HK40005133B - Bivalent inhibitors of iap proteins and therapeutic methods using the same - Google Patents

Description

Bivalent inhibitors of IAP proteins and therapeutic methods using the same

The present application is a divisional application of Chinese patent application No. 201380055219.5, filed on August 16, 2013, entitled “Bivalent inhibitors of IAP protein and treatment methods using the same”.

Government funding

This invention was made with government support under Grant Nos. CA 127551 and CA 109025 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

Field of the Invention

The present invention relates to bivalent inhibitors of inhibitors of apoptosis proteins (IAPs) and to therapeutic methods for treating conditions and diseases in which inhibition of IAP proteins provides benefit. The present inhibitors bind to IAP proteins (including cIAP1, cIAP2, and XIAP) with very high affinity to induce apoptosis in human cancer cell lines, thereby enhancing the anti-tumor activity of other anti-cancer drugs.

Background of the Invention

Apoptosis, or programmed cell death, is a cellular process critical for homeostasis, normal development, host defense, and the suppression of tumorigenesis. Misregulation of apoptosis has been implicated in a variety of human diseases ⁽¹⁾ , including cancer ^{(1), (3)} , and resistance to apoptosis is now recognized as a hallmark of cancer ⁽⁴⁾ . As a result, targeting key apoptosis regulators has become an attractive strategy for developing new approaches to human cancer treatment ⁽¹⁾ .

Most current cancer therapies, including chemotherapeutics, radiation, and immunotherapy, indirectly induce apoptosis in cancer cells. Therefore, the inability of cancer cells to undergo apoptosis due to defects in the normal apoptotic machinery is often associated with increased resistance to chemotherapy-, radiation-, or immunotherapy-induced apoptosis. This type of primary or acquired resistance of human cancers to current therapies due to defects in apoptosis is a major problem in current cancer therapy.

In order to improve the survival and quality of life of cancer patients, current and future efforts in designing and developing new molecular target-specific anticancer therapies include strategies that specifically target cancer cells that are resistant to apoptosis. In this regard, targeting negative regulators that play an important role in directly inhibiting apoptosis in cancer cells represents a very promising therapeutic strategy for the design of new anticancer drugs.

One class of central negative regulators of apoptosis is the inhibitor of apoptosis proteins (IAPs). This class includes proteins such as XIAP, cIAP1, cIAP2, ML-IAP, HIAP, KIAP, TSIAP, NAIP, survivin, livin, ILP-2, apollon, and BRUCE. IAP proteins effectively inhibit apoptosis in cancer cells induced by a wide variety of apoptotic stimuli, including chemotherapeutics, radiation, and immunotherapy.

Although their roles are not limited to the regulation of apoptosis ^{(7), (8)} , IAP proteins are a class of key apoptosis regulators and are characterized by the presence of one or more BIR (baculoviral IAP repeat) domains ^(5)-(6) . Among IAPs, cellular IAP1 (cIAP1) and cIAP2 play key roles in the regulation of death receptor-mediated apoptosis, while X-linked IAP (XIAP) inhibits death receptor-mediated and mitochondrial-mediated apoptosis by binding to and inhibiting caspase-3/7 and caspase-9, three cysteine proteases that are critical for the execution of apoptosis ⁽⁵⁾ . These IAP proteins are highly overexpressed in both cancer cell lines and human tumor tissues, and have low expression in normal cells and tissues ⁽⁹⁾ . Extensive studies have shown that overexpression of IAP proteins renders cancer cells resistant to apoptosis induction by a variety of anticancer drugs ^(10)-(12) . A detailed discussion of IAP proteins and their roles in cancer and apoptosis is described in U.S. Patent No. 7,960,372, which is incorporated herein by reference. Therefore, targeting one or more of these IAP proteins is a promising therapeutic strategy for treating human cancers ^(10)-(12) .

Studies have shown that peptide-based inhibitors are useful tools for elucidating the anti-apoptotic functions of IAPs and the role of IAPs in the response of cancer cells to chemotherapeutic agents. However, peptide-based inhibitors have inherent limitations as useful therapeutic agents, including poor cell permeability and in vivo stability. In published studies using Smac-based peptide inhibitors, the peptides had to be fused to a carrier peptide to render them relatively cell-permeable.

Small molecule inhibitors of IAP proteins are also known. For example, US Patent Publication No. 2005/0197403 and US Patent No. 7,960,372, each of which is incorporated herein by reference in its entirety, disclose dimeric Smac mimetic compounds.

Despite the discovery of small molecule inhibitors of IAP proteins, the design of effective non-peptide inhibitors of IAP proteins remains a major challenge in modern drug discovery. Therefore, there remains a need in the art for IAP inhibitors with physical and pharmaceutical properties that allow the inhibitors to be used in therapeutic applications. The present invention provides compounds designed to bind to IAP proteins and inhibit IAP protein activity.

SUMMARY OF THE INVENTION

It is generally accepted that the inability of cancer cells or their supporting cells to undergo apoptosis in response to genetic insults or exposure to inducers of apoptosis (e.g., chemotherapeutic agents and radiation) is a major factor in the onset and progression of cancer. Induction of apoptosis in cancer cells or their supporting cells (e.g., neovascular cells in tumor vasculature) is believed to be a universal mechanism of action for virtually all effective cancer therapeutics and radiotherapy in current practice. One reason for the inability of cells to undergo apoptosis is increased expression and accumulation of IAPs.

The present invention relates to inhibitors of IAP proteins, compositions comprising the inhibitors, and methods of using the inhibitors in the therapeutic treatment of conditions and diseases in which inhibition of IAP protein activity provides a benefit. The compounds of the present invention are potent inhibitors of IAP protein activation and induce apoptosis in cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph of mean tumor volume ( ^mm3 ) vs. days post-implantation showing the anti-tumor activity of Examples 2 and 24 in the MDA-MB-231 xenograft model in nude mice.

More specifically, the present invention relates to compounds of structural formula (I):

wherein X is selected from the group consisting of and -SO ₂ -;

Y is selected from -NH-, -O-, -S- and absent;

R is selected from the group consisting of: wherein Ring A is a C _4-8 aliphatic ring, wherein Ring B is an aryl group or a heteroaryl group containing a nitrogen atom, and Ring B is optionally substituted; and R ₁ is selected from the group consisting of: -(CH ₂ ) _4-10 -, -(CH ₂ ) _1-3 CH═CH-(CH ₂ ) _1-3 -, wherein Z is O, S or NH and wherein n is 0, 1 or 2, and wherein Ring B is an aryl group or a heteroaryl group containing a nitrogen atom, and the ring is optionally substituted;

or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

In one embodiment, the invention provides compounds that inhibit the activity of IAP proteins and increase the sensitivity of cells to inducers of apoptosis, such as chemotherapeutic agents and radiation therapy.

In other embodiments, compounds of the invention are used in methods of inducing apoptosis in a cell and sensitizing the cell to an inducer of apoptosis.

In yet another embodiment, the present invention provides a method for treating a condition or disease by administering a therapeutically effective amount of a compound of structural formula (I) to an individual in need thereof. The disease or condition of interest, such as cancer, is treatable by inhibition of IAP proteins. Thus, the compounds of the present invention can be used to treat and ameliorate diseases that respond to the induction of apoptotic cell death, such as diseases characterized by dysregulation of apoptosis, including hyperproliferative diseases such as cancer. In certain embodiments, the compounds can be used to treat and ameliorate cancers characterized by resistance to cancer therapy (e.g., chemo-resistance, radiation-resistance, hormone-resistance, etc.). In other embodiments, the compounds of the present invention can be used to treat hyperproliferative diseases characterized by overexpression of IAP.

Another embodiment of the present invention provides a composition comprising (a) an IAP inhibitor of structural formula (I) and (b) an excipient and/or a pharmaceutically acceptable carrier, which is useful for treating diseases or conditions in which inhibition of IAP proteins provides a benefit.

Another embodiment of the invention is the use of a composition comprising a compound of structural formula (I) and a second therapeutically active agent in a method of treating a disease or condition in a subject in which inhibition of an IAP protein provides a benefit.

In a further embodiment, the present invention provides the use of a composition comprising an IAP protein inhibitor of structural formula (I) and optionally a second therapeutic agent for the preparation of a medicament for treating a disease or condition of interest (eg, cancer).

Yet another embodiment of the present invention is to provide a kit for human pharmaceutical use, comprising (a) a container, (b1) a packaged composition comprising an IAP protein inhibitor of structural formula (I), and optionally (b2) a packaged composition comprising a second therapeutic agent useful for treating a disease or condition of interest, and (c) a package insert containing instructions for using the composition or compositions (administered simultaneously or sequentially) in the treatment of the disease or condition.

The IAP protein inhibitor of structural formula (I) and the second therapeutic agent can be administered together as a single unit dose or separately as multiple unit doses, wherein the IAP inhibitor of structural formula (I) is administered before the second therapeutic agent, or vice versa. It is contemplated that one or more doses of the IAP inhibitor of structural formula (I) and/or one or more doses of the second therapeutic agent can be administered.

In one embodiment, the IAP protein inhibitor of structural formula (I) and the second therapeutic agent are administered simultaneously. In a related embodiment, the IAP protein inhibitor of structural formula (I) and the second therapeutic agent are administered from a single composition or from separate compositions. In a further embodiment, the IAP protein inhibitor of structural formula (I) and the second therapeutic agent are administered sequentially. The IAP protein inhibitor of structural formula (I) as used in the present invention can be administered in an amount of about 0.005 to about 500 mg per dose, an amount of about 0.05 to about 250 mg per dose, or an amount of about 0.5 to about 100 mg per dose.

Detailed Description of Preferred Embodiments

The present invention is described in conjunction with preferred embodiments. However, it should be understood that the invention is not limited to the disclosed embodiments. It should be understood that various modifications may be made by those skilled in the art in view of the description of the embodiments of the invention herein. Such modifications are encompassed by the following claims.

Smac/DIABLO (second mitochondrial-derived activator of caspases or direct IAP-binding protein with low PI) is a protein released from mitochondria in response to apoptotic stimuli and acts as an endogenous inhibitor of cIAP1, cIAP2, and XIAP ^{(14), (15)} . The interaction between Smac and IAPs is mediated by the N-terminal AVPI tetrapeptide motif in Smac and one or more BIR domains in these IAP proteins ^{(16), (17)} . Smac is a homodimer that binds to both the BIR2 and BIR3 domains in XIAP and antagonizes XIAP inhibition of caspase-3/-7 and caspase-9 ⁽¹⁸⁾ . In contrast, Smac binds only to the BIR3 domain in cIAP1 and cIAP2 ⁽¹⁹⁾ and induces rapid protein degradation in cells ⁽²⁰⁾ . Smac is a very potent antagonist of these three IAP proteins through two different mechanisms.

