell death pathways (e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g., hyperproliferative diseases, such as cancer.
BCL-2 family proteins are key regulators of the mitochondrial apoptotic pathway in health and disease. The BCL-2 family includes both pro-apoptotic (e.g., BAX) and anti-apoptotic proteins that form a complex protein interaction network of checks and balances that dictate cell fate (see, e.g., Danial, N. N. & Korsmeyer, S. J. Cell death: critical control points. Cell 116, 205-19 (2004)).
The α-helical BCL-2 homology 3 (BH3) domains of pro-apoptotic members (e.g., BAX) function as death ligands. Pro-apoptotic member BAX is an executioner protein of the BCL-2 family that, when activated, undergoes a structural transformation, which converts it from an inactive cytosolic monomer into a lethal mitochondrial pore (see Gavathiotis, E., Reyna, D. E., Davis, M. L., Bird, G. H. & Walensky, L. D. BH3-triggered structural reorganization drives the activation of pro-apoptotic BAX. Mol Cell 40, 481-92 (2010)).
Oligomerization of BAX (and its close homologue BAK) within the mitochondrial outer membrane enables the release of apoptogenic factors such as cytochrome c and smac/diablo that turn on caspases, the enzymatic effectors of apoptosis (see Liu, X., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147-57 (1996); Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479-89 (1997); Du, C., Fang, M., Li, Y., Li, L. & Wang, X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102, 33-42 (2000); Wei, M. C. et al. Preapeptetie Pro-apoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-30 (2001)). The explicit mechanism by which BAX is triggered and how select pro-apoptotic BCL-2 proteins directly engage and activate BAX have been key questions in the apoptosis field (see, e.g., Youle, R. J. & Strasser, A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47-59 (2008)).
The α-helical BCL-2 homology 3 (BH3) domains of activated pro-apoptotic members (e.g., BAX) can, however, be intercepted and sequestered by structurally-defined surface grooves within the anti-apoptotic members (see, e.g., Sattler, M. et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 275, 983-6 (1997)). The relative levels of death-activating (pro-apoptotic) BH3 domains and anti-apoptotic BH3-binding pockets dictate the cellular response to stress. Cancer cells hijack the survival circuitry of the BCL-2 family pathway, exploiting pathologic overexpression of anti-apoptotic proteins to stymie physiologic and pharmacologic pro-apoptotic stimuli. By overexpressing these anti-apoptotic proteins, cancer cells maintain a survival advantage in the face of pro-apoptotic stimuli. Thus, the over-expression of anti-apoptotic members is believed to contribute to cancer pathogenesis.
Whereas the mainstay of developmental BCL-2 family therapeutics has focused on the loss-of-function strategy of inhibiting anti-apoptotic proteins, direct activation of BAX by select pro-apoptotic BCL-2 members that only contain a conserved BH3 domain (“BH3-only” proteins) has also emerged as a physiologically relevant mechanism for inducing mitochondrial apoptosis during development and homeostasis (see Ren, D. et al. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 330, 1390-3 (2010)).
This application features pyrazol-3-one compounds that activate a pro-apoptotic function of BAX, making them therapeutically useful for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diseases, disorders, and conditions associated with deregulated apoptosis of cells (e.g., diseased or damaged cells; e.g., insufficient apoptosis of diseased or damaged cells; or lack of apoptosis of diseased or damaged cells). Examples of such diseases, disorders, and conditions include (but are not limited to) those associated with blockade(s) of cell death pathways (e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g., hyperproliferative diseases, such as cancer (e.g., leukemia, e.g., acute lymphoblastic leukemia (“ALL”) or acute myelogenous leukemia (“AML”); e.g., chronic lymphoblastic leukemia (“CLL”) or chronic myelogenous leukemia (“CML”)). While not wishing to be bound by theory, it is believed that the compounds described herein induce and increase apoptosis in target cells (e.g., pathogenic cells including, but not limited to, cancer cells), thereby suppressing tumor growth and/or proliferation. It is further believed that increasing apoptosis in such target cells reestablishes the normal apoptotic control that, during homeostasis, is associated with a regulated balance between pro- and anti-apoptotic protein functions.
[A] In some embodiments, the compounds described herein directly activate BAX by direct binding to BAX.
In some embodiments, the compounds described herein selectively bind to and activate BAX. For example, the compounds described herein selectively bind to and activate BAX in the presence of one (or more) different BCL-2 proteins, e.g., in the presence of one (or more) other pro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the presence of one (or more) anti-apoptotic BCL-2 proteins.
In some embodiments, the compounds described herein directly activate BAX by direct binding to BAX; and selectively bind to and activate BAX, e.g. selectively bind to and activate BAX in the presence of one (or more) different BCL-2 proteins, e.g., in the presence of one (or more) other pro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the presence of one (or more) anti-apoptotic BCL-2 proteins.
It has been discovered that BAX contains a geographically distinct BH3 binding groove, which has been shown to mediate the direct activation of BAX. Specifically, structural analysis of a BIM BH3 death domain in complex with pro-apoptotic BAX uncovered a BH3 interaction site that, when engaged, results in the direct activation of BAX (see Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076-81 (2008)). A BIM BH3 α-helix, structurally reinforced by hydrocarbon stapling, engages BAX at the opposite side of the protein from the canonical BH3-binding groove of anti-apoptotic proteins (see Gavathiotis, E. et al. BAX activation is initiated at a novel interaction site. Nature 455, 1076-81 (2008)). See
In some embodiments, the compounds described herein directly activate BAX by binding to BAX at the BAX trigger site.
In some embodiments, the compounds described herein selectively activate BAX by binding to BAX at the BAX trigger site; e.g., selectively activate BAX in the presence of one (or more) different BCL-2 proteins, e.g., in the presence of one (or more) other pro-apoptotic BCL-2 proteins (e.g., BAK) and/or in the presence of one (or more) anti-apoptotic BCL-2 proteins.
[C] In some embodiments, the compounds described herein induce or activate BAX-dependent or mediated apoptosis (cell death).
In some embodiments, the methods described herein can include in vitro methods, e.g., contacting a sample containing BAX (e.g., a cell or tissue containing BAX) with a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
In some embodiments, the methods described herein can include administering a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)) to a subject (e.g., a subject in need thereof, e.g., a mammal, such as a human).
[E] Accordingly, in one aspect, methods for activating (e.g., directly, selectively, directly and selectively as defined anywhere herein) BAX and/or inducing or activating BAX-dependent apoptosis are featured, which include contacting BAX with a compound of formula (I) or a pharmaceutically acceptable salt thereof:
wherein:
A is N or CH;
X is heteroaryl, which contains 5 ring atoms, wherein from 1-2 of the ring atoms is/are independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein:
or
X is phenyl optionally substituted with from 1-5 Ra;
or
X is heteroaryl, which contains from 8-10 ring atoms, wherein from 1-4 of the ring atoms is/are independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein:
Y is:
(i) C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb; or
(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rb; or
(iii) C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 thioalkoxy, C1-C6 haloalkoxy, or C1-C6 halothioalkoxy, each of which is optionally substituted with —OH, —NH2, or —SH;
R1 is:
(i) C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Re; or
(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc; or
(iii) —C(O)—(C6-C10 aryl or heteroaryl, which contains from 5-10 ring atoms as defined in R1 definition (i) and (ii), respectively, above); or
(iv) hydrogen;
each of R2 and Ra is, independently, selected from any one of the substituents delineated collectively in (a), (b), (c), (d), and (e) below:
(a) C1-C8 alkyl or C1-C8 haloalkyl, each of which is optionally substituted with from 1-2 Rd;
(b) phenyl that is optionally substituted with from 1-4 Re;
(c) heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re;
(d) C3-C8 cycloalkyl or C3-C8 cycloalkenyl, each of which is optionally substituted with from 1-4 independently selected C1-C4 alkyl groups; or
(e) —NHC(O)(C1-C6 alkyl), —C(O)(C1-C6 alkyl); or —C(O)O(C1-C6 alkyl);
Rb, at each occurrence, is independently selected from any one the substituents delineated collectively in (aa), (bb) and (cc) below:
(aa) C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, C1-C6 haloalkyl, —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, or —NHC(O)(C1-C6 alkyl), each of which is optionally substituted with —OH, —NH2, azido (—N3), or —SH;
(bb) halo; —OH; —CN; nitro; —NH2; azido; C2-C4 alkenyl; C2-C4 alkynyl; —C(O)H; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); —OC(O)(C1-C6 alkyl); —SO2(C1-C6 alkyl); —SO2(C1-C6 haloalkyl); —C(O)NH2; —C(O)NH(C1-C6 alkyl); C(O)N(C1-C6 alkyl)2; —SO2(C1-C6 alkyl); —SO2NH2; —SO2NH(C1-C6 alkyl); —SO2N(C1-C6 alkyl)2; —NHCO(C1-C6 alkyl), or —NHSO2(C1-C6 alkyl); and
(cc) C3-C6 cycloalkyl, C3-C6 cycloalkoxy, or heterocyclyl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S; and each of said ring systems is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups;
each occurrence of Rc and Re is, independently, selected from any one the substituents delineated collectively in (aaa), (bbb), (ccc), and (ddd) below:
(aaa) C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, C1-C6 haloalkyl, —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, or —NHC(O)(C1-C6 alkyl), each of which is optionally substituted with —OH, —NH2, or —SH; (and optionally benzyloxy);
(bbb) halo; —OH; —CN; nitro; —NH2; azido; C2-C4 alkenyl; C2-C4 alkynyl; —C(O)H; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); —OC(O)(C1-C6 alkyl); —SO2(C1-C6 alkyl); —SO2(C1-C6 haloalkyl); —C(O)NH2; —C(O)NH(C1-C6 alkyl); C(O)N(C1-C6 alkyl)2; —SO2(C1-C6 alkyl); —SO2NH2; —SO2NH(C1-C6 alkyl); —SO2N(C1-C6 alkyl)2; —NHCO(C1-C6 alkyl), —NHSO2(C1-C6 alkyl); —C(O)O—(CH2)1-3(e.g., 1)—C(O)-(phenyl optionally substituted as defined in (ddd) below (e.g., —C(O)O—CH2—C(O)-(phenyl);
(ccc) L-C3-C8 cycloalkyl, C3-C6 cycloalkoxy, or L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of said ring systems is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups; and wherein L is a bond or C1-C6 alkylene; and
(ddd) phenyl, —O-(phenyl), or heteroaryl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heteroaryl is independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein each of said phenyl and heteroaryl is optionally substituted with from 1-3 substituents independently selected from halo; hydroxyl; cyano; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); nitro; —NH2; —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, —NHC(O)(C1-C6 alkyl), C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, and C1-C6 haloalkyl, wherein said alkyl or alkyl portion is optionally substituted with —OH, —NH2, or —SH; and
Rd at each occurrence is, independently, selected from hydroxyl, C1-C6 alkoxy; C1-C6 thioalkoxy; C1-C6 haloalkoxy; C1-C6 thiohaloalkoxy; —NH2; —NH(C1-C6 alkyl); N(C1-C6 alkyl)2; —NHC(O)(C1-C6 alkyl); cyano; —C(O)H; —C(O)(C1-C6 alkyl); —C(O)(C1-C6 haloalkyl); C(O)OH; —C(O)O(C1-C6 alkyl); —C(O)NH2; —C(O)NH(C1-C6 alkyl); C(O)N(C1-C6 alkyl)2; —SO2(C1-C6 alkyl); —SO2NH2; —SO2NH(C1-C6 alkyl); and —SO2N(C1-C6 alkyl)2.
