Small molecule inhibitors of BCL-2 proteins

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...

Reexamination Certificate

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C514S299000, C514S312000, C514S313000, C514S434000, C514S454000, C514S456000

Reexamination Certificate

active

06492389

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to the field of oncology and inhibitors of Bcl-2 proteins, and more particularly to small molecule inhibitors of Bcl-2 proteins involved in mediating the death of cancer cells, virally infected cells and self-reactive lymphocytes.
BACKGROUND OF THE INVENTION
Bcl-2 (B cell lymphoma/leukemia 2) was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas. Bcl-2 is now known to belong to a growing family of proteins which regulate programmed cell death or apoptosis. The Bcl-2 family includes both death antagonists (Bcl-2, Bcl-x
L
, Bcl-w, Bfl-1, Brag-l, Mcl-l and Al) and death agonists (Bax, Bak, Bcl-x
5
, Bad, Bid, Bik and Hrk) (Thompson,
Science
267:1456-62 (1992); Reed,
J. Cell Biol.
124:1-6 (1994); Yang et al.,
Blood
88:386-401 (1996)). This family of molecules shares four homologous regions termed Bcl homology (BH) domains BH 1, BH2, BH3, and BH4. All death antagonist members contain the BH4 domain while the agonist members lack BH4. It is known that the BH1 and BH2 domains of the death antagonists such as Bcl-2 are required for these proteins to heterodimerize with death agonists, such as Bax, and to repress cell death. On the other hand, the BH3 domain of death agonists is required for these proteins to heterodimerize with Bcl-2 and to promote apoptosis.
Programmed cell death or apoptosis plays a fundamental role in the development and maintenance of cellular homeostasis. Homologous proteins and pathways in apoptosis are found in a wide range of species, indicating that cellular demise is critical for the life and death cycle of the cell in all organisms. When extracellular stimuli switch on the cell-death signal, the response of the cell to such stimuli is specific for the particular cell type (Bonini et al.,
Cell
72:379-95 (1993)). The pathway to cellular suicide is controlled by certain checkpoints (Oltvai,
Cell
79:189-92 (1994)). The Bcl family proteins, including both antagonists of apoptosis (such as Bcl-2) and agonists of apoptosis (such as Bax), constitute the primary checkpoint. As such, the transmission of a cell-death signal can be either promoted or blocked by the different combinations of the Bcl-2 family members. The three-dimensional structure of a death antagonist, Bcl-X
L
, as determined by X-ray crystallography and NMR spectroscopy, provides a structural basis for understanding the biological functions of Bcl-2 family members and for developing novel therapeutics targeting Bcl-2 mediated apoptotic pathways (Muchmore et al.,
Nature
381:335-41 (1996)).
The detailed mechanism of Bcl-2 proteins in mediating molecular pathways of apoptosis has been the subject of intensive investigation. It is known that the apoptotic signaling pathway involves the activation of caspases which, once activated, cleave several cellular substrates such as poly(adenosine diphosphate-ribose) polymerase (PARP) and lead to final events of apoptosis. Bcl-2 plays a crucial role in regulating the process of apoptosis. One possible mechanism for Bcl-2 function is that Bcl-2 inhibits the release of cytochrome c from mitochondria. Cytochrome c is important for the activation of caspases. As such, Bcl-2 blocks caspase activation and subsequent events leading to apoptosis.
Being able to block apoptosis, Bcl-2 is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. High levels and aberrant patterns of Bcl-2 gene expression are found in a wide variety of human cancers, including ~30-60% of prostate, ~90% of colorectal, ~60% of gastric, ~20% of non-small cell lung cancers, ~30% of neuroblastomas, and variable percentages of melanomas, renal cell, and thyroid cancers, as well as acute and chronic lymphocytic and non-lymphocytic leukemias (Ellis et al.,
Cell Biol.
7, 663 (1991); Henkart,
Immunity
1, 343 (1994)); Kägi et al.,
Science
265, 528 (1994); Kägi et al.,