Crystal and NMR structures of XIAP BIR3 in complex with Smac protein or Smac peptides show that the AVPI tetrapeptide motif in Smac binds to a well-defined surface groove in XIAP and that this interaction represents an attractive site for designing small molecule XIAP inhibitors ^(16)-(18) . By using the AVPI tetrapeptide as a lead structure, several classes of small molecule Smac mimetics have been designed as antagonists of XIAP and cIAP1/2 ^(21)-(38) . Two different types of Smac mimetics have been designed ^(21)-(23) . The first type, designed to mimic a single AVPI binding motif, is referred to as a monovalent Smac mimetic ^(21)-(23) . The second type, a bivalent Smac mimetic, consists of two AVPI mimetics tethered by a linker to mimic the dimeric form of the Smac protein ^(21)-(23) .

One advantage of monovalent Smac mimetics as potential drugs is oral bioavailability, but a disadvantage is their modest potency against full-length XIAP in functional assays. The main advantage of bivalent Smac mimetics is that they are much more potent XIAP antagonists than monovalent Smac mimetics by simultaneously targeting both the BIR2 and BIR3 domains in XIAP ⁽³⁰⁾ . Bivalent Smac mimetics are generally 2-3 orders of magnitude more effective than their monovalent Smac mimetic counterparts in inducing apoptosis in cancer cells ⁽²¹⁾ . Currently, three monovalent and two bivalent Smac mimetics have entered clinical trials for the treatment of human cancer ⁽²¹⁾ .

Because bivalent Smac mimetics are significantly more effective than monovalent Smac mimetics in targeting XIAP and cIAP1/2, inducing apoptosis in cancer cells in vitro and in vivo, and inhibiting tumor growth, the present bivalent compounds have been designed for cancer therapy and for treating other diseases and conditions mediated by IAP protein activity.

As used herein, the term "IAP protein" refers to any known member of the inhibitor of apoptosis protein family, including, but not limited to, XIAP, cIAP-1, cIAP-2, ML-IAP, HIAP, TSIAP, KIAP, NAIP, survivin, livin, ILP-2, apollon, and BRUCE.

As used herein, the term "IAP overexpression" refers to elevated (e.g., abnormal) levels of mRNA encoding an IAP protein and/or elevated levels of an IAP protein in a cell, compared to a basal level of mRNA encoding the IAP protein or a comparable, corresponding, non-diseased cell expressing the IAP protein, or a comparable, non-diseased cell with a basal level of the IAP protein. Methods for detecting the levels of IAP protein mRNA or IAP protein in cells include, but are not limited to, Western blotting using antibodies against IAP proteins, immunohistochemistry, and nucleic acid amplification or direct RNA detection. Equally important as determining the absolute level of IAP protein in cells overexpressing IAP protein is determining the relative level of IAP protein in such cells compared to other pro-apoptotic signaling molecules (e.g., pro-apoptotic Bcl-2 family proteins). A cell becomes dependent on IAP proteins for survival when the balance between these two factors is such that, if not for the IAP protein level, the pro-apoptotic signaling molecules would be sufficient to cause the cell to execute the apoptotic program and die. In such cells, exposure to an inhibitory effective amount of an IAP protein inhibitor would be sufficient to cause the cell to execute the apoptotic program and die. Thus, the term "overexpression of an IAP protein" also refers to the fact that due to the relative levels of pro- and anti-apoptotic signals, a cell undergoes apoptosis in response to an inhibitory effective amount of a compound that inhibits IAP protein function.

The term "disease or condition in which inhibition of an IAP protein provides a benefit" relates to conditions in which an IAP protein, and/or the effects of an IAP protein, are important or necessary, e.g., for the onset, progression, or manifestation of the disease or condition, or a disease or condition known to be treated by an IAP protein inhibitor. Examples of such conditions include, but are not limited to, cancer. One of ordinary skill in the art can readily determine whether a compound treats a disease or condition mediated by an IAP protein in any particular cell type, e.g., by using assays that can be readily used to assess the activity of a particular compound.

The term "second therapeutic agent" refers to a therapeutic agent other than an IAP inhibitor of formula (I) that is known to treat the disease or condition of interest. For example, when cancer is the disease or condition of interest, the second therapeutic agent may be, for example, a known chemotherapeutic agent (such as taxol) or radiation.

The term "disease" or "disorder" refers to a disorder and/or abnormality that is generally considered a pathological state or function and that can manifest itself in the form of specific signs, symptoms, and/or functional impairments. As demonstrated below, the compounds of structural formula (I) are potent inhibitors of IAP proteins and can be used to treat diseases and disorders in which inhibition of IAP proteins provides a benefit.

As used herein, the terms "treat," "treating," "treatment," and the like refer to eliminating, reducing, or ameliorating a disease or disorder, and/or the symptoms associated therewith. Although not excluded, treating a disease or disorder does not require that the disease, disorder, or the symptoms associated therewith be completely eliminated. As used herein, the terms "treat," "treating," "treatment," and the like may include "prophylactic treatment," which refers to reducing the likelihood of redevelopment of a disease or disorder or recurrence of a previously controlled disease or disorder in a subject who does not have the disease or disorder but is at risk of redeveloping the disease or disorder or recurring the disease or disorder or is susceptible to redeveloping the disease or disorder or recurring the disease or disorder. The terms "treatment" and synonyms contemplate administering a therapeutically effective amount of a compound of the present invention to an individual in need of such treatment.

Within the meaning of the present invention, "treatment" also includes relapse prevention or stage prevention, as well as the treatment of acute or chronic signs, symptoms and/or functional disorders. Treatment can be symptomatic, for example, to suppress symptoms. It can be achieved over a short period of time, directed over a medium period of time, or can be long-term treatment, for example in the context of maintenance therapy.

As used herein, the terms "sensitize" and "sensitizing" refer to making an animal or cells within an animal more sensitive or responsive to the biological effects of a second active agent (e.g., promoting or blocking aspects of cellular function, including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, or apoptosis) by administering a first therapeutic agent (e.g., a compound of Structural Formula I). The sensitizing effect of a first agent on a target cell can be measured as the difference in a specified biological effect (e.g., promoting or blocking aspects of cellular function, including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed after administration of the second agent with and without the administration of the first agent. The response of the sensitized cells can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 350%, at least 300%, at least 350%, at least 400%, at least 450% or at least 500% compared to the response in the absence of the first agent.

As used herein, the term "hyperproliferative disease" refers to any condition in which a localized population of proliferating cells in an animal is not subject to the usual normal growth restrictions. Examples of hyperproliferative diseases include, but are not limited to, tumors, neoplasms, lymphomas, and the like. A neoplasm is considered benign if it does not undergo invasion or metastasis; if one of these two conditions occurs, it is considered malignant. "Metastatic" cells mean that the cells can invade and destroy nearby body structures. Hyperplasia is a form of cell proliferation that involves an increase in the number of cells in a tissue or organ without significant changes in structure or function. Tissue deformation is a form of controlled cell growth in which one type of fully differentiated cell replaces another type of differentiated cell.

The pathological growth of activated lymphoid cells usually leads to autoimmune diseases or chronic inflammatory diseases. As used herein, the term "autoimmune disease" refers to any disease in which an organism produces antibodies or immune cells that recognize its own molecules, cells or tissues. Non-limiting examples of autoimmune diseases include autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease or IgA nephropathy, sprue, chronic fatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graft-versus-host disease, Graves' disease, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, lichen planus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo, etc.

As used herein, the term "neoplastic disease" refers to any abnormal cell growth, either benign (non-cancerous) or malignant (cancerous).

As used herein, the term "anti-tumor agent" refers to any compound that blocks the proliferation, growth, or spread of a targeted (eg, malignant) neoplasm.

As used herein, the term "apoptosis regulator" refers to an agent that participates in the regulation (e.g., inhibition, reduction, increase, promotion) of apoptosis. Examples of apoptosis regulators include proteins comprising a death domain, such as, but not limited to, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DR5, DR6, FADD, and RIP. Other examples of apoptosis regulators include, but are not limited to, TNFα, Fas ligand, antibodies to Fas/CD95 and other TNF family receptors, TRAIL (also known as Apo2 ligand or Apo2L/TRAIL), agonists (e.g., monoclonal or polyclonal agonist antibodies) of TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinases, PP1, and caspase proteins. Regulators broadly include agonists and antagonists of TNF family receptors and TNF family ligands. Apoptosis regulators can be soluble or membrane-bound (e.g., ligand or receptor). Preferred modulators of apoptosis are inducers of apoptosis, such as TNF or a TNF-related ligand, in particular TRAMP ligand, Fas/CD95 ligand, TNFR-1 ligand or TRAIL.

As used herein, the term "dysregulation of apoptosis" refers to any abnormality in the ability of a cell to undergo cell death (e.g., a predisposition) by apoptosis. Dysregulation of apoptosis is associated with or induced by a variety of conditions, including, for example, autoimmune diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjogren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma, or Crohn's disease), hyperproliferative diseases (e.g., tumors, B-cell lymphomas, or T-cell lymphomas), viral infections (e.g., herpes, papilloma, or HIV), and other conditions, such as osteoarthritis and atherosclerosis. It should be noted that when the disorder is induced by or associated with a viral infection, the viral infection may be detected at the time the disorder occurs or is observed, or the viral infection may not be detected. That is, a viral-induced disorder may occur even after the symptoms of the viral infection have disappeared.

As used herein, the term "therapeutically effective amount" or "effective dose" refers to an amount of one or more active ingredients that, when administered by the methods of the present invention, is sufficient to effectively deliver one or more active ingredients for treating the condition or disease of interest to a subject in need thereof. In the case of cancer or other proliferative disorders, a therapeutically effective amount of an agent can reduce (i.e., delay to some extent and preferably stop) unwanted cell proliferation; reduce the number of cancer cells; reduce tumor size; inhibit (i.e., delay to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., delay to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; reduce IAP protein signaling in target cells to increase survival time; and/or alleviate one or more cancer-related symptoms to some extent by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. To the extent that the administered compound or composition prevents the growth of and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.

The term "container" means any container and its closure suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.

The term "insert" means the information that accompanies a drug product and provides instructions on how to administer the product, along with the safety and effectiveness data needed to allow physicians, pharmacists, and patients to make informed decisions about the product's use. Package inserts are generally considered the "labeling" for drug products.

"Simultaneous administration," "administered in combination," "administered concurrently," and similar phrases mean that two or more agents are administered to the subject being treated at the same time. "Concurrently" means that each agent is administered simultaneously or sequentially in any order at different time points. However, if the administration is not simultaneous, it means that they are administered to the subject in sequence and sufficiently close in time to provide the desired therapeutic effect and to act synergistically. For example, an IAP protein inhibitor of structural formula (I) can be administered sequentially with a second therapeutic agent at the same time or at different time points in any order. The IAP protein inhibitor of the present invention and the second therapeutic agent can be administered separately in any suitable form and by any suitable route. When the IAP protein inhibitor of the present invention and the second therapeutic agent are not administered simultaneously, it will be understood that they can be administered to the subject in need thereof in any order. For example, an IAP protein inhibitor of the invention can be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or following (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapeutic modality (e.g., radiation therapy). In various embodiments, the IAP protein inhibitor of structural formula (I) and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 to 2 hours apart, 2 to 3 hours apart, 3 to 4 hours apart, 4 to 5 hours apart, 5 to 6 hours apart, 6 to 7 hours apart, 7 to 8 hours apart, 8 to 9 hours apart, 9 to 10 hours apart, 10 to 11 hours apart, 11 to 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart. In one embodiment, the components of the combination therapy are administered 1 minute to 24 hours apart.