One or more of the following can apply.
In some embodiments, X is not phenyl optionally substituted with from 1-5 Ra.
In some embodiments, R1 is not substituted directly or indirectly with one or more hydroxyl (—OH) groups.
In some embodiments, X is not phenyl optionally substituted with from 1-5 Ra; and R1 is not substituted directly or indirectly with one or more hydroxyl (—OH) groups.
In some embodiments, when R1 is 2-methoxyphenyl, and R2 is C1-C8 alkyl (e.g., CH3), then Y cannot be substituted phenyl, e.g., monosubstituted phenyl, e.g., phenyl monosubstituted at the para position, e.g., 4-chlorophenyl).
In some embodiments, when R1 is 2-carboxyphenyl, and R2 is C1-C8 alkyl (e.g., CH3), then Y cannot be substituted phenyl, e.g., monosubstituted phenyl, e.g., phenyl monosubstituted at the para position, e.g., 4-methoxyphenyl).
In some embodiments, R1 is other than 3-nitro-4-chlorophenyl. In certain embodiments, R1 is other than 3-nitro-4-chlorophenyl when R2 and Y are both unsubstituted phenyl.
In some embodiments, the compound is other than the compound sometimes referred to herein as “BAM7.”
In another aspect, compounds having formula (I-A), or a pharmaceutically acceptable salt thereof, are featured:
In some embodiments of formula (I-A):
X′ is S;
X′″ is unsubstituted phenyl,
X″ is H or C1-C4 alkyl;
R2 is:
R12 is:
each of R13 and R14 is H; and
each occurrence of Re is, independently, halo; cyano; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); nitro; —NH2; —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, —NHC(O)(C1-C6 alkyl), C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, C3-C6 cycloalkyl; and C1-C6 haloalkyl.
One or more of the following can apply.
In certain embodiments, it is provided that R12 cannot be —C(O)OH when R2 is unsubstituted phenyl.
In certain embodiments, it is provided that R12 cannot be —OCH2CH3 when R2 is unsubstituted phenyl.
In certain embodiments, it is provided that R12 cannot be —OCH2CH3 when R2 is CH3.
In other embodiments of formula (I-A):
X′ is NH;
X′″ is unsubstituted phenyl,
X″ is H or C1-C4 alkyl;
R2 is:
R12 is:
each of R13 and R14 is H; and
each occurrence of Re is, independently, halo; cyano; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); nitro; —NH2; —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, —NHC(O)(C1-C6 alkyl), C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, C3-C6 cycloalkyl; and C1-C6 haloalkyl.
[F] In one aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diseases, disorders, and conditions associated with deregulated apoptosis of cells (e.g., diseased or damaged cells; e.g., insufficient apoptosis of diseased or damaged cells; or the lack of apoptosis of diseased or damaged cells) in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
[G] In another aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diseases, disorders, and conditions associated with blockade(s) of cell death pathways (e.g., over-expression of anti-apoptotic proteins BCL-2 proteins) in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
[H] In a further aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) a hyperproliferative disease in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
In an aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) a hyperproliferative disease in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
[I] In still another aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) cancer (e.g., leukemia, e.g., ALL or AML; e.g., CLL or CML) in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
In an aspect, methods for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) cancer (e.g., leukemia, e.g., ALL or AML) in a subject in need thereof are featured. The methods include administering to the subject (e.g., an effective amount of) a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)).
[J] In yet another aspect, methods of modulating (e.g., increasing) apoptosis in vitro or in vivo are featured. Also featured are methods of modulating (e.g., decreasing) cell division in vitro or in vivo are featured. The methods can include contacting a sample containing BAX (e.g., a cell or tissue containing BAX) with a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I)); or administering a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)) to a subject (e.g., a subject in need thereof, e.g., a mammal, such as a human).
[K] In some embodiments, the methods described above and throughout this disclosure can include one or more of the following features.
The cancer can include carcinomas (originating in the outer layer of cells of the skin and internal membranes, e.g., breasts, lungs, intestines, skin, prostate, etc.); sarcomas (arising from connective tissue such as bone, muscle, cartilage and blood vessels), and hematologic malignancies (e.g., lymphomas and leukemias, which arise in the blood or blood-forming organs such as the spleen, lymph nodes and bone marrow, e.g., leukemias, e.g., ALL or AML; e.g., CLL or CML). Cancer cells can include, for example, tumor cells, neoplastic cells, malignant cells, metastatic cells, and hyperplastic cells.
Non-limiting examples of cancers include breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.
In some embodiments, the patient has not been treated with an agent that causes a thrombocytopenia-associated condition. In some embodiments the patient is not suffering from and/or is not a risk from developing a thrombocytopenia-associated condition.
In some embodiments, the methods further include administering one or more additional therapeutic agents (e.g., anticancer/chemotherapeutic agents) and/or techniques (e.g., radiation therapies, surgical interventions, and the like) to a subject or in vitro cells, tissues, and organs. In certain embodiments, the methods further include administering one or more additional therapeutic agents such as: agents that induce apoptosis; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics (BH3 mimetics); agents that bind to and inhibit anti-apoptotic proteins (e.g., agents that inhibit anti-apoptotic BCL-2 proteins); alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins, etc.), toxins, radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteasome inhibitors: NF kappa .beta. modulators; anti-CDK compounds; HDAC inhibitors; and the like.
In certain embodiments, the methods further include administering one or more additional therapeutic agents that bind to and inhibit anti-apoptotic proteins (e.g., agents that inhibit anti-apoptotic BCL-2 proteins), such as ABT-263, obatoclax, gossypol derivatives, IAP inhibitors, and stapled peptides that target anti-apoptotic proteins (MCL-1 SAHB (see, Stewart et al, Nature Chem Biol, 2010), BID SAHB (Walensky et al Science 2004), BAD SAHB (Danial et al Nature Medicine 2008), BIM SAHB (Gavathiotis et al Nature 2008), etc.).
In certain embodiments, the methods further include administering one or more additional therapeutic agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); kinase inhibitors (e.g., Epidermal Growth Factor Receptor (EGFR) kinase inhibitor, Vascular Growth Factor Receptor (VGFR) kinase inhibitor, Fibroblast Growth Factor Receptor (FGFR) kinase inhibitor, Platelet-derived Growth Factor Receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors such as GLEEVEC); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethimide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; and staurosporine, and the like.
In some embodiments, the subject can be a subject in need thereof (e.g., a subject identified as being in need of such treatment, such as a subject having, or at risk of having, one or more of the diseases or conditions described herein). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). In some embodiments, the subject can be a mammal. In certain embodiments, the subject can be a human.
In certain embodiments, the subject has not previously undergone chemotherapy. In certain embodiments, the subject is not suffering from, or at risk of, thrombocytopenia, such as thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transplantation as treatment for cancer or lymphoma.
[L] In another aspect, methods of screening for (thereby identifying) compounds that activate BAX are featured. Said methods generally include screening a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)) and a test compound. The methods include providing: a compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I)); a test compound; a first group of cells; and contacting the first group of cells with the formula (I) compound and the test compound; and observing the effects of contacting the first group of cells with the formula (I) compound and the test compound. In some of these embodiments, the methods further provide the additional step of comparing the effects observed in the first cells against a second group of the cells contacted with the formula (I) compound alone, or with the test compound alone. Effects that may be observed include, but are not limited to, those described in the Examples section.