Nature
369, 31 (1994); Heusel et al.,
Cell
76, 977 (1994)).
The expression levels of Bcl-2 protein also correlate with relative resistance to a wide spectrum of current chemotherapeutic drugs and &ggr;-irradiation (Hanada et al., Cancer Res. 53:4978-86 (1993); Kitada et al.,
Antisense Res. Dev.
4:71-9 (1994); Miyashita et al.,
Cancer Res.
52:5407-11 (1992); Miyashita et al.,
Blood
81:151-7 (1993)). Since Bcl-2 can protect against such a wide variety of drugs which have very different mechanisms of action, it is possible that all these drugs use a common final pathway for the eventual induction of cell death which is regulated by Bcl-2. This notion is supported by the findings that chemotherapeutic drugs induce cell death through a mechanism consistent with apoptosis as opposed to necrosis. Therefore, Bcl-2 can inhibit the cell killing effect of currently available anticancer drugs by blocking the apoptotic pathway.
Because of its role in blocking apoptosis, Bcl-2 plays an important role in many types of cancer. As noted above, Bcl-2 blocks apoptosis, thereby preventing normal cell turnover. As a result, neoplastic cell expansion occurs unimpeded by the normal cellular turnover process. Prostate cancer is one particular example where Bcl-2 has important implication in the pathogenesis and treatment for a disease. Approximately 100,000 new cases of prostate cancer are diagnosed each year in the United States and about 30,000 deaths per year are attributable to this disease (Lynn et al.,
JNCI
87:867 (1995)). It has recently been found that hormone therapy-resistant prostate cancers express Bcl-2 (McDonnell et al.,
Cancer Res.
52:694-04 (1992)), while the normal prostate cells from which prostate cancers originate lack Bcl-2 (Colombel et al.,
Am J Pathol
143:390-400 (1993)). This indicates that Bcl-2 may protect prostate cancer cells from undergoing apoptosis induced by the anticancer drugs, such as Taxol (Haldar et al.,
Cancer Res.,
56:1235-5 (1996)). The clinical efficacy of nearly every cytotoxic anticancer drug currently available depends directly or indirectly on the assumption that tumor cells grow more rapidly than normal cells. However, this may not apply to human prostate cancer cells, which show very slow growth kinetics. Tumor kinetics studies have indicated that prostate cancer may be the consequence of the imbalance in cell turnover mechanisms more so than an increase in cell cycle rates. Thus, current anticancer drugs may not be effective in eradicating these nonproliferative prostate cancer cells.
The understanding of the biology of Bcl-2 in cancer and chemoresistance has opened new avenues in the development of novel anticancer strategies. One effective approach to overcome the chemoresistance of prostate cancers is to inhibit the protective function of Bcl-2 proteins. New drugs that modulate Bcl-2 mediated apoptotic response would represent a novel mechanism-based strategy for the treatment of prostate cancers and other cancers. Because the function of Bcl-2 is not absolutely necessary in many normal cell types (Veis et al.,
Cell,
75:229-40(1993)), a systematic inhibition of Bcl-2 may not affect the normal cellular function. This notion is supported by recent encouraging data from the clinical trial that antisense oligonucleotides targeted against the Bcl-2 gene can specifically inhibit non-Hodgkin's lymphoma in humans (Webb et al,.
Lancet
349:1137-41 (1997)). However, the clinical value of such antisense oligonucleotides is limited by their lack of enzymatic stability, cell permeability, and oral activity. As discussed above, currently available anticancer drugs may not be effective due to the chemoresistance of prostate cancer cells. Therefore, there is an impending need for highly potent, cell permeable, and orally active Bcl-2 inhibitors as a new generation of effective therapeutics for the treatment of prostate cancer, as well as other cancers.
Compared to other therapeutics such as antibodies, peptides or antisense oligonucleotides, small organic drugs may possess several advantages in the clinical applic

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