In the context of describing the present invention (especially in the context of the claims), the use of the terms "a", "an", "the" and similar designators should be understood to cover both the singular and the plural, unless otherwise indicated. Unless otherwise indicated herein, the recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referencing each individual value that falls within the range, and each individual value is incorporated into this specification as if it were individually enumerated herein. The use of any and all examples or exemplary language (such as, for example) provided herein is intended to better illustrate the present invention, rather than to limit the scope of the present invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present invention.

The present invention relates to compounds of structural formula (I), which are mimetics of Smac and act as inhibitors of IAP proteins. The compounds of the present invention sensitize cells to inducers of apoptosis and, in certain cases, themselves induce apoptosis by inhibiting IAP proteins. Therefore, the present invention relates to methods for sensitizing cells to inducers of apoptosis and methods for inducing apoptosis in cells, comprising contacting the cells with a compound of structural formula I alone or in combination with an inducer of apoptosis. The present invention further relates to methods for treating or ameliorating diseases in animals that respond to the induction of apoptosis, comprising administering to the animals a compound of structural formula I and an inducer of apoptosis. Such diseases include diseases characterized by dysregulation of apoptosis and diseases characterized by overexpression of IAP proteins.

The present invention relates to potent inhibitors of IAP proteins. These IAP protein inhibitors are non-peptide, bivalent Smac mimetics that bind XIAP, cIAP1, and cIAP2 with subnanomolar affinity and are highly potent XIAP antagonists in cell-free functional assays. At low concentrations, the compounds of this invention effectively induce the degradation of cIAP1 and cIAP2, activate caspases-3 and -8, and cleave PARP in cancer cells. The compounds of this invention exhibit low _IC50 inhibition of cell growth in both MDA-MB-231 and SK-OV-3 cell lines.

Thus, the IAP protein inhibitors of the present invention can be used to treat unwanted proliferating cells (including cancer and precancerous conditions) in a subject in need of such treatment. Also provided are methods of treating a subject having unwanted proliferating cells, comprising administering a therapeutically effective amount of a compound of the present invention to a subject in need of such treatment. Also provided are methods of preventing the proliferation of unwanted proliferating cells (e.g., cancer and precancerous conditions) in a subject, comprising the step of administering a therapeutically effective amount of a compound of structural formula (I) to a subject at risk of developing a condition characterized by unwanted proliferating cells. In some embodiments, the compounds of structural formula (I) reduce the proliferation of unwanted cells by inducing apoptosis in those cells.

The present invention relates to an IAP protein inhibitor having the structural formula (I):

wherein X is selected from the group consisting of and -SO ₂ -;

Y is selected from -NH-, -O-, -S- and absent;

R is selected from the group consisting of: wherein Ring A is a C _4-8 aliphatic ring, wherein Ring B is an aryl group or a heteroaryl group containing a nitrogen atom, and Ring B is optionally substituted; and R ₁ is selected from the group consisting of: -(CH ₂ ) _4-10 -, -(CH ₂ ) _1-3 CH═CH-(CH ₂ ) _1-3 -, wherein Z is O, S or NH and wherein n is 0, 1 or 2, and wherein Ring B is an aryl group or a heteroaryl group containing a nitrogen atom, and the ring is optionally substituted;

or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof.

As used herein, the term " _C4-8 aliphatic ring" refers to cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups that are unsubstituted or substituted with 1 to 3 groups (e.g., _C1-4 alkyl, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino or amino).

As used herein, the term "alkyl" refers to a linear and branched saturated _C1-10 hydrocarbon group, non-limiting examples of which include methyl, ethyl, and linear and branched propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. The term _Cn means that the alkyl group has "n" carbon atoms.

The term " _C3-6 cycloalkylene" refers to a disubstituted cycloalkane having 3 to 6 carbon atoms, for example, " _C3-6 cycloalkylene" may be unsubstituted or substituted with 1 to 3 groups, for example, _C1-4 alkyl, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino or amino.

The term "alkenyl" has the same meaning as "alkyl" except that it contains a carbon-carbon double bond, for example, ethenyl, propenyl, and butenyl.

The term "halo," as used herein, is defined as fluorine, chlorine, bromine, and iodine.

The term "hydroxy" is defined as -OH.

The term "alkoxy" is defined as -OR, where R is alkyl.

The term "amino" is defined as _-NH2 , and the term "alkylamino" is defined as _-NR2 , wherein at least one R is alkyl and the second R is alkyl or hydrogen.

The term "nitro" is defined as _-NO2 .

The term "cyano" is defined as -CN.

The term "trifluoromethyl" is defined as _-CF3 .

The term "trifluoromethoxy" is defined as _-OCF3 .

As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, such as phenyl or naphthyl. Unless otherwise indicated, an aryl group may be unsubstituted or substituted by one or more, and in particular one to four, groups independently selected from, for example, halogen, alkyl, alkenyl, -OCF ₃ , -NO ₂ , -CN, -NC, -OH, alkoxy, amino, alkylamino, -CO ₂ H, -CO ₂ alkyl, alkynyl, cycloalkyl, nitro, thiol, imino, amido, phosphonate, phosphinate, silyl, alkylthio, sulfonyl, sulfonamide, aldehyde, heterocycloalkyl, trifluoromethyl, aryl and heteroaryl.

As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic ring system containing one or two aromatic rings and at least one and up to four nitrogen atoms in one aromatic ring. Unless otherwise indicated, a heteroaryl group may be unsubstituted or substituted by one or more, and in particular one to four, substituents selected from, for example, halogen, alkyl, alkenyl, -OCF ₃ , -NO ₂ , -CN, -NC, -OH, alkoxy, amino, alkylamino, -CO ₂ H, -CO ₂ alkyl, alkynyl, cycloalkyl, nitro, thiol, imino, amido, phosphonate, phosphinate, silyl, alkylthio, sulfonyl, sulfonamide, aldehyde, heterocycloalkyl, trifluoromethyl, aryl, and heteroaryl.

The term "arylene" refers to a bidentate aryl group that is bonded to two other groups and serves to link these groups, for example, the term "heteroarylene" is similarly defined.

Non-limiting examples of aryl groups are

Non-limiting examples of heteroaryl groups are

Compounds of structural formula (I) inhibit IAP proteins and are useful in treating various diseases and conditions. Specifically, compounds of structural formula (I) are used in methods for treating diseases or conditions in which inhibition of IAP proteins provides a benefit (e.g., cancer, autoimmune diseases, and chronic inflammatory conditions). The methods comprise administering a therapeutically effective amount of a compound of structural formula (I) to a subject in need thereof. In addition to the compound of structural formula (I), the methods of the present invention also encompass administering a second therapeutic agent to the subject. The second therapeutic agent is selected from drugs known to be useful for treating the disease or condition afflicting the subject in need thereof, such as chemotherapeutic agents and/or radiation known to be useful for treating specific cancers.

In some preferred embodiments, Ring B is phenyl, naphthyl, pyridyl, pyridazinyl, pyrazinyl, or pyrimidinyl.

In some preferred embodiments, R includes, but is not limited to:

wherein p is 0 to 4, wherein q is 0 to 2, and —(CH ₂ ) _2-4 —C ₆ H ₅ .

Specific R groups include, but are not limited to:

In some preferred embodiments, R ₁ is, but is not limited to, -(CH ₂ ) _4-8 -, -(CH ₂ ) _4-8 -, -(CH ₂ ) _1-2 -CH=CH-(CH ₂ ) _1-2 -, wherein n is 0 or 1.

Specific _R1 groups include, but are not limited to, -( _CH2 ) _4- , -( _CH2 ) _6- , -(CH2) _8- , -( _{CH2 ) 2} -CH=CH-(CH) _1-2- ,

In some preferred embodiments, X is and Y is -NH-.

In other preferred embodiments, X is _SO2 , and Y is absent.

In another preferred embodiment, X is and Y is absent.

In yet another preferred embodiment, X is and Y is -NH-.

In yet another preferred embodiment, X and X' are and Y is -O-.

In addition, salts, hydrates, solvates and prodrugs of the compounds of the present invention are also included in the present invention and can be used in the methods disclosed herein. The present invention further includes all possible stereoisomers and geometric isomers of the compounds of structural formula (I). The present invention includes both racemic compounds and optical isomers. When it is desired that the compound of structural formula (I) is a single enantiomer, it can be obtained by splitting the final product or by stereospecific synthesis from isomerically pure starting materials or using chiral auxiliary agents, for example, see Z.Ma et al., Tetrahedron: Asymmetry, 8 (6), pages 883-888 (1997). The splitting of the final product, intermediate or starting material can be achieved by any suitable method known in the art. In addition, where tautomers of the compounds of structural formula (I) are possible, the present invention is intended to include all tautomeric forms of the compound.

The compounds of the present invention may exist as salts. Pharmaceutically acceptable salts of the compounds of the present invention are often preferred in the methods of the present invention. As used herein, the term "pharmaceutically acceptable salt" refers to any salt of the compound of the present invention that is physiologically tolerated in the target animal (e.g., mammal) (e.g., obtained by reacting with an acid or base). The salts of the compounds of the present invention may be derived from inorganic or organic acids and bases. The term "pharmaceutically acceptable salt" also refers to the zwitterionic form of the compound of formula (I). The salt of the compound of formula (I) may be prepared during the final separation and purification process of the compound, or separately by reacting the compound with an acid having a suitable cation. The pharmaceutically acceptable salt of the compound of formula (I) may be an acid addition salt formed with a pharmaceutically acceptable acid. Examples of acids that can be used to form pharmaceutically acceptable salts include inorganic acids (e.g., nitric acid, boric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid) and organic acids (e.g., oxalic acid, maleic acid, succinic acid, and citric acid). Non-limiting examples of salts of the compounds of the present invention include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-isothionate, phosphate, hydrogenphosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthenate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, edisylate, benzenesulfonate, and p-toluenesulfonate. Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of the formula NW ₄ ⁺ , wherein W is C _1-4 alkyl, etc. Additionally, available amino groups present in the compounds of the present invention may be quaternized with chlorides, bromides, and iodides of methyl, ethyl, propyl, and butyl; sulfates of dimethyl, diethyl, dibutyl, and dipentyl; chlorides, bromides, and iodides of decyl, dodecyl, myristyl, and steryl; and bromides of benzyl and phenethyl.

The compounds of formula (I) may contain one or more asymmetric centers and may therefore exist as stereoisomers. The present invention includes both mixtures and individual stereoisomers. Specifically, the compounds of formula (I) include individual cis and trans isomers as well as mixtures of cis and trans isomers, for example,

As used herein, the term "prodrug" refers to a pharmaceutically inactive derivative of a parent "drug" molecule that requires biotransformation (e.g., spontaneous or enzymatic) within the target physiological system to release or convert (e.g., by enzymatic, physiological, mechanical, electromagnetic means) the prodrug into an active drug. Prodrugs are designed to overcome problems associated with stability, toxicity, lack of specificity, or limited bioavailability.

