Use of the function of caseinolytic protease P as a biomarker for drug response to imipridone-like agents

Abstract

Use of caseinolytic protease P (ClpP) function and / or concentration as a biomarker for predicting response to compounds of formula I in neoplastic diseases, preferably cancer or diseases in which enhancing ClpP activity may provide therapeutic benefit. In another aspect, it relates to methods of treatment, as well as methods and kits, comprising the use of said biomarkers.

Description

Technical Field

[0001] The present invention relates to the use of the function and / or concentration of caseinolytic protease P (ClpP) as a biomarker for predicting the response of compounds of formula I in neoplastic diseases, preferably cancer or other diseases in which enhancement of ClpP activity may provide a therapeutic benefit. In other aspects, the present invention relates to methods and kits, and also to therapeutic methods involving the use of biomarkers. Further, chemical substances that activate ClpP are described.

Background Art

[0002] Mammalian mitochondria contain a serine protease complex, (ClpP), which is the proteolytic component of the ClpXP proteolytic complex. This complex plays a central role in the quality control of mitochondrial proteins (Houry, W.A. et al, Cell Chemical Biology 2018, 25, 1017 - 1030 and references cited therein) and in regulating the bioenergetic activity of cells. Houry, W.A. et al have also reported that ClpP is highly expressed in multiple cancers and plays an important role in cell metastasis. Furthermore, mitochondrial dysfunction is central to disease mechanisms and is likely a causative factor in many neurodegenerative diseases (Beal and Johri, J Pharmcol Exp Thera. 2012, 342(3), 619 - 630 and references cited therein). Deficiency of ClpP induces an overload of misfolded / unfolded proteins in mitochondria, suppresses mitochondrial respiratory activity, increases oxidative damage to mitochondria, and causes cell death (Qi et al, Acta Neuropathologica, 2019, 137, 939 - 960 and references cited therein).

[0003] Agents that modulate the function of ClpP have been identified. Direct activation of proteases by small molecules is rare in drug discovery. Agents that activate ClpP have been reported (Sieber, S.A. et al, Angew. Chem. Int. Ed. 2018, 57, 14, 602 - 14607 and references cited therein). Additionally, agents that inhibit ClpP have also been reported (Schimmer, A.D. et al, Cancer Cell 2015, 27, 864 - 876 and references cited therein). Both Schimmer, A.D. et al and Sieber, S.A. et al have described using those agents for treating cancer. Oral active agents for treating cancer have potential in the market as they are easy to administer upon repeated dosing. However, very potent small molecule up - regulators of ClpP activity are not known. The larger macrocyclic activators of ClpP are known as "ADEP" but lack structural properties regarding oral bioavailability (Lipinski's rules, Oprea et al., Adv. Drug Deliv. Rev. 2016, 101, 89 - 98 and references cited therein).

[0004] Proteases very similar to human ClpP have been found to be encoded in the genomes of some bacteria and several viruses. Agents that modulate ClpP function have been shown to be useful for treating bacterial infections. Kao R.Y.T. et al have described small molecule inhibitors of ClpP and their effects against Staphylococcus aureus (Kao, R.Y.T. et al., PNAS 2018, 115, 8003 - 8008 and references cited therein). Additionally, ClpP activators have been described (Lee R.E. et al, ACS Infect Dis 2019, November 8; 5(11):1915 - 1925 and references cited therein).

[0005] Mitochondria have multiple quality control systems to ensure homeostasis (proteostasis). Deficiencies in these systems cause mitochondrial dysfunction such as aging, various neurodegenerative diseases, cardiovascular diseases, and cancer (Li R. et al., Ann Rev Biophy, 2020, January 13. doi: 10.1146 / annurev-biophys-121219-081604 and references cited therein, Martins L.M., J Mol Med, 2013, 91, 665-671 and references cited therein, and Jeong Y.Y., Cells, 2020, 9(1), 150 and references cited therein). The accumulation of α-synuclein and mitochondrial dysfunction are associated with the pathology of Parkinson's disease and Alzheimer's disease (Qi et al, Acta Neuropathologica, 2019, 137, 939-960 and references cited therein, and Nielsen and Twohig, Mol Neurodegener, 2019, 14(1), 23 and references cited therein). Furthermore, α-synuclein may decrease the protein level of ClpP. Improvement of ClpP activity in the cell system significantly reduced α-synuclein-related pathology.