[M] In one aspect, pharmaceutical compositions are featured, which include a compound of formula (I), or a pharmaceutically acceptable salt thereof (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)) and a pharmaceutically acceptable carrier. In some embodiments, the compositions can include one or more additional therapeutic agents (e.g., anticancer/chemotherapeutic agents) as defined anywhere herein.
In another aspect, methods of making the pharmaceutical compositions described herein are featured. In some embodiments, the methods include taking any one or more of the compounds of formula (I) (e.g., including any subgenera or specific compound thereof of formula (I), e.g., formula (I-A)) or a salt (e.g., a pharmaceutically acceptable salt) thereof as defined anywhere herein, and mixing said compound(s) with one or more pharmaceutically acceptable carriers.
[N] In one aspect, methods of making the compounds described herein are featured. In some embodiments, the methods include taking any one of the intermediate compounds described herein and reacting it with one or more chemical reagents in one or more steps to produce a compound of formula (I) (and/or a compound of any of the other formulae described herein) or a salt (e.g., a pharmaceutically acceptable salt) thereof as defined anywhere herein.
[O] In one aspect, the compounds of formula (I) themselves (e.g., including any subgenera or specific compound thereof of formula (I)) or a salt (e.g., a pharmaceutically acceptable salt) thereof as defined anywhere herein are featured. In another aspect, any of the formula (I) compounds specifically described herein are featured.
In some embodiments, the compounds of formula (I) are other than those described in the following printed publications:
In some embodiments, the foregoing exclusions can be combined with any one or more of the exclusions described in section [E] above.
[P] Embodiments can include any one or more of the following features.
X can contain 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S; e.g., N and S or N and NH.
One of the two ring atoms can be independently selected from N, NH, and N(C1-C3 alkyl) (e.g., N), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S (e.g., S or NH, e.g., S).
X can have formula X-1:
in which:
X′ is NH, O, or S; and
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra.
X′ can be S.
X′ can be NH.
X′″ can be Y.
X″ can be H or Ra.
X″ can be H.
X″ can be Ra. In embodiments, Ra can be C1-C8 alkyl (e.g., CH3). In other embodiments, Ra can be phenyl that is optionally substituted with from 1-4 Re; or C3-C8 cycloalkyl which is optionally substituted with from 1-4 independently selected C1-C4 alkyl groups.
Y can be C6-C10 aryl (e.g., phenyl), which is optionally substituted with from 1-5 independently selected Rb. In embodiments, Y can be unsubstituted phenyl.
R1 can be C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rc.
R1 can be phenyl, which is substituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently selected Rc.
Each occurrence of R can be, independently, selected from any one the substituents delineated collectively in (aaa), (bbb), (ccc), and (ddd) below:
(aaa) C1-C6 alkoxy, C1-C6 alkyl, C1-C6 haloalkyl, —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, or —NHC(O)(C1-C6 alkyl), each of which is optionally substituted with —OH, —NH2, or —SH;
(bbb) C(O)OH; —C(O)O(C1-C6 alkyl); —OC(O)(C1-C6 alkyl); —SO2NH2; —SO2NH(C1-C6 alkyl); or —SO2N(C1-C6 alkyl)2; or —C(O)O—(CH2)1-3(e.g., 1)—C(O)-(phenyl optionally substituted as defined in (ddd) below (e.g., —C(O)O—CH2—C(O)-(phenyl);
(ccc) C3-C6 cycloalkoxy or L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups; and wherein L is a bond or C1-C6 alkylene; and
(ddd) phenyl or heteroaryl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heteroaryl is independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein each of said phenyl and heteroaryl is optionally substituted with from 1-3 substituents independently selected from halo; hydroxyl; cyano; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); nitro; —NH2; —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, —NHC(O)(C1-C6 alkyl), C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, and C1-C6 haloalkyl, wherein said alkyl or alkyl portion is optionally substituted with —OH, —NH2, or —SH.
In embodiments, each occurrence of Rc is, independently, selected from:
In embodiments, Rc can be C1-C6 alkoxy (e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that is substituted with —NH2.
R1 can have formula A:
The following definitions apply to any formula described herein that contains formula (A).
One or two of R12, R13, and R14 is(are) an independently selected Rc, and the other(s) is(are) hydrogen.
R12 can be Rc (as defined anywhere herein).
R3 can be H.
R14 can be H.
R14 can be Rc.
R12 can be Rc (as defined anywhere herein), and each of R13 and R14 can be H.
R14 can be Rc (as defined anywhere herein), and each of R12 and R13 can be H.
In embodiments, Rc can be C1-C6 alkoxy (e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that is substituted with —NH2.
Rc can be —C(O)OH.
Rc can be L-heterocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups; and wherein L is a bond or C1-C6 alkylene (e.g., a bond); e.g., Rc is morpholino or piperazinyl. In embodiments, L can be a bond or CH2 (e.g., L can be a bond).
R13 can be H, and each of R12 and R14 can be Rc (each independently as defined anywhere herein).
One of R12 and R14 can be C1-C6 alkoxy (e.g., ethoxy), and the other of R12 and R14 can be independently selected from:
R1 can be heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc.
R1 can be heteroaryl, which contains from 5-6 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1) independently selected Rc. For example, R1 can be thiazolyl.
R1 can be heteroaryl, which contains from 8-10 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1) independently selected Rc. For example, R1 can be indazolyl.
R2 can be C1-C8 alkyl. For example, R2 can be CH3.
R2 can be phenyl that is optionally substituted with from 1-4 Re. In embodiments, R2 can be unsubstituted phenyl.
R2 can be C3-C8 cycloalkyl which is optionally substituted with from 1-4 independently selected C1-C4 alkyl groups.
R2 is heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re. For example, R2 can be furanyl, thienyl, or thiazolyl.
A can be N.
In some embodiments:
A is N;
X contains 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S, and one of the ring atoms is independently selected from N, NH, and N(C1-C3 alkyl), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and
R1 is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rc (e.g., R1 can be phenyl, which is substituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently selected Rc).
In certain embodiments, the compound can have formula I-A:
wherein:
X′ is NH, O, or S;
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra;
one or two of R12, R13, and R14 is(are) an independently selected Rc, and the other(s) is(are) hydrogen; and
R2 can be as defined anywhere herein.
X′ can be S or NH (e.g., S), X′″ is Y, and X″ is H or Ra (e.g., X″ can be H or C1-C3 alkyl, e.g., CH3).
Y can be C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb (e.g., Y can be unsubstituted phenyl).
R12 can be Rc, and each of R13 and R14 is H; or R14 is Rc, and each of R12 and R13 is H.
Each occurrence of R is, independently, selected from:
Rc can be C1-C6 alkoxy (e.g., ethoxy or iso-propoxy); or Rc can be —COOH; or Rc can be morpholino or piperazinyl.
Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy or iso-propoxy) that is optionally substituted with —NH2; or Rc can be —COOH; or Rc can be an optionally substituted morpholino or optionally substituted piperazinyl ring.
Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy or iso-propoxy) that is optionally substituted with —NH2.
R2 can be C1-C8 alkyl (e.g., CH3).
R2 can be phenyl or heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re (e.g., R2 is heteroaryl as defined above, e.g., thienyl, furanyl, or thiazolyl).
X″ can be H or CH3.
R2 can be C1-C4 alkyl, such as CH3. R2 can be unsubstituted phenyl. R2 can be heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re, such as thienyl, furanyl, or thiazolyl.
R12 can be —C(O)OH. R12 can be C2-C6 alkoxy that is optionally substituted with —NH2, such as —OCH2CH3 or —OCH2CH2NH2. R12 can be heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups, such as an optionally substituted piperazinyl ring.
X″ can be H or CH3; and R2 can be C1-C4 alkyl, such as CH3; or R2 can be unsubstituted phenyl; or R2 can be heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re, such as thienyl, furanyl, or thiazolyl.
X″ can be H or CH3; and
R2 can be C1-C4 alkyl, such as CH3; or R2 can be unsubstituted phenyl; or R2 can be heteroaryl containing from 5-6 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re, such as thienyl, furanyl, or thiazolyl; and
R12 can be —C(O)OH; or R12 can be C2-C6 alkoxy that is optionally substituted with —NH2, such as —OCH2CH3 or —OCH2CH2NH2; or R12 can be heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups, such as an optionally substituted piperazinyl ring.
X″ can be H or CH3; and
R2 can be C1-C4 alkyl, such as CH3; and
R12 can be C2-C6 alkoxy that is optionally substituted with —NH2, such as —OCH2CH3 or —OCH2CH2NH2;
In some embodiments:
A is N;
X contains 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S, and one of the ring atoms is independently selected from N, NH, and N(C1-C3 alkyl), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and
R1 is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc.
In certain embodiments, the compound can have formula I-B:
wherein:
X′ is NH, O, or S; and
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra;
X′ can be S, X′″ can be Y, and X″ can be H or Ra.
X″ can be H.
R1 can be thiazolyl.
Y can be C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb (e.g., unsubstituted phenyl).
R2 can be phenyl that is optionally substituted with from 1-4 Re; or heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re.
R2 can be unsubstituted phenyl.
The compound can be selected from compounds delineated in
The contacting can be in vitro.