Prodrugs generally provide advantages in solubility, tissue compatibility, or delayed release in mammals (see, e.g., Bundgard, "Design of Prodrugs", pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, "The Organic Chemistry of Drug Design and Drug Action", pp. 352-401, Academic Press, San Diego, CA (1992)). Exemplary prodrugs comprise the active drug molecule itself and a chemical masking group (e.g., a group that reversibly inhibits the activity of the drug). Some preferred prodrugs are modifications or derivatives of compounds having groups that are cleavable under metabolic conditions. Exemplary prodrugs become pharmaceutically active in vivo or in vitro when they undergo solvolysis under physiological conditions or undergo enzymatic degradation or other biochemical transformations (e.g., phosphorylation, hydrogenation, dehydrogenation, glycosylation). Commonly used prodrugs include acid derivatives such as esters prepared by reacting the parent acid with a suitable alcohol (e.g., a lower alkanol), amides prepared by reacting the parent acid compound with an amine, or basic groups (e.g., lower alkyl amides) that react to form acylated base derivatives.

Specific compounds of the present invention include, but are not limited to, compounds having the structures listed below.

The present invention provides IAP protein inhibitors, as exemplified by compounds of structural formula (I), for use in treating a variety of diseases and conditions in which inhibition of IAP proteins has a beneficial effect. In one embodiment, the present invention relates to a method of treating a subject suffering from a disease or condition in which inhibition of an IAP protein provides a benefit, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of structural formula (I).

The present IAP protein inhibitors address unmet needs for the treatment of a variety of cancer types, either when administered as monotherapy to induce apoptosis in cancer cells that rely on IAP function, or when administered in a temporal relationship with other anti-cancer treatments such that a greater proportion of cancer cells become susceptible to the apoptotic program compared to the corresponding proportion of cells in animals treated with the cancer therapeutic drug or radiation therapy alone.

As used herein, the term "anti-cancer therapy" refers to therapeutic agents (eg, chemotherapeutic compounds and/or molecular therapeutic compounds), radiation therapy, and surgical intervention used to treat a hyperproliferative disease, such as cancer in a mammal.

The methods of the present invention can be accomplished by administering a compound of formula (I) as a pure compound or as a pharmaceutical composition. Administration of the pharmaceutical composition or pure compound of formula (I) can be performed during or after the onset of the disease or condition of interest. Typically, the pharmaceutical composition is sterile and does not contain toxic, carcinogenic, or mutagenic compounds that may cause adverse reactions upon administration. Further provided are kits comprising: a compound of formula (I) and, optionally, a second therapeutic agent useful for treating diseases and conditions in which inhibition of IAP proteins provides a benefit, packaged separately or together, and an insert with instructions for use of these active agents.

In many embodiments, a compound of structural formula (I) is administered in conjunction with a second therapeutic agent useful for treating a disease or condition in which inhibition of an IAP protein provides a benefit. The second therapeutic agent is different from the compound of structural formula (I). The compound of structural formula (I) and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect. In addition, the compound of structural formula (I) and the second therapeutic agent can be administered from a single composition or two separate compositions.

The second therapeutic agent is administered in an amount that provides its desired therapeutic effect. Effective dosage ranges for each second therapeutic agent are known in the art, and the second therapeutic agent is administered to an individual in need thereof within this established range.

In certain embodiments, combination therapy comprising administering a therapeutically effective amount of a compound of formula (I) and a second therapeutic agent results in a greater tumor response and greater clinical benefit compared to treatment with either the compound of formula (I) or the second therapeutic agent alone.

The compounds of structural formula (I) can also be used to achieve the administration of a lower, and therefore less toxic and more tolerable, dose of a second therapeutic agent to produce the same tumor response/clinical benefit as a conventional dose of the second therapeutic agent. In addition, because the compounds of the present invention act at least in part by inhibiting IAP proteins, exposure of cancer cells and supporting cells to therapeutically effective amounts of the present IAP protein inhibitors can be temporally linked to coincide with attempts to program cells to undergo apoptosis in response to the second therapeutic agent. Thus, in some embodiments, administering the compounds of the present invention in conjunction with a second therapeutic agent in a certain temporal relationship provides particularly effective therapeutic results.

Thus, the compound of formula (I) and the second therapeutic agent can be administered together as a single unit dose or separately as multiple unit doses, wherein the compound of formula (I) is administered before the second therapeutic agent, or vice versa. One or more doses of the compound of formula (I) and/or one or more doses of the second therapeutic agent can be administered. Thus, the compound of formula (I) can be used in combination with one or more second therapeutic agents (such as, but not limited to, anticancer agents).

Diseases or conditions that can be treated according to the present invention include, for example, cancer. A variety of cancers can be treated, including, but not limited to: carcinomas, including bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer, kidney cancer, and skin cancer (including squamous cell carcinoma); hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, and skin cancer (including squamous cell carcinoma); hematopoietic tumors of the lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, and The invention relates to hematopoietic neoplasms of the myeloid lineage, including acute and chronic myeloid leukemias, myelodysplastic syndromes, myeloid leukemias, and promyelocytic leukemias; tumors of the central and peripheral nervous systems, including astrocytomas, neuroblastomas, gliomas, and schwannomas; tumors of mesenchymal origin, including fibrosarcomas, rhabdomyosarcomas, and osteosarcomas; and other tumors, including melanomas, xenoderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, teratocarcinoma, renal cell carcinoma (RCC), pancreatic cancer, myeloma, myeloid and lymphoblastic leukemias, neuroblastomas, and glioblastomas.

Additional forms of cancer treatable by the IAP protein inhibitors of the invention include, for example, adult and pediatric tumors, growths of solid tumors/malignancies, mucous cell and round cell carcinomas, locally advanced tumors, metastatic carcinomas, human soft tissue sarcomas (including Ewing sarcoma), carcinoma metastases (including lymphatic metastases), squamous cell carcinomas (particularly of the head and neck), esophageal squamous cell carcinoma, oral cancer, blood cell malignancies (including multiple myeloma, leukemias (including acute lymphocytic leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia)), effusion lymphomas (body cavity-based lymphomas), thymic lymphomas, lung cancer (including small cell carcinomas), cutaneous T-cell lymphomas, Hodgkin lymphoma , non-Hodgkin lymphoma, adrenocortical carcinoma, ACTH-producing tumors, non-small cell carcinoma, breast cancer (including small cell carcinoma and ductal carcinoma), gastrointestinal cancer (including gastric cancer, colon cancer, colorectal cancer, and polyps associated with colorectal neoplasia), pancreatic cancer, liver cancer, urinary tract cancer (including bladder cancer, such as primary superficial bladder tumors, invasive bladder transitional cell carcinoma, and muscle-invasive bladder cancer), prostate cancer, malignancies of the female genital tract (including ovarian cancer, primary peritoneal epithelial tumors, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine cancer, and solid tumors in the ovarian follicles), malignancies of the male genital tract (including testicular cancer and penile cancer), kidney cancer (including renal cell carcinoma), brain cancer (including intrinsic brain tumors brain tumor), neuroblastoma, astrocytic brain tumor, glioma, and metastatic tumor cell infiltration in the central nervous system), bone cancer (including osteoma and osteosarcoma), skin cancer (including malignant melanoma, tumor progression of human skin keratinocytes, and squamous cell carcinoma), thyroid cancer, retinoblastoma, neuroblastoma, ascites, malignant pleural effusion, mesothelioma, Wilms' tumor, gallbladder cancer, trophoblastic tumor, hemangiopericytoma, and Kaposi's sarcoma.

Another embodiment of the present invention is to induce apoptosis and enhance the induction of apoptosis in response to apoptosis-inducing signals by using an IAP protein inhibitor of structural formula (I). The present IAP protein inhibitor also sensitizes cells to apoptosis inducers, including cells resistant to such inducers. The IAP protein inhibitors of the present invention can be used to induce apoptosis in any disease that can be treated, improved, or prevented by inducing apoptosis. Therefore, the present invention provides compositions and methods for targeting animals characterized by overexpression of IAP proteins. In some embodiments, the cells (e.g., cancer cells) show elevated expression levels of IAP proteins compared to non-pathological samples (e.g., non-cancerous cells). In other embodiments, the cells operatively exhibit elevated expression levels of IAP proteins by virtue of executing an apoptotic program and dying in response to a therapeutically effective amount of a compound of structural formula (I), the response being at least in part due to the dependence of such cells on the function of IAP proteins for their survival.

In another embodiment, the present invention relates to regulating a state associated with apoptosis, which state is associated with one or more apoptosis regulators. Examples of apoptosis regulators include, but are not limited to, antibodies to Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DR5, DR6, FADD, RIP, TNFα, Fas ligand, TRAIL, TRAIL-RI or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase, PP1, and caspase proteins. Other agents involved in the initiation, determination, and degeneration stages of apoptosis are also included. Examples of apoptosis regulators include agents whose activity, presence, or concentration changes can regulate apoptosis in a subject. Preferred apoptosis regulators are apoptosis inducers, such as TNF or TNF-related ligands, particularly TRAMP ligand, Fas/CD95 ligand, TNFR-1 ligand, or TRAIL.

These treatments can be used in a variety of settings for the treatment of various cancers. In a specific embodiment, the individual in need of treatment has previously undergone treatment for the cancer. Such prior treatments include, but are not limited to, prior chemotherapy, radiation therapy, surgery, or immunotherapy, such as cancer vaccines.

In one embodiment, the present invention provides a method of treating cancer comprising: (a) administering to a subject in need thereof a therapeutically effective amount of an IAP protein inhibitor of structural formula (I); and (b) administering to the subject a therapeutically effective amount of one or more of radiation therapy, chemotherapy, and immunotherapy. The amounts administered are each effective for treating the cancer. In another embodiment, the amounts together are effective for treating the cancer.

In another embodiment, the present invention provides a method of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition comprising an IAP protein inhibitor of structural formula (I).

In another embodiment, the present IAP protein inhibitors are used in methods for treating the following diseases: T and B cell-mediated autoimmune diseases; inflammatory diseases; infections; hyperproliferative diseases; AIDS; degenerative disorders; vascular diseases; etc. In some embodiments, infections suitable for treatment with the compositions and methods of the present invention include, but are not limited to, infections caused by viruses, bacteria, fungi, mycoplasmas, viruses, etc.

The compounds of the present invention and methods can also be used to treat autoimmune diseases or chronic inflammatory conditions. As used herein, the term "autoimmune disease" refers to any condition in which an organism produces antibodies or immune cells that recognize its own molecules, cells, or tissues. Non-limiting examples of autoimmune diseases include autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease or IgA nephropathy, sprue, chronic fatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graft-versus-host disease, Graves' disease, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, lichen planus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo, etc.