[0006] ONC201, a small molecule drug for treating cancer, is in clinical trials and is being evaluated for the treatment of several cancers. Some published reports describe various aspects of the mechanism of action of ONC201. The publications describe that ONC201 functions via G protein-coupled receptors (GPCRs) (El-Deiry W. S., Neoplasia 2018, 20, 80-91 and references cited therein). Furthermore, the reports describe that cell functions, including mitochondrial function, change with treatment with ONC201 (Lipkowitz S., Oncotarget 2018, 9, 18, 454-18, 479 and references cited therein).

[0007] Perrault syndrome is a disorder characterized by ovarian dysgenesis in females and sensorineural hearing loss in both sexes. In more severe cases, additional symptoms may include ataxia, neuropathy, and intellectual disability (Dougan, D.A. Sci Rep 2018, 8(1), 12862 and references cited therein). Mutations in six different genes are associated with this disorder, and in type 3 Perrault syndrome, mutations in ClpP are the cause. Y229D has been shown to inhibit ClpP-peptidase activity, and two mutations, Y229D and I208M, are thought to alter peptidase activity. Loss-of-function mutations in genes encoding heme biosynthetic enzymes can cause congenital porphyrias. ClpX promotes heme biosynthesis, and a mutation in ClpX (Gly298Asp) results in pathological accumulation of the heme biosynthetic intermediate protoporphyrin (PPIX) (Paw B.H., Proc. Natl. Acad. Sci. USA. 114:E8045-E8052 (2017) and references cited therein). Non-dividing hepatocytes in end-stage liver disease exhibit idiopathic cirrhosis with permanent growth arrest (Ramakrishna, G. et al, Cell Mol Gastroenterol Hepatol. 2019, 8(1):73-94 and references cited therein). A common cause of idiopathic cirrhosis is fatty liver disease. The modern drug development process, often referred to as the translational medicine approach, focuses on identifying the right patients for treatments involving specific interventions in important aspects of the disease process. This requires multiple inputs, such as an understanding of specific molecular events important for individual disease processes and a clear understanding of how specific treatments intervene in those individual disease processes (Rossetti L., Drug Dis. Today 2016, 21, 517-526 and references cited therein). The center of this approach is the development and use of biomarkers and companion diagnostics involving specific treatments.

Summary of the Invention

[0008] In the present invention, we report that human ClpP (hClpP or HSClpP) is a biomarker for the chemical action of ONC201 and related chemical analogs, and that this biomarker can be used to determine whether a patient is a candidate for this drug treatment and whether the drug treatment has the expected molecular effects. In particular, we show that these compounds directly bind and activate the peptidase activity of hClpP. The binding and activation effects on hClpP occur within a certain time and are dose-dependent, and parallel the growth inhibitory effects of these compounds on cancer cells. Thus, our findings demonstrate that the biological action of ONC201 (and related compounds) depends on the physical activity of hClpP. Our findings are relevant to hClpP and ClpP (ClpP) in other mammalian species. In addition, ONC201 and related chemical analogs directly bind and activate the peptidase activity of bacterial ClpP (bClpP). This is relevant to Staphylococcus aureus and other bacterial species. The effects on bClpP occur within a certain time and are dose-dependent and are thought to be the cause of the growth inhibitory effects of these compounds on bacterial cells. Also, the antibacterial action of ONC201 and structurally related compounds is thought to be due to the physical activity of bClpP. The present invention also enables the molecular means-based evaluation of susceptible bacteria and chemically related compounds to ONC201.

[0009] Many neurodegenerative diseases are characterized by the relative selective death of neuron subtypes. Mitochondrial dysfunction can contribute to multiple neurodegenerative diseases, not limited to these, such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, and Alzheimer's disease. The present invention involves the use of the agents described herein for treating these diseases, a method of selecting patients who would benefit from such treatment, and a method of monitoring a patient's response to the treatment.