The contacting is in vivo.
In some embodiments, any compound, composition, or method described herein can also include or further include any one or more of the other features delineated in the detailed description and/or in the claims.
[Q] Definitions
The term “mammal” includes organisms, which include mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and humans.
In embodiments, an amount of a compound of formula (I) or salt thereof can be an effective amount. “An effective amount” refers to an amount of a compound that confers a therapeutic effect (e.g., treats, e.g., controls, relieves, ameliorates, alleviates, or slows the progression of; or prevents, e.g., delays the onset of or reduces the risk of developing, a disease, disorder, or condition or symptoms thereof) on the treated subject. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of the compound described above may range from about 0.01 mg/kg to about 1000 mg/kg, (e.g., from about 0.1 mg/kg to about 100 mg/kg, from about 1 mg/kg to about 100 mg/kg). Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.
In general, and unless otherwise indicated, substituent (radical) prefix names are derived from the parent hydride by either (i) replacing the “ane” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl,” etc.; or (ii) replacing the “e” in the parent hydride with the suffixes “yl,” “diyl,” “triyl,” “tetrayl,” etc. (Here the atom(s) with the free valence, when specified, is (are) given numbers as low as is consistent with any established numbering of the parent hydride). Accepted contracted names, e.g., adamantyl, naphthyl, anthryl, phenanthryl, furyl, pyridyl, isoquinolyl, quinolyl, and piperidyl, and trivial names, e.g., vinyl, allyl, phenyl, and thienyl are also used herein throughout. Conventional numbering/lettering systems are also adhered to for substituent numbering and the nomenclature of fused, bicyclic, tricyclic, and polycyclic rings.
The following definitions are used unless otherwise described. Specific and general values listed below for radicals, substituents, and ranges are for illustration only. They do not exclude other defined values or other values within defined ranges for the radicals and substituents. Unless otherwise indicated, alkyl, alkylene, alkoxy, alkenyl, and the like denote both straight and branched groups.
The term “alkyl” refers to a saturated hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C6 alkyl indicates that the group may have from 1 to 6 (inclusive) carbon atoms in it. Any atom can be optionally substituted, e.g., by one or more substituents. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, and tert-butyl.
As used herein, the term “Cn-m alkylene,” employed alone or in combination with other terms, refers to a non-branched divalent alkyl linking group having n to m carbon atoms.
The term “haloalkyl” refers to an alkyl group in which at least one hydrogen atom is replaced by halo. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) is replaced by halo. In these embodiments, the hydrogen atoms can each be replaced by the same halogen (e.g., fluoro) or the hydrogen atoms can be replaced by a combination of different halogens (e.g., fluoro and chloro). “Haloalkyl” also includes alkyl moieties in which all hydrogens have been replaced by halo (sometimes referred to herein as perhaloalkyl, e.g., perfluoroalkyl, such as trifluoromethyl). Any atom can be optionally substituted, e.g., by one or more substituents.
As referred to herein, the term “alkoxy” refers to a group of formula —O(alkyl). Alkoxy can be, for example, methoxy (—OCH3), ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexyloxy. Likewise, the term “thioalkoxy” refers to a group of formula —S(alkyl). The terms “haloalkoxy” and “thio-haloalkoxy” refer to —O(haloalkyl) and —S(haloalkyl), respectively. Finally, the terms “cycloalkoxy” and “heterocyclyloxy” refer to a group of the formula —O(cycloalkyl) and —O(heterocyclyl), respectively.
The term “alkenyl” refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom can be optionally substituted, e.g., by one or more substituents. Alkenyl groups can include, e.g., vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double bond carbons can optionally be the point of attachment of the alkenyl substituent.
The term “alkynyl” refers to a straight or branched hydrocarbon chain containing the indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alkynyl groups can be optionally substituted, e.g., by one or more substituents. Alkynyl groups can include groups such as ethynyl, propargyl, and 3-hexynyl. One of the triple bond carbons can optionally be the point of attachment of the alkynyl substituent.
The term “heterocyclyl” refers to a fully saturated monocyclic, bicyclic, tricyclic or other polycyclic ring system having one or more constituent heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and/or form a salt), or S. The heteroatom or ring carbon can be the point of attachment of the heterocyclyl substituent to another moiety. Any atom can be optionally substituted, e.g., by one or more substituents. Heterocyclyl groups can include groups such as tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. By way of example, a phrase such as “heterocyclic ring containing from 5-6 ring atoms”, wherein from 1-2 of the ring atoms is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heterocyclic ring is optionally substituted with from 1-3 independently selected Ra would include (but not be limited to) tetrahydrofuryl, tetrahydropyranyl, piperidyl (piperidino), piperazinyl, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl.
The term “cycloalkyl” refers to a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom can be optionally substituted, e.g., by one or more substituents. A ring carbon serves as the point of attachment of a cycloalkyl group to another moiety. Cycloalkyl moieties can include groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclo[2.2.1]heptyl).
The term “cycloalkenyl” refers to partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon groups. A ring carbon (e.g., saturated or unsaturated) is the point of attachment of the cycloalkenyl substituent. Any atom can be optionally substituted e.g., by one or more substituents. Cycloalkenyl moieties can include, e.g., cyclohexenyl, cyclohexadienyl, or norbornenyl.
The term “aryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (>3 fused rings) hydrocarbon ring system. One or more ring atoms can be optionally substituted by one or more substituents for example. Aryl moieties include groups such as phenyl and naphthyl.
The term “heteroaryl” refers to an aromatic monocyclic, bicyclic (2 fused rings), tricyclic (3 fused rings), or polycyclic (>3 fused rings) hydrocarbon groups having one or more heteroatom ring atoms independently selected from O, N (it is understood that one or two additional groups may be present to complete the nitrogen valence and/or form a salt), or S. One or more ring atoms can be optionally substituted, e.g., by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl, benzothiazolyl, 3-carbolinyl, carbazolyl, coumarinyl, chromenyl, cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, and xanthenyl.
As used herein, the descriptor “—CN” represents the cyano group (and vice versa), wherein the carbon and nitrogen atoms are bound together by a triple bond. As used herein, the descriptor “—OH” represents the hydroxy group (and vice versa). The descriptors “C═O” or “C(O)” refers to a carbon atom that is doubly bonded to an oxygen atom.
In general, when a definition for a particular variable includes hydrogen and non-hydrogen (halo, alkyl, aryl, etc.) possibilities, the term “substituent(s) other than hydrogen” refers collectively to the non-hydrogen possibilities for that particular variable. The term “substituent” refers to a group “substituted” on groups such as an alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group at any atom of that group. In one aspect, the substituent(s) on a group are independently any one single or any combination of two or more of the permissible atoms or groups of atoms delineated for that substituent. In another aspect, a substituent may itself be substituted with any one of the above substituents.
Further, as used herein, the phrase “optionally substituted” means unsubstituted (e.g., substituted with hydrogen (H)) or substituted. As used herein, the term “substituted” means that a hydrogen atom is removed and replaced by a substituent. It is understood that substitution at a given atom is limited by valency.
Descriptors such as “C6-C10 aryl that is optionally substituted with from 1-4 independently selected Rb (and the like) is intended to include both an unsubstituted C6-C10 aryl group and a C6-C10 aryl group that is substituted with from 1-4 independently selected Rb. The use of a substituent (radical) prefix name such as alkyl without the modifier “optionally substituted” or “substituted” is understood to mean that the particular substituent is unsubstituted. However, the use of “haloalkyl” without the modifier “optionally substituted” or “substituted” is still understood to mean an alkyl group, in which at least one hydrogen atom is replaced by halo.
[R] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
In
This application features pyrazol-3-one compounds that activate a pro-apoptotic function of BAX, making them therapeutically useful for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diseases, disorders, and conditions associated with deregulated apoptosis of cells (e.g., diseased or damaged cells; e.g., insufficient apoptosis of diseased or damaged cells; or lack of or reduced apoptosis of diseased or damaged cells). Examples of such diseases, disorders, and conditions include (but are not limited to) those associated with blockade(s) of cell death pathways (e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g., hyperproliferative diseases, such as cancer. While not wishing to be bound by theory, it is believed that the compounds described herein induce and increase apoptosis in target cells (e.g., pathogenic cells including, but not limited to, cancer cells), thereby suppressing tumor growth and/or proliferation. It is further believed that increasing apoptosis in said target cells reestablishes the normal apoptotic control that, during homeostasis, is associated with a regulated balance between pro- and anti-apoptotic protein functions.
Compounds
This application features pyrazol-3-one compounds having formula (I) below as well as compositions and methods that include the formula (I) compounds.
Here and throughout this specification, the attendant variables R1, R2, A, X, and Y (and any sub-variables) can be as defined anywhere herein.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination.
Thus, for ease of exposition, it is also understood that where in this specification, a variable (e.g., R1) is defined by “as defined anywhere herein” (or the like), the definitions for that particular variable include the first occurring and broadest generic definition as well as any sub-generic and specific definitions delineated anywhere in this specification.
Variable X
In some embodiments, X is heteroaryl, which contains 5 ring atoms, wherein from 1-2 of the ring atoms is/are independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein:
or
X is phenyl optionally substituted with from 1-5 Ra.
In some embodiments, X is heteroaryl, which contains 5 ring atoms, wherein from 1-2 of the ring atoms is/are independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein:
In some embodiments, X contains 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S (e.g., selected from N and S; e.g., selected from N and NH).