Additional diseases and conditions, including cancer, that can be treated by administering the IAP protein inhibitors of the invention are disclosed in US Patent 7,960,372; incorporated herein by reference in its entirety.

In the methods of the present invention, a therapeutically effective amount of one or more compounds (I), typically formulated in accordance with pharmaceutical practice, is administered to a person in need thereof. Whether such treatment is indicated depends on the individual case and is subject to medical evaluation (diagnosis) that takes into account the presence of signs, symptoms, and/or disorders, the risk of developing specific signs, symptoms, and/or disorders, and other factors.

The compounds of structural formula (I) can be administered by any suitable route, for example, by oral, buccal, inhalation, sublingual, rectal, vaginal, intracisternal or intradural administration via lumbar puncture, transurethral, nasal, transdermal (i.e., transdermal), or parenteral (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a specific site). Parenteral administration can be accomplished using a needle and syringe or using high-pressure techniques.

Pharmaceutical compositions include those in which the compound of formula (I) is administered in an effective amount to achieve its intended purpose. The precise formulation, route of administration, and dosage are determined by the individual physician in view of the condition or disease being diagnosed. The dosage amount and interval can be adjusted individually to provide a level of the compound of formula (I) sufficient to maintain the therapeutic effect.

The toxicity and therapeutic efficacy of the compounds of structural formula (I) can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, for determining the maximum tolerated dose (MTD) of the compound (defined as the highest dose that does not cause any toxicity in animals). The dose ratio between the maximum tolerated dose and the therapeutic effect (e.g., inhibition of tumor growth) is the therapeutic index. The dose can vary within this range depending on the dosage form employed and the route of administration utilized. The determination of a therapeutically effective amount, especially in view of the detailed disclosure provided herein, is well within the capabilities of those skilled in the art.

The therapeutically effective amount of a compound of formula (I) required for use in therapy varies with the nature of the condition being treated, the desired duration of activity, and the age and condition of the patient, and is ultimately determined by the attending physician. The number of doses and the interval between doses can be individually adjusted to provide a plasma level of the IAP protein inhibitor sufficient to maintain the desired therapeutic effect. The desired dose can be conveniently administered as a single dose or as multiple doses administered at appropriate intervals, for example, as one, two, three, four, or more sub-doses per day. Multiple doses are often desirable or necessary. For example, an IAP protein inhibitor of the invention can be administered at the following frequencies: four doses, delivered as one dose per day, at four-day intervals (q4d×4); four doses, delivered as one dose per day, at three-day intervals (q3d×4); one dose per day, at five-day intervals (qd×5); one dose per week for three weeks (qwk3); five daily doses, two days off, and an additional five daily doses (5/2/5); or any dosage regimen determined to be appropriate for the situation.

The compound of formula (I) used in the methods of the present invention can be administered in an amount of about 0.005 to about 500 mg per dose, in an amount of about 0.05 to about 250 mg per dose, or in an amount of about 0.5 to about 100 mg per dose. For example, the compound of formula (I) can be administered in an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg per dose (including all doses between 0.005 mg and 500 mg).

The dosage of a composition containing an IAP protein inhibitor of structural formula (I) or a composition containing the same can be about 1 ng/kg to about 200 mg/kg, about 1 μg/kg to about 100 mg/kg, or about 1 mg/kg to about 50 mg/kg. The dosage of the composition can be any dosage, including but not limited to about 1 μg/kg. The dosage of the composition can be any dosage, including but not limited to about 1 μg/kg, 10 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 1 mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40 mg/kg, 45mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, 100mg/kg, 125 mg/kg, 150mg/kg, 175mg/kg, or 200mg/kg. The above dosages are examples of average cases, but there may be individual cases where higher or lower dosages are warranted, and this is within the scope of the invention. In practice, the physician determines the actual dosage regimen that is most suitable for an individual patient, which may vary with the age, weight, and response of the particular patient.

In the treatment of cancer, compounds of formula (I) may be administered together with chemotherapeutic agents and/or immunotherapeutic agents and/or radiation or in combination with another therapeutic technique such as surgery. The term chemotherapeutic agent as used herein includes anticancer agents, antitumor agents, apoptosis regulators.

Embodiments of the present invention employ the following electromagnetic radiation: gamma radiation ( ^10-20 to ^10-13 m), X-ray radiation ( ^10-12 to ^10-9 m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1 mm), and microwave radiation (1 mm to 30 cm).

Many cancer treatment regimens currently utilize radiosensitizers that are activated by electromagnetic radiation (e.g., X-rays). Examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, nidazole, demethylnidazole, pimonidazole, etanerazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives thereof.

Photodynamic therapy (PDT) of cancer uses visible light as a radioactive sensitizer. Examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, benzoporphyrin derivatives, NPe6, tin protoporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and therapeutically effective analogs and derivatives thereof.

In addition to the IAP protein inhibitors of the present invention, radiosensitizers can be administered in combination with a therapeutically effective amount of one or more compounds, including, but not limited to, compounds that promote the incorporation of radiosensitizers into target cells, compounds that control the flow of therapeutic agents, nutrients, and/or oxygen to target cells, chemotherapeutic agents that act on tumors with or without additional radiation, or other therapeutically effective compounds for the treatment of cancer or other diseases. Examples of additional therapeutic agents that can be used in combination with radiosensitizers include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red blood cell transfusions, perfluorocarbons (e.g., 2,3-DPG, BW12C), calcium channel blockers, pentoxifylline, anti-angiogenic compounds, hydralazine, and L-BSO.

The chemotherapeutic agent can be any pharmacologically active agent or compound that induces apoptosis. The pharmacologically active agent or compound can be, for example, a small organic molecule, a peptide, a polypeptide, a nucleic acid, or an antibody. Chemotherapeutic agents that can be used include, but are not limited to, alkylating agents, antimetabolites, hormones and their antagonists (natural products and their derivatives), radioisotopes, antibodies, and natural products, and combinations thereof. For example, the IAP protein inhibitors of the present invention can be administered in combination with antibiotics (e.g., doxorubicin and other anthracycline analogs), nitrogen mustards (e.g., cyclophosphamide), pyrimidine analogs (e.g., 5-fluorouracil), cisplatin, hydroxyurea, taxol, and its natural and synthetic derivatives. As another example, in the case of mixed tumors, such as breast adenocarcinoma, where the tumor includes both gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in combination with leuprorelin or goserelin (synthetic peptide analogs of LH-RH). Other anti-tumor regimens include the use of inhibitor compounds in conjunction with another treatment modality (e.g., surgery or radiation), the latter also referred to herein as "adjuvant anti-tumor modalities." Additional chemotherapeutic agents useful in the present invention include hormones and their antagonists, radioisotopes, antibodies, natural products, and combinations thereof.

Examples of chemotherapeutic agents useful in the methods of the invention are listed in the table below.

Table 1

Agents that affect microtubules interfere with cell mitosis and are well known in the art for their cytotoxic activity. Agents that affect microtubules useful in the present invention include, but are not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (NSC 125973), derivatives (e.g., NSC 608832), thiocolchicine (NSC 361792), tritylcysteine (NSC 83265), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones (including but not limited to epothilone A, epothilone B, and discodermolide (see Service, (1996) Science, 274:2009), estramustine, nocodazole, MAP4, etc. Examples of such agents are also described in Bulinski (1997) J. Cell Sci. 110:3055-3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou (1997) Nature 397:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985; and Panda (1996) J. Biol. Chem. 271:29807-29812.

Cytostatic agents that can be used include, but are not limited to, the following hormones and steroids (including synthetic analogs): 17-α-ethinylestadiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminogluthimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, and Zoladex.

Other cytostatic agents are anti-angiogenic agents, such as matrix metalloproteinase inhibitors, and other VEGF inhibitors (e.g., anti-VEGF antibodies and small molecules such as ZD6474 and SU668). Anti-Her2 antibodies can also be used. An EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are antibodies C225 and Src inhibitors that are immunospecific for EGFR.

Also suitable for use as a cytostatic is bicalutamide (Astra Zeneca), which causes androgen-dependent carcinomas to become non-proliferative. Yet another example of a cytostatic is an anti-estrogen that inhibits the proliferation or growth of estrogen-dependent breast cancer. Inhibitors of the transduction of cell proliferation signals are cytostatics. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.

Antimicrobial therapeutic agents can also be used as the second therapeutic agent in the present invention. Any agent that can kill, inhibit or reduce the function of a microorganism, as well as any agent considered to have such activity, can be used. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disrupting agents, etc., which can be used alone or in combination. In fact, any type of antibiotic can be used, including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, etc.

Additional second therapeutic agents that can be administered with the IAP protein inhibitors of the present invention are disclosed in US Pat. No. 7,960,372, which is incorporated herein by reference in its entirety.

The compounds of the present invention are typically administered in admixture with a pharmaceutical carrier selected with respect to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use according to the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers (comprising excipients and adjuvants that facilitate the processing of the compounds of formula (I)).

These pharmaceutical compositions can be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. Appropriate formulations depend on the selected route of administration. When a therapeutically effective amount of a compound of formula (I) is administered orally, the composition is typically in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition can additionally contain a solid carrier, such as gelatin or an adjuvant. Tablets, capsules, and powders contain approximately 0.01% to approximately 95%, and preferably approximately 1% to approximately 50%, of a compound of formula (I). When administered in liquid form, a liquid carrier (e.g., water, petroleum, or an oil of animal or plant origin) can be added. The liquid form of the composition can further contain physiological saline solution, glucose or other sugar solutions, or glycols. When administered in liquid form, the composition contains approximately 0.1% to approximately 90%, and preferably approximately 1% to approximately 50%, of a compound of formula (I) by weight.

When a therapeutically effective amount of a compound of formula (I) is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions (taking into account pH, isotonicity, stability, etc.) is within the skill of the art. Preferred compositions for intravenous, cutaneous, or subcutaneous injection typically contain an isotonic vehicle.

The compound of structural formula (I) can be easily combined with pharmaceutically acceptable carriers well known in the art. Such carriers enable activating agents to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspending agents, etc. for oral ingestion by patients to be treated. Pharmaceutical preparations for oral use can be obtained by the following: the compound of structural formula (I) is added to a solid excipient, optionally the resulting mixture is ground, and if necessary, after adding a suitable auxiliary agent, the particle mixture is processed to obtain tablets or lozenge cores. Suitable excipients include, for example, fillers and cellulose preparations. If necessary, disintegrants can be added.

The compound of structural formula (I) can be formulated for parenteral administration by injection (e.g., by single bolus injection or continuous infusion). The preparation for injection can be presented in unit dosage form (e.g., in an ampoule or in a multidose container) with an added preservative. The composition can take the form of a suspension, solution, or emulsion, for example, in an oily or aqueous vehicle, and can contain formulating agents, such as suspending agents, stabilizers, and/or dispersants.

The pharmaceutical composition for parenteral administration includes an aqueous solution of an active agent in a water-soluble form. In addition, the suspending agent of the compound of structural formula (I) can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. Optionally, the suspending agent can also contain a suitable stabilizer or reagent that increases the compound solubility and allows the preparation of a highly concentrated solution. Alternatively, the composition of the present invention can be in a powder form, which is used for preparation with a suitable vehicle (e.g., sterilized pyrogen-free water) before use.