[0010] Fluorescence, positron emission tomography (F 18-PET), near-infrared, and other small molecule probes can be chemically conjugated to the compounds described herein as a direct method for imaging ClpP expression in tumors or other tissues (Liu H-W., Chem Soc. 2018, 47, 7140-7180 and references cited therein; Pantel A.R., Cancer Lett, 2017, 387, 25-31 and references cited therein). This provides a basis for detecting ClpP expression in tumors as a biomarker for cancer. Second, the use of these probes can be directly used to measure the efficacy of ClpP binding by drugs such as ONC201 and chemical agents or other ClpP binders described herein by assays for measuring competitive reduction of ClpP binding. Target (ClpP) binding by ONC201 or other compounds described herein can be directly measured in live animals, humans, or in vitro screening assays. When combined with a ClpP enzyme activity assay, this ClpP binding by small molecules can be directly measured. Third, the development of ClpP-dependent activity probes can be applied to measure the activity of ClpP in tumors or cell lysates. Applying the principle of the enzyme-activated fluorescent probes described in (Liu H-W., Chem Soc Review. 2018, 47, 7140-7180 and references cited therein), we propose to attach a reactive chemical group to the compounds of the present invention for this purpose. The amine-reactive TR compounds target these compounds to ClpP in unperturbed tumors and are conjugated to a chemical fluorescent substrate for the purpose of directly measuring ClpP using this approach. Fourth, the use of the TR compound probes can be used to discover novel ClpP-binding molecules by taking advantage of the displacement of the TR-probe compounds by unknown compounds in high-throughput assays using time-resolved fluorescence assays or other assays. We further disclose that the use of our TR-compound probes can similarly be used to identify novel small molecule binders of the bacterial ClpP (bClpP) enzyme.When combined with the bClpP activity assay, the effects of these small molecules on bClpP activity can be directly determined. This provides a unique TR-probe compound for the discovery of bClpP binders as potential antibacterial agents. Furthermore, novel chemical substances useful as anticancer agents are described.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] The present invention relates to a method for determining whether an individual responds to a drug described by formula I, and a method for determining whether an individual maintains responsiveness to a drug described by formula I, which method comprises assaying a biological sample for the level of at least one biomarker. The present invention further relates to a kit for carrying out the method. The present invention further describes a novel chemical substance and the use of the novel chemical substance for treating cancer, various proliferative diseases, various immunological diseases, various inflammatory diseases, bacterial infections, neurodegenerative diseases, viral diseases such as HIV, acquired immunodeficiency syndrome (AIDS), hereditary spastic paraplegia, cystic fibrosis (CF) and Pelizaeus-Merzbacher disease. The relationship of ClpP with cancer and other diseases and targeted therapeutic agents has been described by Wong, KS and Houry, WA (ACS Chem. Biol., 2019: DIO: 10.102.1021 / acschembio.9b00347 and references cited therein). All publications, patents and patent applications cited herein are incorporated herein by reference as if each individual publication, patent or patent application was specifically and individually indicated as being incorporated by reference. The phrase "and references cited therein" following a particular citation indicates that all citations within that particular citation, whether a publication, patent or patent application, are also incorporated herein by reference, regardless of whether it is a publication, patent or patent application. Reduction in the expression of ClpP in both RNA and the expressed protein (ClpP) in cells derived from patients with hereditary spastic paraplegia can be modulated by treatment with the compounds and methods according to the present invention (Bross, P et al, Neuroscience, 2008, 153, 474-482).

[0013] One aspect of the present invention relates to the treatment of hereditary spastic paraplegia by administration of the compounds of the present invention.

[0014] One aspect of the present invention relates to a novel method for detecting ClpP as a biomarker for cancer and other diseases. This is based on our unique discovery that ONC201 and chemically related compounds defined by Formula I are high-affinity binders and activators of ClpP enzyme activity. Another aspect of the present invention relates to the use of the agents described herein as activity probes for detecting ClpP protein and activity levels in tumors and cells containing biological samples taken from mammals. These biological samples can be obtained from mammals before or after treatment with a compound represented by Formula I. Additionally, these samples can also be treated with the compounds described by Formula I, and the response to the compound can be determined by changes in ClpP activity levels and protein levels, or other related markers for ClpP activity.

[0015] Another aspect of the present invention relates to the regulation of the complex ClpXP and its components ClpP and ClpX, which is an AAA+ ATPase. This regulation of these components can be used in the treatment of diseases.

[0016] One aspect of the present invention relates to a novel method for detecting ClpXP as a biomarker for cancer and other diseases.

[0017] One aspect of the present invention relates to a novel method for detecting ClpX as a biomarker for cancer and other diseases.

[0018] Another aspect of the present invention relates to the identification of other chemical substances as binders to ClpP. The compounds of the present invention can be used in assays for screening a library of compounds to identify novel chemical substances.