In certain embodiments, one of the two ring atoms is independently selected from N, NH, and N(C1-C3 alkyl) (e.g., N), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S (e.g., the other ring atom is O or S, e.g., S; or e.g., NH).
As an example, X can have formula X-1:
in which:
X′ is NH, O, or S; and
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra.
Embodiments in which X has formula X-1 can include one or more of the following features.
X′ is S. X′ is NH.
X′″ is Y, which can be as defined anywhere herein, and X″ is H or Ra.
X′ is S; and X′″ is Y, which can be as defined anywhere herein, and X″ is H or Ra (e.g., X″ is H).
X′ is NH; and X′″ is Y, which can be as defined anywhere herein, and X″ is H or Ra (e.g., X″ is H).
X″ is H.
X″ is Ra. In certain embodiments, Ra is C1-C8 (e.g., C1-C6, C1-C3) alkyl (e.g., CH3). In other embodiments, Ra is phenyl that is optionally substituted with from 1-4 Re. In still other embodiments, Ra is or C3-C8 cycloalkyl which is optionally substituted with from 1-4 independently selected C1-C4 alkyl groups.
X″ is Y, which can be as defined anywhere herein; and X′″ is H or Ra. In embodiments, X′ is S or NH (e.g., S).
Variable Y
In some embodiments, Y is:
(i) C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb; or
(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rb; or
(iii) C1-C6 alkyl or C1-C6 haloalkyl, each of which is optionally substituted with —OH, —NH2, or —SH.
In some embodiments, Y is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb. In certain embodiments, Y is phenyl, which is optionally substituted with from 1-5 independently selected Rb. In certain embodiments, Y is unsubstituted phenyl.
In some embodiments, Y is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rb.
In some embodiments, Y is heteroaryl, which contains from 5-6 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rb.
In some embodiments, Y is C1-C6 alkyl or C1-C6 haloalkyl, each of which is optionally substituted with —OH, —NH2, or —SH (e.g., C1-C6 alkyl, which is optionally substituted with —OH, —NH2, or —SH; e.g., C1-C6 alkyl, such as CH3).
Variable R1
R1 is:
(i) C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rc; or
(ii) heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc; or
(iii) —C(O)—(C6-C10 aryl or heteroaryl, which contains from 5-10 ring atoms as defined in R1 definition (i) and (ii), respectively); or
(iv) hydrogen.
In some embodiments, R1 is any one, two, or three of the above (e.g., (i) and/or (ii) and one of (iii) or (iv); e.g., (i) and/or (ii); e.g., (iii) and/or (iv)).
In some embodiments, R1 is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rc.
In certain embodiments, R1 is phenyl, which is substituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently selected Rc.
In certain embodiments, each occurrence of Rc is, independently, selected from any one the substituents delineated collectively in (aaa), (bbb), (ccc), and (ddd) below:
(aaa) C1-C6 alkoxy, C1-C6 alkyl, C1-C6 haloalkyl, —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, or —NHC(O)(C1-C6 alkyl), each of which is optionally substituted with —OH, —NH2, or —SH;
(bbb) C(O)OH; —C(O)O(C1-C6 alkyl); —OC(O)(C1-C6 alkyl); —SO2NH2; —SO2NH(C1-C6 alkyl); or —SO2N(C1-C6 alkyl)2; or —C(O)O—(CH2)1-3(e.g., 1)—C(O)-(phenyl optionally substituted as defined in (ddd) below (e.g., —C(O)O—CH2—C(O)-(phenyl);
(ccc) C3-C6 cycloalkoxy or L-heterocyclyl containing from 5-7 ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups; and wherein L is a bond or C1-C6 alkylene (e.g., a bond); e.g., Rc is morpholino or piperazinyl; and
(ddd) phenyl or heteroaryl containing from 5-6 ring atoms, wherein from 1-2 of the ring atoms of the heteroaryl is independently selected from N, NH, N(C1-C3 alkyl), O, and S; wherein each of said phenyl and heteroaryl is optionally substituted with from 1-3 substituents independently selected from halo; hydroxyl; cyano; —C(O)(C1-C6 alkyl); C(O)OH; —C(O)O(C1-C6 alkyl); nitro; —NH2; —NH(C1-C6 alkyl), N(C1-C6 alkyl)2, —NHC(O)(C1-C6 alkyl), C1-C6 alkoxy; C1-C6 haloalkoxy; C1-C6 thioalkoxy; C1-C6 thiohaloalkoxy; C1-C6 alkyl, and C1-C6 haloalkyl, wherein said alkyl or alkyl portion is optionally substituted with —OH, —NH2, or —SH.
In certain embodiments, each occurrence of Rc is, independently, selected from:
In certain embodiments, Rc is C1-C6 alkoxy (e.g., ethoxy e.g., containing branched alkyl, such as iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that is substituted with —NH2.
In certain embodiments, R1 has formula A:
wherein one or two of R12, R13, and R14 is(are) an independently selected Rc, and the other(s) is(are) hydrogen.
Embodiments in which R1 has formula A can include one or more of the following features.
R12 is Rc.
R13 is H.
R14 is H.
R14 is Rc.
R12 is Rc, and each of R13 and R14 is H.
R14 is Rc, and each of R12 and R13 is H.
In some of the above described embodiments, Rc is C1-C6 alkoxy (e.g., ethoxy or iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
In embodiments, Rc can be C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that is substituted with —NH2.
In some of the above described embodiments, Rc is —C(O)OH (or a salt thereof, e.g., sodium salt thereof).
In some of the above described embodiments, Rc is L-heterocyclyl containing from 5-7 (e.g., 6) ring atoms, wherein from 1-2 of the ring atoms of the heterocyclyl is independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), NC(O)O(C1-C6 alkyl), O, and S; and each of which is optionally substituted with from 1-3 independently selected C1-C4 alkyl groups; and wherein L is a bond or C1-C6 alkylene (e.g., L is a bond or CH2; e.g., a bond); e.g., Rc is morpholino or piperazinyl.
R13 is H, and each of R12 and R14 is Rc. In certain embodiments, one of R12 and R14 is C1-C6 alkoxy (e.g., ethoxy), and the other of R12 and R14 is independently selected from:
In some embodiments, R1 is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc.
In certain embodiments, R1 is heteroaryl, which contains from 5-6 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1) independently selected Rc. For example, R1 can be thiazolyl.
In other embodiments, R1 is heteroaryl, which contains from 8-10 ring atoms, wherein from 1-4 (e.g., 1-2) of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 (e.g., 1-2 or 1) independently selected Rc. For example, R1 can be indazolyl.
In some embodiments, R1 is hydrogen.
In some embodiments, R1 is —C(O)—(C6-C10 aryl or heteroaryl, which contains from 5-10 ring atoms as defined in R1 definition (i) and (ii), respectively); e.g., —C(O)-(heteroaryl, which contains from 5-10 (e.g., 5-6, e.g., 5) ring atoms as defined in R1 definition (i) and (ii), respectively), such as —C(O)-(thiazolyl).
Variable R2
In some embodiments, R2 is C1-C8 alkyl (e.g., C1-C6 alkyl, e.g., C1-C3 alkyl, e.g., CH3).
In some embodiments, R2 is phenyl that is optionally substituted with from 1-4 Re. In certain embodiments, R2 is unsubstituted phenyl.
In some embodiments, R2 is heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re. In certain embodiments, R2 is heteroaryl containing from 5 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re; e.g., thienyl or furyl.
In some embodiments, R2 is C3-C8 cycloalkyl which is optionally substituted with from 1-4 independently selected C1-C4 alkyl groups.
Variable A
In some embodiments, A is N.
Non-Limiting Combinations of Attendant Variables
In some embodiments:
A is N;
X contains 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S, and one of the ring atoms is independently selected from N, NH, and N(C1-C3 alkyl) (e.g., N), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S (e.g., the other ring atom is S or NH, e.g., S); and
R1 is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rc (e.g., R1 is phenyl, which is substituted with from 1-5 (e.g., 1-3, 1-2, or 1) independently selected Rc), which Rc can be as defined anywhere herein.
In some embodiments the compound has formula I-A:
wherein:
X′ is NH, O, or S (e.g., S or NH, e.g., S);
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra; and
one or two of R12, R13, and R14 is(are) an independently selected Rc, and the other(s) is(are) hydrogen; R2 can be as defined anywhere herein.
Embodiments in which the compound has formula I-A can include one or more of the following features.
X′ is S, X′″ is Y, and X″ is H or Ra (e.g., X″ is H).
X′ is NH, X′″ is Y, and X″ is H or Ra (e.g., X″ is H).
Ra is C1-C8 alkyl (e.g., C1-C6 alkyl, e.g., C1-C3 alkyl, e.g., CH3).
Y is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb (e.g., Y is unsubstituted phenyl).
R12 is Rc, and each of R13 and R14 is H; or R14 is Rc, and each of R12 and R13 is H.
Each occurrence of R is, independently, selected from:
Rc is C1-C6 alkoxy (e.g., ethoxy; e.g., containing branched alkyl, such as iso-propoxy).
Rc is C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy).
Rc is C1-C6 alkoxy (e.g., C2-C6 alkoxy, e.g., ethoxy; e.g., containing branched alkyl, such a iso-propoxy) that is substituted with —NH2.