The compound of structural formula (I) can also be formulated as a rectal composition, such as, for example, a suppository or retention enema containing a conventional suppository base. In addition to the preparations previously described, the compound of structural formula (I) can also be formulated as a depot preparation. Such depot preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the compound of structural formula (I) can be formulated with a suitable polymeric material or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin.

Specifically, compounds of structural formula (I) can be administered orally, buccally, or sublingually in the form of tablets containing excipients (e.g., starch or lactose), or in capsules or ovules, alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavorings or coloring agents. Such liquid formulations can be prepared with pharmaceutically acceptable additives (e.g., suspending agents). Compounds of structural formula (I) can also be injected parenterally, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the IAP protein inhibitor is preferably administered in the form of a sterile aqueous solution, which may contain other substances (e.g., salts or monosaccharides (e.g., mannitol or glucose)) to render the solution isotonic with blood.

As another embodiment, the present invention includes a kit comprising one or more compounds or compositions packaged in a manner promoting them to practice the purposes of the method of the present invention. In a simple embodiment, the kit includes a compound as herein described or a composition (for example, a composition comprising a compound of structural formula (I) and an optional second therapeutic agent) useful for practicing the method, which is packaged in a container (such as a sealed bottle or container), with a label attached to a container or included in the kit, which describes the purposes of the compound or composition practicing the method of the present invention. Preferably, the compound or composition are packaged as unit dosage form. The kit may further include a device suitable for administering the composition according to the intended route of administration.

Previous IAP protein inhibitors have properties that have hindered their development as therapeutic agents. According to an important feature of the present invention, compounds of structural formula (I) are synthesized and evaluated as inhibitors of IAP proteins. For example, the compounds of the present invention generally have binding affinities ( _IC50 ) for IAP proteins of less than 100 nM, less than 50 nM, less than 25 nM, and less than 10 nM.

Synthesis of compounds

The compounds of the present invention are prepared as follows. The following synthetic scheme represents the reactions used to synthesize the compounds of structural formula (I). Modifications and alternative schemes for preparing the IAP proteins of the present invention are readily within the capabilities of those skilled in the art.

Solvents and reagents were obtained commercially and used without further purification.Chemical shifts (δ) for NMR spectra are reported as δ values (ppm) downfield relative to an internal standard, with multiplicities reported in the usual manner.

Unless otherwise indicated, all temperatures are in degrees Celsius.

In the synthetic methods, examples and throughout the specification, the abbreviations have the following meanings: MS Mass spectrometry CbC Benzyl chloroformate LiOH lithium hydroxide HCl hydrochloric acid Deuterated methanol NMR Nuclear magnetic resonance spectroscopy Hz hertz EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride HOBT 1-Hydroxybenzotriazole Pd/C Palladium on carbon

Synthesis Scheme 1

Each compound of structural formula (I), except those having a cyclopropyl ring in R, was synthesized according to the method shown in Synthesis Scheme 1 above. Compound 2 was synthesized according to the method disclosed in Q. Cai et al., J. Med. Chem. , 2011, 2714-26. Protection of the amino group in compound 2 with Cbz afforded carbamate 3. Hydrolysis of the methyl ester in carbamate 3 produced acid 4. Condensation of acid 4 with a series of amines afforded amide 5. Removal of the Boc protecting group in amide 5 afforded amine 6. Condensation of amine 6 with LN-Boc-N-methyl-alanine afforded amide 7. Cleavage of the Cbz protecting group in amide 7 afforded amine 8.

Condensation of amine 8 with a series of diisocyanates (9) and subsequent removal of the Boc protecting group yields bisureas containing Smac mimetics. Condensation of amine 8 with a series of diisothiocyanates (10) and subsequent removal of the Boc protecting group yields bis-thioureas containing Smac mimetics. Condensation of amine 8 with a series of dicarbonochloridates (12) and subsequent removal of the Boc protecting group yields bis-carbamates containing Smac mimetics. Condensation of amine 8 with a series of disulfonyl chlorides and subsequent removal of the Boc protecting group yields bis-sulfonamides containing Smac mimetics.

Synthesis Scheme 2

The synthesis of compounds of general formula (I) having a cyclopropyl ring in R is shown in the above-mentioned synthetic scheme 2. Condensation of compound 2 with diisocyanates, diisothiocyanates, dichloroformates (dicarbonochloridate) or disulfonyl chlorides respectively gives intermediates 13. Removal of the Boc protecting group in compound 13 and subsequent condensation with L-N-Boc-N-methyl-Ala gives amide 14. Hydrolysis of the methyl ester in amide 14 gives a series of acids. Condensation of the acids with a series of amines and subsequent deprotection of the Boc protecting group provides the final compounds.

¹ H NMR (300MHz, CD ₃ OD): δ7.37-7.24 (m, 20H), 6.16 (s, 2H), 4.72-4.60 (m, 4H), 4.10(m, 2H), 4.00-3.85(m, 6H), 3.25-3.04(m, 8H), 2.69(s, 6H), 2.34(m, 2H), 2.14- 2.03 (m, 6H), 1.77-1.48 (m, 8H), 1.54 (d, J=6.9Hz, 6H), 1.35 (m, 8H); ESI MS: m/z1151.8 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.23 (m, 20H), 6.15 (s, 2H), 4.70-4.60 (m, 4H), 4.10(m, 2H), 3.97-3.80(m, 6H), 3.25-3.03(m, 8H), 2.69(s, 6H), 2.34(m, 2H), 2.10- 2.03 (m, 6H), 1.78-1.57 (m, 8H), 1.52 (d, J=7.2Hz, 6H), 1.39 (m, 4H); ESI MS: m/z1123.6 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.53 (s, 4H), 7.37 (m, 20H), 6.18 (s, 2H), 4.84 (m, 2H), 4.67(t, J=8.4Hz, 2H), 4.27(m, 2H), 4.09-3.80(m, 6H), 3.30-3.05(m, 4H), 2.71(s, 6H), 2.37(m, 2H), 2.35-1.80(m, 4H), 1.70-1.55(m, 6H), 1.45(d, J=6.9Hz, 6H); ESI MS: m/z1115.9(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.36-7.15 (m, 24H), 6.15 (s, 2H), 4.84 (m, 2H), 4.63(m, 4H), 4.32-4.14(m, 4H), 3.99-3.81(m, 6H), 3.16-3.06(m, 4H), 2.63(s, 6H), 2.34(m, 2H), 2.18-2.85 (m, 6H), 1.85-1.60 (m, 4H), 1.50 (d, J=7.2Hz, 6H); ESI MS: m/z1143.67 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.36 (m, 20H), 6.14 (s, 2H), 4.82 (m, 2H), 4.60 (t, J=8.4Hz, 2H), 4.44(m, 2H), 3.92-3.80(m, 4H), 3.70(m, 2H), 3.42(m, 2H), 3.16-3.03(m, 6H), 2.66(s, 6H), 2.36(m, 2H), 2.16(m, 2H), 2.00(m, 4H), 1.73(m, 8H), 1, 52-1.43(m, 10H); ESIMS: m/z 1165.4(M+H) ⁺ .

¹ H NMR (300MHz. CD ₃ OD): δ7.36-7.22 (m, 20H), 6.14 (s, 2H), 4.82 (m, 2H), 4.60(t, J=8.4Hz, 2H), 4.44(m, 2H), 3.91-3.85(m, 4H), 3.65(m, 2H), 3.48(m, 2H), 3.15- 3.03 (m, 6H), 2.66 (s, 6H), 2.32 (m, 2H), 2.14 (m, 2H), 2.00 (m, 4H), 1.85-1.70 (m, 8H), 1.52 (d, J=8.7Hz, 6H), 1.42-1.33 (m, 6H); ESI MS: m/z 1193.7(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.48 (d, J=8.1Hz, 4H), 7.33 (m, 20H), 7.11 (d, J=8.1Hz, 4H), 6.17(s, 2H), 4.82(m, 2H), 4.63(m, 2H), 4.25(m, 2H), 4.08-4.03(m, 6H), 3. 88(s, 2H), 3.30-3.20(m, 4H), 2.70(s, 6H), 2.34(m, 2H), 2.20-1.80(m, 6H), 1.75-1.60(m, 4H), 1.55 (d, J=6.9Hz, 6H); ESI MS: m/z 1206.4 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ8.14 (m, 1H), 7.34-7.18 (m, 23H), 6.17 (s, 2H), 4.84(m, 2H), 4.67(t, J=8.4Hz, 2H), 4.22(m, 2H), 4.07(m, 6H), 3.24(m, 4H), 2.73(s, 6H), 2.34(m, 2H), 2.14-2.04(m, 6H), 1.77-1.66(m, 4H), 1.57(d, J=6.9Hz, 6H); ESI MS: m/z 1115.9(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.55 (d, J=9.0Hz.4H), 7.36-7.24 (m, 20H), 6.91 (d, J=9.0Hz, 4H), 6.17 (m, 2H), 4.84 (m, 2H), 4.64 (t, J=8.1Hz, 2H), 4.23 (m, 2H), 4.09 (m, 6H), 3.21 (m, 4H), 2.71 (s, 6H), 2.34 (m, 2H), 2.14-2.02 (m, 6H), 1.80-1.73 (m, 4H), 1.56 (d, J=6.9Hz, 6H); ESI MS: m/z 1207, 3(M+H) ⁺ .