R2 is C1-C8 alkyl (e.g., C1-C6 alkyl, e.g., C1-C3 alkyl, e.g., CH3). R2 is phenyl that is optionally substituted with from 1-4 Re. R2 is heteroaryl containing from 5-6 (e.g., 5) ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), NC(O)(C1-C6 alkyl), O, and S; and wherein said heteroaryl is optionally substituted with from 1-3 Re.
In some embodiments of formula I-A:
In certain of these formula I-A embodiments:
In certain of these formula I-A embodiments:
In some embodiments of formula I-A:
In certain of these formula I-A embodiments:
In certain of these formula I-A embodiments:
In some embodiments of formula I-A:
In certain of these formula I-A embodiments:
In certain of these formula I-A embodiments:
In some embodiments:
A is N;
X contains 2 ring atoms independently selected from N, NH, N(C1-C3 alkyl), O, and S, and one of the ring atoms is independently selected from N, NH, and N(C1-C3 alkyl), and the other ring atom is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and
R1 is heteroaryl, which contains from 5-10 ring atoms, wherein from 1-4 of the ring atoms is independently selected from N, NH, N(C1-C3 alkyl), O, and S; and wherein said heteroaryl ring is optionally substituted with from 1-3 independently selected Rc.
In some embodiments, the compound has formula I-B:
wherein:
X′ is NH, O, or S; and
one of X″ and X′″ is Y, and the other of X″ and X′″ is H or Ra.
Embodiments in which the compound has formula I-B can include one or more of the following features.
X′ is S, X′″ is Y, and X″ is H or Ra (e.g., X″ is H).
X′ is NH, X′″ is Y, and X″ is H or Ra (e.g., X″ is H).
R1 is thiazolyl.
Y is C6-C10 aryl, which is optionally substituted with from 1-5 independently selected Rb. R2 is phenyl that is optionally substituted with from 1-4 Re. R2 is unsubstituted phenyl.
Compound Forms and Salts
In some embodiments, the compounds described herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures (e.g., including (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (+) (dextrorotatory) forms, (−) (levorotatory) forms, the racemic mixtures thereof, and other mixtures thereof). Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomeric forms, as well as mixtures thereof, of these compounds are expressly included in the present invention. The compounds described herein may also or further contain linkages wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds). Accordingly, all cis/trans and E/Z isomers and rotational isomers are expressly included in the present invention. The compounds of this invention may also be represented in multiple tautomeric forms; in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented. All such isomeric forms of such compounds are expressly included in the present invention. Unless otherwise mentioned or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms of that compound.
Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, and include, but are not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972), each of which is incorporated herein by reference in their entireties. It is also understood that this invention encompasses all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, and include, but are not limited to, column chromatography, thin-layer chromatography, and high-performance liquid chromatography.
The compounds of this invention include the compounds themselves, as well as their salts and their prodrugs, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include C1-6 alkyl esters of carboxylic acid groups, which, upon administration to a subject, are capable of providing active compounds.
Pharmaceutically acceptable salts of the compounds of this invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term “pharmaceutically acceptable salt” refers to a salt formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase “pharmaceutically acceptable” refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient.
Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxy groups (e.g. L-arginine, -lysine, -histidine salts).
Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; Journal of Pharmaceutical Science, 66, 2 (1977); “Pharmaceutical Salts: Properties, Selection, and Use A Handbook; Wermuth, C. G. and Stahl, P. H. (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002 [ISBN 3-906390-26-8]; and Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19; each of which is incorporated herein by reference in its entirety.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the invention.
In addition to salt forms, the invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that undergo chemical changes under physiological conditions to provide the compounds of the invention. Additionally, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be more bioavailable by oral administration than the parent drug. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the invention which is administered as an ester (the “prodrug”), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. In embodiments, the ester can be an alkyl ester (e.g., C1-C3 alkyl, e.g., CH3 or CH2CH3; or C3-C6 alkyl, e.g., C3-C6 branched alkyl, e.g., t-butyl, isopropyl, isobutyl). Additional examples include peptidyl derivatives of a compound of the invention.
The invention also includes various hydrate and solvate forms of the compounds (and salts thereof) described herein.
The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.
Synthesis of Compounds
The compounds described herein can be conveniently prepared in accordance with the procedures outlined herein, from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented may be varied for the purpose of optimizing the formation of the compounds described herein.
Synthetic chemistry transformations (including protecting group methodologies) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. C. Larock, Comprehensive Organic Transformations, 2d.ed., Wiley-VCH Publishers (1999); P. G. M. Wuts and T. W. Greene, Protective Groups in Organic Synthesis, 4th Ed., John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-visible), or mass spectrometry (MS), or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC).
Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
The reactions of the processes described herein can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis.
Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of solvents. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
In some embodiments, the formula (I) compounds can be prepared using the reaction pathways and techniques as shown in
Pharmaceutical Compositions
The term “pharmaceutically acceptable carrier” refers to a carrier or adjuvant that may be administered to a subject (e.g., a patient), together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.
The compositions for administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules, lozenges or the like in the case of solid compositions. In such compositions, the compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
The amount administered depends on the compound formulation, route of administration, etc. and is generally empirically determined in routine trials, and variations will necessarily occur depending on the target, the host, and the route of administration, etc. Generally, the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or 1000 mg, according to the particular application. In a particular embodiment, unit dosage forms are packaged in a multipack adapted for sequential use, such as blisterpack, comprising sheets of at least 6, 9 or 12 unit dosage forms. The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.
Use and Administration
This application features pyrazol-3-one compounds that activate pro-apoptotic BAX, making them therapeutically useful for treating (e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of) or preventing (e.g., delaying the onset of or reducing the risk of developing) diseases, disorders, and conditions associated with deregulated apoptosis of (e.g., diseased or damaged cells; e.g., insufficient apoptosis of diseased or damaged cells; or lack of or reduced apoptosis of diseased or damaged cells). Examples of such diseases, disorders, and conditions include (but are not limited to) those associated with blockade(s) of cell death pathways (e.g., over-expression of anti-apoptotic BCL-2 proteins), e.g., hyperproliferative diseases, such as cancer. While not wishing to be bound by theory, it is believed that the compounds described herein induce and increase apoptosis in target cells (e.g., pathogenic cells including, but not limited to, cancer cells), thereby suppressing tumor growth and/or proliferation. It is further believed that increasing apoptosis in said target cells reestablishes the normal apoptotic control that, during homeostasis, is associated with a regulated balance between pro- and anti-apoptotic protein functions.
The compounds and compositions described herein can, for example, be administered orally, parenterally (e.g., subcutaneously, intracutaneously, intravenously, intramuscularly, intraarticularly, intraarterially, intrasynovially, intrasternally, intrathecally, intralesionally and by intracranial injection or infusion techniques), by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, by injection, subdermally, intraperitoneally, transmucosally, or in an ophthalmic preparation, with a dosage ranging from about 0.01 mg/kg to about 1000 mg/kg, (e.g., from about 0.01 to about 100 mg/kg, from about 0.1 to about 100 mg/kg, from about 1 to about 100 mg/kg, from about 1 to about 10 mg/kg) every 4 to 120 hours, or according to the requirements of the particular drug. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 50, 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970). In certain embodiments, the compositions are administered by oral administration or administration by injection. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
In some embodiments, the compounds described herein can be coadministered with one or more other therapeutic agents. In certain embodiments, the additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention (e.g., sequentially, e.g., on different overlapping schedules with the administration of one or more compounds of formula (I) (including any subgenera or specific compounds thereof)). In other embodiments, these agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition. In still another embodiment, these agents can be given as a separate dose that is administered at about the same time that one or more compounds of formula (I) (including any subgenera or specific compounds thereof) are administered (e.g., simultaneously with the administration of one or more compounds of formula (I) (including any subgenera or specific compounds thereof)). When the compositions of this invention include a combination of a compound of the formulae described herein and one or more additional therapeutic or prophylactic agents, both the compound and the additional agent can be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
The compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form.
The compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and/or suspensions. Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the compositions of this invention is useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation.
In some embodiments, topical administration of the compounds and compositions described herein may be presented in the form of an aerosol, a semi-solid pharmaceutical composition, a powder, or a solution. By the term “a semi-solid composition” is meant an ointment, cream, salve, jelly, or other pharmaceutical composition of substantially similar consistency suitable for application to the skin. Examples of semi-solid compositions are given in Chapter 17 of The Theory and Practice of Industrial Pharmacy, Lachman, Lieberman and Kanig, published by Lea and Febiger (1970) and in Remington's Pharmaceutical Sciences, 21st Edition (2005) published by Mack Publishing Company, which is incorporated herein by reference in its entirety.
Topically-transdermal patches are also included in this invention. Also within the invention is a patch to deliver active chemotherapeutic combinations herein. A patch includes a material layer (e.g., polymeric, cloth, gauze, bandage) and the compound of the formulae herein as delineated herein. One side of the material layer can have a protective layer adhered to it to resist passage of the compounds or compositions. The patch can additionally include an adhesive to hold the patch in place on a subject. An adhesive is a composition, including those of either natural or synthetic origin, that when contacted with the skin of a subject, temporarily adheres to the skin. It can be water resistant. The adhesive can be placed on the patch to hold it in contact with the skin of the subject for an extended period of time. The adhesive can be made of a tackiness, or adhesive strength, such that it holds the device in place subject to incidental contact, however, upon an affirmative act (e.g., ripping, peeling, or other intentional removal) the adhesive gives way to the external pressure placed on the device or the adhesive itself, and allows for breaking of the adhesion contact. The adhesive can be pressure sensitive, that is, it can allow for positioning of the adhesive (and the device to be adhered to the skin) against the skin by the application of pressure (e.g., pushing, rubbing) on the adhesive or device.
The compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
A composition having the compound of the formulae herein and an additional agent (e.g., a therapeutic agent) can be administered using any of the routes of administration described herein. In some embodiments, a composition having the compound of the formulae herein and an additional agent (e.g., a therapeutic agent) can be administered using an implantable device. Implantable devices and related technology are known in the art and are useful as delivery systems where a continuous, or timed-release delivery of compounds or compositions delineated herein is desired. Additionally, the implantable device delivery system is useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563 (2001). Timed-release technology involving alternate delivery methods can also be used in this invention. For example, timed-release formulations based on polymer technologies, sustained-release techniques and encapsulation techniques (e.g., polymeric, liposomal) can also be used for delivery of the compounds and compositions delineated herein.
The invention will be further described in the following examples. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
We generated a diverse in silico compilation of 750,000 small molecules from commercially available libraries and docked the database of 3-dimensional molecules on average minimized BAX structures using Glide 4.019,20 in standard precision mode (SPVS) (
To evaluate the capacity of candidate BAMs to bind BAX, we developed a screening competitive fluorescence polarization assay (FPA) based on the interaction between recombinant BAX and the fluoresceinated Stabilized Alpha-Helix of BCL-2 domain (SAHB) modeled after BIM BH3 (EC50, 283 nM) (
The BH3 binding pocket of anti-apoptotic targets shares topographic similarities with the BH3 trigger site on BAX, including a hydrophobic groove that engages the hydrophobic face of BH3 helices and a perimeter of similarly oriented charged and polar residues that are complementary to discrete residues of the hydrophilic BH3 interface. The two BH3 binding sites differ in their geographic location, pocket depth, and functionality. Whereas BIM BH3 is compatible with both the BAX trigger site and anti-apoptotic pockets, we examined whether BAM7 was selective for BCL-2 family targets by competitive FPA. As demonstrated for BAX, direct FPA analyses documented high affinity interactions between FITC-BIM SAHB and the anti-apoptotic proteins BCL-XL (EC50, 13.6 nM), MCL-1 (EC50, 19.8 nM), and BFL-1/A1 (EC50, 17.9 nM), which represent the structural diversity of the pro-survival arm of the BCL-2 family (
To determine if BAM7 selectively competed with FITC-BIM SAHB for binding to BAX through a direct trigger site interaction or an indirect allosteric effect, we performed NMR analysis of 15N-BAX upon BAM7 titration. As observed for BIM SAHBA17, the addition of BAM7 up to a 1:1 ratio induced significant backbone amide chemical shift changes in those BAX residues concentrated in the region of the α1/α6 trigger site (
We next performed molecular docking analysis to examine the predicted interactions between BAM7 and the BAX trigger site. Interestingly, BAM7 appears to insinuate itself along a crevice formed by residues located at (1) the junction between the α1-α2 loop's C-terminus and the N-terminus of α2, (2) the N-terminus of al, and (3) the C-terminus of α6 (
In order to transform from an inactive cytosolic monomer into a toxic mitochondrial oligomer, BAX undergoes a major conformational change upon BH3 triggering. We recently demonstrated using correlative structural and biochemical methods that these essential changes include “opening” of the α1/α2 loop, mobilization of the C-terminal α9 for mitochondrial translocation, and BAX BH3 exposure for propagating BAX activation11. To determine if the selective binding interaction we documented for BAM7 results in functional BAX activation, we performed a series of structural, biochemical, and cellular studies. First, we conducted an NMR analysis of 15N-BAX upon titration with higher concentrations of BAM7 to examine secondary structural changes that ensue upon ligand binding. We observed that increasing the ratio of BAM7/BAX from 1:1 to 2:1 caused additional chemical shift changes in the α1-α2 loop, the BH3 domain (α2), and in the C-terminal α9 helix, three discrete regions that also undergo allosteric changes in response to increased BIM SAHB exposure11 (
Finally, we investigated whether this prototype BAX activator molecule that directly, selectively, and functionally activates BAX in vitro could induce BAX-dependent cell death. For these studies, we employed genetically-defined mouse embryo fibroblasts (MEFs) that either express only BAX (Bak−/−), only BAK (Bax−/−) or neither death effector (Bax−/−Bak−/−). Thus, to undergo apoptosis, Bak−/− MEFs rely on BAX and Bax−/− MEFs rely on BAK, whereas Bax−/−Bak−/− MEFs are profoundly resistant to apoptosis15. Strikingly, BAM7 dose-responsively impaired the viability of Bak−/− MEFs that exclusively express BAX, but had no effect on Bax−/− MEFs that contain BAK but lack BAX (
To assess the anti-cancer activity of BAM7, DHL5 DLBCL cells, which are relatively resistant to ABT-737, were exposed to BAM7 and dose-responsive impairment of cell viability was observed at 24 hours, as assessed by CellTiterGlo (
Thus, we find that BAM7 directly binds to the BAX trigger site and initiates the characteristic structural changes that lead to functional BAX activation. When applied to genetically-defined MEFs, BAM7 only kills the cell line that contains BAX, inducing the morphologic features of apoptosis; in the context of imaging Bax−/−/Bak−/− MEFs that express EGFP-BAX, BAX translocation from cytosol to mitochondria, and attendant cellular shrinkage and membrane blebbing, is also observed. BAM7 impairs the viability of DHL5 lymphoma cells and can sensitize the cells to the BCL-2/BCL-XL inhibitor ABT-737. Taken together, these studies demonstrate the feasibility of targeting BAX with a selective small molecule to trigger its pro-apoptotic activity.
Methods
In Silico Screening.
A diverse in silico library was generated from the following commercially available libraries downloaded from the ZINC database34: ACB Blocks, Asinex, Chembridge, Maybridge, Microsource, NCI, Peakdale, and FDA-approved. The in silico library was filtered for drug-like features, ADME properties, and appropriate functional groups with Qikprop. Molecules were converted to 3D all-atom structures, generating a maximum of 4 stereoisomers, ionization states for pH 7.0 and pH 2.0, and different tautomers with Ligprep. The database of in silico 3D molecules totaled approximately 750,000 compounds. BAX structures for docking were prepared using an averaged BAX closed-loop structure and an averaged BAX open-loop structure with GROMACS software. The two structures were generated in suitable format for docking with Maestro. Docking was performed using Glide, with the small molecule database for each BAX structure in standard precision mode (SPVS)19. The top 20,000 hits based on Glidescore function were selected and redocked to the BAX structures using extra precision docking mode (XPVS)20. The top 1000 hits from each docking calculation were visualized on the structure and then analyzed for interactions with key BAX residues, leading to selection of 100 compounds for experimental screening.
BCL-2 Family Protein Production.
Recombinant and tagless full-length BAX, BCL-XLΔC, MCL-1ΔNΔC, and BFL-1/A1ΔC were expressed and purified as previously reported17,35. Transformed Escherichia coli BL21 (DE3) were cultured in ampicillin-containing Luria Broth and protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The bacterial pellets were resuspended in buffer (250 mM NaCl, 20 mM Tris, complete protease inhibitor tablet, pH 7.2), sonicated, and after centrifugation at 45,000×g for 45 min, the supernatants were applied to glutathione-agarose columns (Sigma) for GST-BCL-XLΔC, MCL-1ΔNΔC, and BFL-1/A1ΔC, or a chitin column (BioLabs) for Intein-BAX. On-bead digestion of GST-tagged protein was accomplished by overnight incubation at room temperature in the presence of thrombin (75 units) in PBS (3 mL), whereas the intein tag was cleaved from BAX by overnight incubation of the chitin beads at 4° C. with 50 mM DTT. BCL-XLΔC, MCL-1ΔNΔC, and BFL-1/A1ΔC were purified by size exclusion chromatography (SEC) using 150 mM NaCl, 50 mM Tris, pH 7.4 buffer conditions, and full-length monomeric BAX protein isolated by SEC using a Superdex-75 column (GE Healthcare) and 20 mM Hepes pH 7.2, 150 mM KCl buffer conditions.
Fluorescence Polarization Binding Assays.
Fluorescence polarization assays (FPA) were performed as previously described35,36. Briefly, direct binding curves were first generated by incubating FITC-BIM SAHB (50 nM) with serial dilutions of full-length BAX, BCL-XLΔC, MCL-1ΔNΔC, or BFL-1/Δ1ΔC and fluorescence polarization measured at 20 minutes on a SpectraMax M5 microplate reader (Molecular Devices). For competition assays, a serial dilution of small molecule or acetylated BIM SAHB (Ac-BIM SAHB) was combined with FITC-BIM SAHB (50 nM), followed by the addition of recombinant protein at ˜EC75 concentration, as determined by the direct binding assay (BAX: 500 nM; BCL-XLΔC, MCL-1ΔNΔC, BFL-1/Δ1ΔC: 200 nM). Fluorescence polarization was measured at 20 minutes and IC50 values calculated by nonlinear regression analysis of competitive binding curves using Prism software (Graphpad).
BAM7 Characterization by Mass Spectrometry and 1H-NMR Spectroscopy.