¹ H NMR (300MHz. CD ₃ OD): δ7.46-7.25 (m, 26H), 6.17 (s, 2H), 4.84 (m, 2H), 4.65(m, 2H), 4.32(m, 2H), 4.19-4.02(m, 6H), 3.22(m, 4H), 2.66(s, 6H), 2.37(s, 6H), 2.24- 2.02 (m, 8H), 1.83-1.70 (m, 4H), 1.53 (d, J=6.6Hz, 6H); ESI MS: m/z 1220.2 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.47 (d, J=8.4Hz, 4H), 7.36 (rn, 20H), 7.06 (d, J=8.4Hz, 4H), 6.16 (s, 2H), 4.94 (m, 2H), 4.67 (t, J=8.4Hz, 2H), 4.25 (m, 2H), 4.09-4.04 (m, 6H), 3.17-3.28 (m, 4H), 2.84 (s, 4H), 2.66 (s, 6H), 2.37 (m, 2H), 2.15-2.02 (m, 6H), 1.79-1.67 (m, 4H), 1.56 (d, J=6.6Hz, 6H); ESI MS: m/z 1220.25(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.83 (d, J=8.4Hz, 4H), 7.63 (d, J=8.4Hz, 4H), 7.36-7.16(m, 20H), 6.17(s, 2H), 5.01(m, 2H), 4.67(m, 2H), 4.17(m, 2H), 4.00-3.94(m, 4H), 3.73 (s, 4H), 3.59-3.40 (m, 4H), 2.64 (s, 6H), 2.55-2.37 (m, 4H), 2.06 (m, 4H), 1.80-1.67 (m, 4H), 1.53 (d, J=6.9Hz, 6H); ESI MS: m/z 1237.6(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.60 (d, J=8.4Hz, 4H), 7.36-7.21 (m, 24H), 6.17 (s, 2H), 4.82 (m, 2H), 4.64 (t, J=8.1Hz, 2H), 4.21 (m, 2H), 4.08-4.02 (m, 6H), 3.24 (m, 4H), 2.70 (s, 6H), 2.24 (m, 2H), 2.14-2.03 (m, 6H), 1.78-1.71 (m, 4H), 1.56 (d, J=6.9Hz, 6H); ESI MS: m/z 1223.3(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.33-7.21 (m, 20H), 6.12 (s, 2H), 5.11 (m, 2H), 4.84(m, 2H).4.56(t, J=8.4Hz, 2H), 4.25(m, 2H), 3.93(m, 2H), 3.66-3.53(m, 6H), 3.22- 3.15(m, 8H), 2.67(s, 6H), 2.34(m, 2H), 2.15-1.96(m, 4H), 1.83-1.77(m, 6H), 1.54(d, J=6.9Hz, 6H); ESI MS: m/z 1093.7(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.34-7.23 (m, 20H), 6.14 (s, 2H), 4.92 (m, 2H), 4.70(m, 4H), 4.08-3.86(m, 8H), 3.59(m, 2H), 3.16-3.05(m, 4H), 2.70(s, 6H), 2.36(m, 2H), 2.10-1.92(m, 10H), 1.79-1.71(m, 4H), 1.60-1.40(m, 8H), 1.40-1.25(m, 2H); ESI MS: m/z 1121.7(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.27-7.02(m, 28H), 6.12(m, 2H), 5.07-4.97(m, 2H), 4.60(m, 2H), 4.39(m, 2H), 3.89-3.85(m, 4H), 3.73-3.54(m, 6H), 2.66(s, 6H), 2.31(m, 2H), 2.11-1.81(m, 10H), 1.65(m, 6H) 1.56(d, J=6.9Hz, 6H); ESI MS: m/z 1236.2(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.40 (m, 2H), 7.14-7.06 (m, 6H), 5.06 (m, 2H), 4.84 (m, 2H), 4.72 (m, 2H), 4.50 (t, J=8.4Hz, 2H), 4.12 (m, 2H), 4.02-3.93 (m, 6H), 3.27- 3.10(m, 6H), 2.80(m, 4H), 2.67(s, 6H), 2.34(m, 2H), 2.14-1.90(m, 10H), 1.81-1.72(m, 8H), 1.58 (m, 4H), 1.53 (d, J=6.9Hz, m, 6H), 1.35 (m, 8H); ESI MS: m/z 1151.8 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.55 (s, 4H), 7.43 (m, 2H), 7.17-7.07 (m, 6H), 5.09 (m, 2H), 4.83 (m, 2H), 4.52 (t, J=8.4Hz, 2H), 4.25 (m, 2H), 4.16-4.05 (m, 6H), 3.39-3.34 (m, 4H), 2.81 (m, 4H), 2.73 (s, 6H), 2.32 (m, 4H), 2.05-1.93 (m, 8H), 1.82-1.74 (m, 8H), 1.57 (d, J=6.9Hz, m, 6H); ESI MS: m/z 1044.0 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.38-7.09 (m, 12H), 5.03 (m, 2H), 4.85 (m, 2H), 4.78(m, 2H), 4.60(m, 2H), 4.55(t, J=8.4Hz, 2H), 4.35-4.17(m, 4H), 4.05-3.92(m, 6H), 3.61 (m, 2H), 2.80 (m, 4H), 2.66 (s, 6H), 2.31 (m, 2H), 2.15-1.91 (m, 10H), 1.78-1.72 (m, 8H), 1.51 (d, J=6.9Hz, m, 6H); ESI MS: m/z 1071.63(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.51 (d, J=8.4Hz, 4H), 7.43 (m, 2H), 7.14-7.07 (m, 10H), 5.08 (m, 2H), 4.82 (m, 2H), 4.51 (t, J=8.4Hz, 2H), 4.28 (m, 2H), 4.15-4.04 (m, 6H), 3.89 (s, 2H), 3.38-3.33 (m, 4H), 2.87 (m, 4H), 2.71 (s, 6H), 2.31 (m, 2H), 2.10-1.73 (m, 18H), 1.56 (d, J=6.9Hz, m, 6H); ESI MS: m/z 1134.1(M+H) ⁺ ，

¹ H NMR (300MHz, CD ₃ OD): δ8.21 (m, 3H), 7.85 (m, 1H), 7.34-7.18 (m, 20H), 6.10(s, 2H), 4.85(m, 2H), 4.58(t, J=8.4Hz, 2H), 4.31(m, 2H), 3.93(m, 4H), 3.73(m, 2H), 3.21 (m, 2H), 2.96 (m, 2H), 2.67 (s, 6H), 2.33 (m, 2H), 2.06-1.93 (m, 6H), 1.84-1.76 (m, 4H), 1.51 (d, J=6.9Hz, 6H); ESI MS: m/z 1157.6 (M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.50-6.70 (m, 10H), 4.90 (m, 2H), 4.70 (m, 2H), 4.45- 4.10 (m, 4H), 3.95-3.40 (m, 10H), 2.60 (m, 2H), 2.55 (s, 6H), 2.30-1.60 (m, 12H), 1.45 (brd, J=7.0Hz, 6H), 1.40-1.05 (m, 4H); ESI MS: m/z 1187.3(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.50-6.70 (m, 10H), 4.92 (m, 2H), 4.80 (m, 2H), 4.45- 4.20 (m, 4H), 3.95 (m, 2H), 3.80-3.40 (m, 8H), 2.60 (m, 2H), 2.55 (s, 6H), 2.30-1.60 (m, 12 H), 1.45 (brd, J=7.0Hz, 6H), 1.40-1.05 (m, 4H); ESI MS: m/z 1187.3(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.65-7.45 (m, 4H), 7.35-6.90 (m, 8H), 5.05 (m, 2H), 4.80(m, 2H), 4.50-4.30(m, 4H), 4.05(m, 2H), 3.90-3.40(m, 8H), 2.60(m, 2H), 2 .50(s, 6H), 2.40-1.60(m, 12H), 1.45(brd, J=7.0Hz, 6H), 1.40-1.05(m, 4H); ESI MS: m/z 1151.2 (M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.05 (m, 14H), 4.75 (m, 2H), 4.20-3.90 (m, 4H), 3.90-3.65(m, 6H), 3.35-3.10(m, 4H), 2.90(m, 2H), 2.60(s, 6H), 2.30(m, 2H), 2.05-1.55(m, 8H), 1.45 (brd, J=7.2Hz, 6H), 1.40-1.05 (m, 6H), 0.80 (m, 2H); ESI MS: m/z 1015.5 (M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.05 (m, 14H), 4.75 (m, 2H), 4.30-3.95 (m, 4H), 3.95-3.65(m, 6H), 3.40-3.10(m, 4H), 2.90(m, 2H), 2.60(s, 6H), 2.25(m, 2H), 2.05-1.55(m, 8H), 1.45 (brd, J=7.2Hz, 6H), 1.40-1.05 (m, 6H), 0.80 (m, 2H); ESI MS: m/z 1015.5 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.60 (s, 4H), 7.30-7.10 (m, 10H), 4.80 (m, 2H), 4.45(m, 2H), 4.25(m, 2H), 4.20-4.02(m, 6H), 3.50-3.30(m, 4H), 2.95(m, 2H), 2.70(s, 6H), 2.40-2.05(m, 10H), 1.90-1.70(m, 4H), 1.55(d, J=7.2Hz, 6H), 1.30-1.10(m, 4H); ESI MS: m/z1015.5(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.60 (s, 4H), 7.30-7.10 (m, 10H), 4.80 (m, 2H), 4.45(m, 2H), 4.30(m, 2H), 4.20-4.02(m, 6H), 3.50-3.30(m, 4H), 2.90(m, 2H), 2.70(s, 6H), 2.35-2.05(m, 10H), 1.90-1.70(m, 4H), 1.55(d, J=7.2Hz, 6H), 1.30-1.10(m, 4H); ESI MS: m/z1015.5(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.30-6.90 (m, 12H), 4.90 (m, 2H), 4.70 (m, 2H), 4.40- 4.20 (m, 4H), 3.95 (m, 2H), 3.90-3.30 (m, 8H), 2.65 (m, 2H), 2.60 (s, 6H), 2.30-1.75 (m, 12H), 2.50 (d, J=7.0Hz, 6H), 1.20 (m, 4H); ESI MS: m/z 1051.2(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.30-6.80 (m, 12H), 4.85 (m, 2H), 4.70 (m, 2H), 4.30-4.20(m, 4H), 4.05-3.60(m, 6H), 3.50-3.30(m, 4H), 2.65(m, 2H), 2.55(s, 6H), 2.30- 1.70 (m, 12H), 2.50 (d, J=7.0Hz, 6H), 1.20 (m, 4H); ESI MS: m/z 1051.2 (M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.40-7.20 (m, 10H), 5.99 (s, 2H), 4.75 (m, 2H), 4.45 (m, 2H), 4.10 (m, 2H), 3.95 (m, 2H), 3.80 (m, 2H), 3.65 (m, 2H), 3.25-3.05 (m, 8H), 2.62 (m, 6H), 2.30(m, 2H), 2.20-1.70(m, 12H), 1.45(m, 2H), 1.40(d, J=7.2Hz, 6H); ESI MS: m/z 1095.4(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.48-7.08 (m, 18H), 4.92 (m, 2H), 4.42 (m, 2H), 4.21-4.03(m, 8H), 3.87(m, 2H), 3.36-3.20(m, 4H), 2.85(m, 2H), 2.70(s, 6H ), 2.30-2.02 (m, 10H), 1.76 (m, 4H), 1.56 (d, J=6.9Hz, 6H), 1.23 (m, 4H); ESI MS: m/z 1105.4(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD) δ7.35-7.15 (m, 10H), 4.84 (m, 2H), 4.40-3.90 (m, 8H), 3.75-3.50(m, 6H), 3.40-3.20(m, 8H), 2.71(s, 6H), 2.65(m, 2H), 1.90-1.43(m, 42H); ESIMS: m/z 1107.9(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.20 (m, 10H), 4.84 (m, 2H), 4.61 (d, J＝ 9.0Hz, 2H), 4.20 (t, J=9.0Hz, 2H), 3.97-3.81 (m, 10H), 3.30-2.95 (m, 6H), 2.68 (s, 6H), 2.51 (m, 2H), 2.01-1.31 (m, 42H); ESI MS: m/z 1107.6(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.10 (m, 10H), 4.84 (m, 2H), 4.66 (m, 2H), 4.43(m, 2H), 4.22(m, 2H), 4.04-3.72(m, 8H), 3.10-2.85(m, 6H), 2.68(s, 6H), 2.24-1.37(m, 40H); ESI MS: m/z 1079.5(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ CD): δ7.35-7.15 (m, 10H), 4.81 (m, 2H), 4.65 (m, 2H), 4.35(m, 2H), 4.22(m, 2H), 3.98-3.80(m, 8H), 3.25-2.87(m, 6H), 2.68(s, 6H).2.20-1.33(m, 40H); ESI MS: m/z 1079.9(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.30-7.10(m, 10H), 4.84(m, 2H), 4.61(m, 4H), 4.24(t, J=9.0Hz, 2H), 3.97-3.81(m, 8H), 3.41-3.02(m, 6H), 2.64(s, 6H), 2.17-1.37(m, 40H); ESIMS: m/z 1079.3(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.10 (m, 10H), 4.84 (m, 2H), 4.67-4.24 (m, 6H), 3.97-3.81(m, 8H), 3.41-3.02(m, 6H), 2.68(s, 6H), 2.17-1.37(m, 40H); ESI MS: m/z 1079.5(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.19-7.11 (m, 8H), 4.84 (m, 2H), 4.70 (m, 2H), 4.57(m, 2H), 4.42(m, 2H), 4.10(m, 2H), 4.00(m, 6H), 3.22-3.06(m, 6H), 2.92-2.75(m, 4H), 2.66(s, 6H), 2.26-1.37(m, 30H); ESI MS: m/z 1023.7(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.15 (m, 10H), 4.84 (m, 2H), 4.71 (m, 2H), 4.41(m, 2H), 4.11(m, 2H), 3.98-3.88(m, 6H), 3.48-3.08(m, 10H), 2.82(m, 4H), 2.69(s, 6H), 2.22-1.39(m, 26H); ESI MS: m/z 999.7(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.15 (m, 10H), 4.84 (m, 2H), 4.69 (m, 2H), 4.50-4.30(m, 4H), 4.11-3.86(m, 8H), 3.48(m, 2H), 3.25-3.06(m, 6H), 2.68(s, 6H), 2.31- 1.28(m, 34H); ESI MS: m/z 1051.4(M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.10 (m, 10H), 4.82 (m, 2H), 4.70 (d, J＝ 8.4Hz, 2H), 4.43-4.34(m, 4H), 4.12(m, 2H), 4.01-3.90(m, 6H), 3.65(m, 2H), 3.25-3.06(m, 6H), 2.67(s, 6H), 2.52-2.34(m, 10H), 2.10(m, 6H), 1.80-1.39(18H); ESI MS: m/z 1051.9 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.35-7.15 (m, 10H), 4.82 (m, 2H), 4.70 (d, J＝ 9.0Hz, 2H), 4.43-4.28(m, 4H), 4.12(m, 2H), 4.01-3.90(m, 6H), 3.25-3.06(m, 6H), 2.77(m, 2H), 2.70(s, 6H), 2.51(m, 2H), 2.30(m, 2H), 2.20-1.90(m, 10H), 1.90-1.45(m, 16H), 1.45- 1.35(m, 4H); ESI MS: m/z 1051.7(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.10 (m, 14H), 4.80 (m, 2H), 4.40-4.25 (m, 4H), 4.20(m, 2H), 4.15-4.05(m, 4H), 3.90(m, 2H), 3.40-3.30(m, 4H), 2.90(m, 2H), 2 .70(s, 6H), 2.30-1.90(m, 10H), 1.90-1.55(m, 14H), 1.55(d, J=7.2Hz, 6H); ESI MS: m/z 1071.5 (M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.10 (m, 14H), 4.75 (m, 2H), 4.40-4.25 (m, 4H), 4.20(m, 2H), 4.15-4.05(m, 4H), 3.90(m, 2H), 3.40-3.30(m, 4H), 2.90(m, 2H), 2.70(s, 6H), 2.30-1.90(m, 10H), 1.90-1.35(m, 20H); ESI MS: m/z 1071.7(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.10 (m, 14H), 4.80 (m, 2H), 4.40 (m, 2H), 4.25- 4.05 (m, 4H), 4.05-3.85 (m, 4H), 3.80 (m, 2H), 3.30-3.15 (m, 6H), 2.70 (s, 6H), 2.30-1.60 (m, 24H), 1.55 (d, J=7.2Hz, 6H); ESI MS: m/z 1071.7(M+H) ⁺ .