4-(2-(2-ethoxyphenyl)hydrazono)-3-methyl-1-(4-phenylthiazol-2-yl)-1H-pyrazol-5(4H)-one. LC-MS: ES+ 406 (M+1). 1H NMR (300 MHz, DMSO-d6) δ 7.96 (d, 2H, J=8.1 Hz), 7.86 (s, 1H), 7.75 (d, 1H, J=7.8 Hz), 7.46 (t, 2H), 7.37-7.33 (m, 1H), 7.25-7.20 (m, 2H), 7.13-7.07 (m, 1H), 4.29-4.22 (q, 2H), 2.36 (s, 3H), 1.48 (t, 3H).
NMR Samples and Spectroscopy.
Uniformly 15N-labeled full-length human BAX was generated as previously described17,37. Protein samples were prepared in 25 mM sodium phosphate, 50 mM NaCl solution at pH 6.0 in 5% D2O. BAM7 (10 mM stock) was titrated into a solution of 50 μM BAX to achieve the indicated molar ratios. Correlation 1H-15N HSQC spectra38 were acquired at 25° C. on a Bruker 800 MHz NMR spectrometer equipped with a cryogenic probe, processed using NMRPipe39, and analyzed with NMRView40. The weighted average chemical shift difference A at the indicated molar ratio was calculated as √{square root over ({(ΔH)2+(ΔN/5)2}/2)} in p.p.m. The absence of a bar indicates no chemical shift difference, or the presence of a proline or residue that is overlapped or not assigned. BAX cross-peak assignments were applied as previously reported37. The significance threshold for backbone amide chemical shift changes was calculated based on the average chemical shift across all residues plus the standard deviation, in accordance with standard methods41.
Structure Modeling.
Docked structures of BAX and BAM7 were generated using Glide 4.019,20 (Schrodinger, 2006) and analyzed using PYMOL42.
BAX Oligomerization Assay.
BAM7 was added to a 200 μL solution (20 mM Hepes/KOH pH 7.2, 150 mM KCl, 0.5% CHAPS) containing size exclusion chromatography (SEC)-purified, monomeric BAX at the indicated BAM7:BAX ratios. The mixtures and BAX monomer alone were incubated at 30° C. for the indicated durations and then subjected to analysis by SEC using an SD75 column and 20 mM Hepes/KOH pH 7.2, 150 mM KCl running buffer. The monomeric and oligomeric fractions elute at ˜11.5-12.0 min and ˜6.5-7.5 min, respectively. Protein standards (GE Healthcare) were used to calibrate the molecular weights of gel filtration peaks. Replicates were performed using at least two independent preparations of freshly SEC-purified monomeric BAX protein.
Liposomal Release Assay.
Liposomes were prepared and release assays performed as previously described32,43. Liposomes were composed of the following molar percentages of lipids (Avanti Polar Lipids): phosphatidylcholine, 48%; phosphatidylethanolamine, 28%; phosphatidylinositol, 10%; dioleoyl phosphatidylserine, 10%; and tetraoleoyl cardiolipin, 4% and were loaded with ANTS/DPX (Molecular Probe) upon extrusion. BAX (400 nM) was combined with BAM7 (200 nM, 400 nM) in 96-well format (Corning) and then liposomes were added (10 μL from 50 μM total lipid stock) in assay buffer (10 mM HEPES [pH 7], 200 mM KCl, 5 mM MgCl2, and 0.2 mM EDTA) to a final volume of 100 μl. Liposomal release was quantified based on the increase in fluorescence that occurs when the ANTS fluorophore is separated from the DPX quencher upon release from the liposomes into the supernatant. Fluorescence (λex=355 nm and λem=520 nM) was measured for 2 hours at 30° C. using a Tecan Infinite M1000 plate reader. To measure maximal release, Triton X-100 was added to a final concentration of 0.2% (v/v) after 2 h and fluorescence measured for an additional 10 min. The percentage release of ANTS/DPX is calculated as percentage release=((F−F0)/(F100−F0))×100, where F0 and F100 are baseline and maximal fluorescence, respectively.
Cell Viability Assay.
Mouse embryonic fibroblasts (MEFs) cells were maintained in DMEM high glucose (Invitrogen) supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, 50 mM HEPES, 0.1 mM MEM non-essential amino acids and 50 μM β-mercaptoethanol. MEFs (2.5×103 cells/well) were seeded in 96-well opaque plates for 18-24 hours and then incubated with serial dilutions of BAM7 or vehicle (0.15% DMSO) in DMEM at 37° C. in a final volume of 100 μl. DHL5 cells were cultured as described (Deng et al. Cancer Cell, 12, 171-185, 2007) and subjected to vehicle, BAM7, ABT-737, and combinations thereof at the indicated doses. Cell viability was assayed at 24 hours by addition of CellTiter-Glo reagent according to the manufacturer's protocol (Promega), and luminescence measured using a SpectraMax M5 microplate reader (Molecular Devices). Viability assays were performed in at least triplicate and the data normalized to vehicle-treated control wells. Leukemia cell viability and caspase 3/7 assays were performed as described (Cohen et al, Chem Biol, 2012).
Light Microscopy.
MEFs (5,000 cells/well) were plated for 24 hours on glass bottom culture dishes (MatTek Corp., MA) and then incubated with BAM7 (15 μM) or vehicle (0.15% DMSO). Live cell imaging was performed using a TE2000-E2 Nikon microscopy equipped with a temperature and CO2-controlled chamber that maintained an atmosphere of 3-5% humidified CO2 at 37° C. A Hamamatsu Orca ER digital CCD camera was used to capture images at 20× magnification for 24 hours at 20 min intervals. Acquisition, hardware control, and image analysis was performed using Nikon NIS-Elements software.
BAX Translocation Assay.
MEFs were seeded on uncoated 24-well glass bottom plates at a density of 2.5×105 cells/well in 500 μL of supplemented DMEM. After 6 hours, cells were transfected using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol, using 750 ng of plasmid DNA and 1.0 μL of Lipofectamine™ per well. Plasmid DNA was generated by cloning full-length BAX into the pEGFP-C3 plasmid (Clontech) using 5′ PstI and 3′ XbaI restriction sites. After overnight transfection, cells were treated with the indicated concentrations of BAM7 or vehicle (0.3% DMSO) in supplemented DMEM for 6 hours. Mitochondria were labeled with MitoTracker® Red CMXRos (Invitrogen) according to the manufacturer's protocol using 20 nM probe in 500 μL supplemented, phenol red-free DMEM for 15 minutes. The cells were then incubated in fresh media for an addition 15 minutes prior to imaging. Confocal microscopy was performed using a Yokogawa spinning disk confocal microscope (Yokogawa Electric Corporation) equipped with a Nikon inverted Ti microscope. Solid state lasers set at 488 nm and 561 nm were used to visualize EGFP and MitoTracker® Red CMXRos, respectively. The plate temperature was maintained at 37° C. using an In Vivo environmental chamber (In Vivo Scientific). Images were collected using an Andor iXon DU-897 EM-CCD camera (Andor Technology) and analyzed with ImageJ software (NIH). Percent EGFP-positive cells was determined by counting EGFP-positive and Mitotracker-positive cells. Percent BAX translocation was calculated by dividing the number of cells containing mitochondrion-localized BAX by the total number of EGFP-positive cells. Each treatment was performed in quadruplicate with >200 cells counted per well.
In some embodiments, compounds can have the 5,6 heteroring structure that is present in compounds 165-90, 165-94, and 165-95 in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This is a divisional of U.S. Utility application Ser. No. 15/048,918, filed Feb. 16, 2016, which is a continuation of U.S. Utility application Ser. No. 14/350,847, filed Apr. 10, 2014, now U.S. Pat. No. 9,303,024, issued on Apr. 5, 2016; which is a U.S. National Phase Application under 35 U.S.C. § 371 of International Patent Application No. PCT/US2012/059799, filed Oct. 11, 2012, which claims the benefit of U.S. Provisional Application No. 61/546,022, filed on Oct. 11, 2011, all of which are incorporated by reference herein.
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| 7160870 | Duffy et al. | Jan 2007 | B2 |
| 9303024 | Walensky | Apr 2016 | B2 |
| 20090012148 | Maxfield et al. | Jan 2009 | A1 |
| 20090163545 | Goldfarb | Jun 2009 | A1 |
| 20110003851 | Zhi et al. | Jan 2011 | A1 |
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| 49472 | Aug 2006 | AR |
| 101343268 | Jan 2009 | CN |
| 101766602 | Jul 2010 | CN |
| 3728278 | Jun 1988 | DE |
| 2305250 | Apr 2011 | EP |
| WO 200174769 | Oct 2001 | WO |
| WO 2003011855 | Feb 2003 | WO |
| WO 2004014902 | Feb 2004 | WO |
| WO 2005077345 | Aug 2005 | WO |
| WO 2005077368 | Aug 2005 | WO |
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| WO 2007053847 | May 2007 | WO |
| WO 2009071701 | Jun 2009 | WO |
| WO 2011029046 | Sep 2009 | WO |
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| WO 2010042225 | Apr 2010 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20180244664 A1 | Aug 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61546022 | Oct 2011 | US |
| Number | Date | Country | |
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| Parent | 15048918 | Feb 2016 | US |
| Child | 15963858 | US |
| Number | Date | Country | |
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| Parent | 14350847 | US | |
| Child | 15048918 | US |