¹ H NMR (300MHz, D ₂ O): δ7.35-7.10 (m, 14H), 4.80 (m, 2H), 4.45 (m, 2H), 4.20- 3.90 (m, 6H), 3.80 (m, 2H), 3.30-3.20 (m, 6H), 2.70 (s, 6H), 2.30-1.60 (m, 24H), 1.55 (d, J=7.2Hz, 6H); ESI MS: m/z 1071.7 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.40-7.20(m, 20H), 6.15(m, 2H), 5.60(m, 2H), 4.85(m, 2H), 4.55(m, 2H), 4.40(m, 2H), 3.95-3.80(m, 4H), 3.65(m, 2H), 3.35-2.05(m, 6H), 2.65 (s, 6H), 2.45-1.70 (m, 16H), 1.55 (d, J=7.2Hz, 6H); ESI MS: m/z 1162.5 (M+H) ⁺ .

¹ H NMR (300MHz, CD ₃ OD): δ7.40-7.20 (m, 20H), 6.15 (m, 2H), 5.45 (m, 2H), 4.82(m, 2H), 4.55(m, 2H), 4.40(m, 2H), 3.95-3.72(m, 4H), 3.65(m, 2H), 3.35-2.95(m, 6H), 2.65 (s, 6H), 2.45-1.70 (m, 20H), 1.55 (d, J=7.2Hz.6H); ESI MS: m/z 1190.6 (M+H) ⁺ .

ESI MS: m/z 1147.6 (M+H) ⁺ .

Binding affinity to XIAP adaptor-BIR2-BIR3, cIAP1-BIR3, and cIAP-2 BIR2

The binding affinity of the compounds of the invention to the XIAP linker-BIR2-BIR3 (residues 120-356), cIAP1-BIR3 (residues 253-363), and cIAP-2 BIR3 (residues 238-349) proteins was determined by a fluorescence polarization (FP)-based competitive assay. For the cIAP-1 BIR3 and cIAP-2 BIR3 assays, a fluorescently labeled Smac mimetic (Smac-2F) was used as a fluorescent probe. The _K values of Smac-2F for cIAP-1 BIR3 and cIAP-2 BIR3 were determined by monitoring the total fluorescence polarization of a mixture composed of a fixed concentration of fluorescent probe and increasing concentrations of the protein up to full saturation. Fluorescence polarization values were measured in a Microfluor 2 96-well black round-bottom plate (Thermo Scientific) using an InfiniteM-1000 microplate reader (Tecan US, Research Triangle Park, NC). 1 nM SMAC-2F and increasing concentrations of protein were added to each well to a final volume of 125 μl in assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/ml bovine gamma globulin, 0.02% sodium azide, Invitrogen, containing 4% DMSO). The plate was incubated at room temperature for 1-2 hours and gently shaken to ensure equilibrium. Polarization values were measured in millipolarization units (mP) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The equilibrium dissociation constant ( _Kd ) was then calculated using Graphpad Prism 5.0 software (Graphpad Software, San Diego, CA) by fitting the sigmoidal dose-dependent FP increase as a function of protein concentration.

Compound _K values were determined by dose-dependent competitive binding experiments, in which serial dilutions of the compound competed with a fixed concentration of fluorescent probe for binding to a fixed concentration of protein (typically 2-3 times the K _value determined above). 5 μl of test compound in DMSO was added to the assay plate along with 120 μl of pre-incubated protein/tracer complex in assay buffer (100 mM potassium phosphate, pH 7.5, 100 μg/ml bovine gamma globulin, 0.02% sodium azide, Invitrogen) and incubated for 2 hours at room temperature with gentle shaking. Final protein and probe concentrations were 3 nM and 1 nM for the cIAP-1 BIR3 and 5 nM and 1 nM for the cIAP-2 BIR3 assays, respectively. A negative control containing only the protein/probe complex (equivalent to 0% inhibition) and a positive control containing only the free probe (equivalent to 100% inhibition) were included in each assay plate. FP values were measured as described above. _IC50 values were determined by nonlinear regression fitting of the competition curves. The Ki values of competitive inhibitors were calculated using the previously described derivation equation based on the measured _IC50 values, the _Kd values of the probe with the different proteins, and the concentrations of protein and probe in the competition assay.

The same procedure was used for the FP-based assay of the XIAP linker-BIR2-BIR3 protein. In this assay, a bivalent fluorescently tagged peptide Smac mimetic (Smac-1F) was used as a fluorescent probe, and its _Kd value for the XIAP linker-BIR2-BIR3 was similarly determined by saturation experiments. 0.01% Triton X-100 was added to the assay buffer to achieve stable fluorescence and polarization values for the dimeric fluorescent probe. The final protein and probe concentrations used in the competitive assay were 3 nM and 1 nM, respectively.

Inhibition of cell growth in MDA-MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines

FIG1 shows the anti-tumor activity of Example 2 and Example 24 in the MDA-MB-231 xenograft model in nude mice. Treatment was started when the tumor reached an average volume of 80 mm ^3. Example 24 was administered intravenously at a weekly dose of 10 mg/kg for 4 weeks (qwkx4, iv). Example 2 was administered at a weekly dose of 3 mg/kg for 4 weeks (qwkx4, iv). Vehicle control was used for control treatment. Each group had 8-10 mice, each with one tumor. Tumor regression was achieved for Examples 2 and 24.

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Claims (15)

1. Compounds having the following structures Where X is selected from _-SO₂- ; Y is selected from –NH-, -O-, and does not exist when X is _-SO2- ; R is selected from and _R1 is selected from phenyl ring B; Or its pharmaceutically acceptable salt. 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R is 3. The compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof, wherein _R1 is 4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is and Y is -NH-. 5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is and Y is -NH-. 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is and Y is -O-. 7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, selected from... And pharmaceutically acceptable salts of any of the above compounds. 8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is... Or its pharmaceutically acceptable salt. 9. A pharmaceutical composition comprising a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or mediator. 10. The pharmaceutical composition of claim 9, further comprising a second therapeutic agent for treating a disease or condition in which inhibition of the IAP protein provides a benefit. 11. A composition comprising (a) a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, (b) a second therapeutic agent for treating a disease or condition in which inhibition of the IAP protein provides benefit, and (c) optional excipients and/or a pharmaceutically acceptable carrier, wherein the compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof and the second therapeutic agent are administered simultaneously or separately. 12. A cassette for human pharmaceutical use, the cassette comprising (a) a container, (b) a packaged composition comprising a compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, (c) a packaged composition comprising a second therapeutic agent for treating a disease or condition in which inhibition of the IAP protein provides benefit, and (d) a package insert containing instructions for use in the treatment of the disease or condition with concurrent or sequential administration of the composition. 13. Use of any compound of claims 1 to 8 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease or condition in which inhibition of the IAP protein provides a benefit. 14. The use of claim 13, wherein the disease or condition is cancer. 15. The use of claim 14, wherein the cancer is breast cancer or ovarian cancer.