[0019] A. Development of a high-affinity ClpP binding probe for detecting ClpP in live animals, patients, or intact cells.

[0020] We have found that hClpP binds directly to TR79, TR80, and TR81, which are compounds of the present invention, when bound to sepharose beads. Further, we determined that ONC201, ONC212 (TR31), and others (TR57) compete dose-dependently with hClpP (human - ClpP) released from the functionalized sepharose beads (Figure 4). This provides that ONC201 and other analogs and related chemicals of the present invention bind to hClpP (Graves L. M. et al, ACS Chem Biol., 2019, 14(5), 1020 - 1029 and references cited therein). The present invention further describes the attachment of fluorescent, infrared, PET, and other imaging moieties to a subset of compounds of Formula I using chemically reactive functional groups. These imaging moieties in the disclosure of the present invention are collectively known as "dyes". Examples of compounds having these properties are TR79, TR80, and TR81. These probes are used as cell - permeable imaging probes for the detection of ClpP as a biomarker for cancer or other diseases.

[0021] B. Measuring probe displacement to evaluate small - molecule therapeutic binding to the biomarker protein ClpP.

[0022] As described herein, the probes of the present invention are used to measure the efficacy of target (ClpP) binding by therapeutic agents, which are directed to this enzyme. This would include ONC201, ONC206, ONC212, and other compounds of Formula I for the treatment of diseases in mammals. An animal or human is exposed to these probes and tumors imaged by fluorescence, PET, or other imaging modalities. Exposure to ONC201 or a related compound is carried out, and the amount of probe remaining bound to ClpP is determined by imaging. By determining the signals before and after such exposure, it is possible to directly measure the extent to which this biomarker target (ClpP) is effectively bound to ONC201 or other ClpP - binding related therapeutic agents.

[0023] Develop a ClpP activity-based probe for the detection of ClpP activity in tumors, cells or cell lysates

[0024] Using a subset of the compounds of Formula I, an activity-dependent probe selective for ClpP is prepared. To prepare the ClpP activity probe, a cleavable fluorescence broad array or other such chemical moiety known to those skilled in the art is used. Examples of suitable compounds of Formula I are TR79, TR80 and TR81, each of which has a chemically reactive amine suitable for binding to a wide range of agents (resulting in a "binder"). These binders are applied for 1) directing the binding of these molecules to ClpP, and 2) measuring ClpP activity by hydrolysis of the fluorescent molecule. These agents are also used for imaging ClpP activity in tumors, tissues or cell lysates.

[0025] D. Development of ClpP probes for high-throughput screens for ClpP binding and modulation

[0026] The various probes / binders described in the present invention are diagnostic reagents for evaluating compound binding to ClpP from mammalian and bacterial sources. This assay is based on fluorescence displacement from ClpP (or otherwise probe displacement). Time-resolved fluorescence anisotropy (or a similar assay) is used to measure the displacement of the probe compound from ClpP by the said compound. This forms the basis of an HTS screening procedure for discovering new small molecule interactors of ClpP from human or bacterial sources.

[0027] Definition

[0028] The terms used herein have their ordinary meanings, and the meanings of such terms are independent at each occurrence thereof. Nevertheless, also except where otherwise provided, the following definitions apply throughout the specification and claims.

[0029] a) Definitions related to biology

[0030] Neoplastic disease: A neoplasm is an abnormal growth and proliferation of abnormal cells or an abnormal amount of cells due to a benign or malignant process.

[0031] Biological sample. The term "sample" with respect to an individual includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens and their products. This definition includes samples that have been manipulated in some way after acquisition, such as treatment with reagents, washing, or enrichment for a particular cell population such as cancer cells. This definition also includes samples that have been enriched for a particular type of molecule, such as nucleic acids, polypeptides, etc.

[0032] The term "biological sample" includes clinical samples. The types of "biological samples" include, but are not limited to, tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirates, lymph node aspirates, cyst aspirates, punch samples, thoracentesis samples, etc. A "biological sample" may contain cells (e.g., target cells, normal cells, blood cells, tissue cells, etc.), may be suspected of containing such cells, or may be considered to be lacking cells. A biological sample may contain a biological fluid derived from cells (e.g., cancer cells, infected cells, etc.), and may include, for example, a sample containing polynucleotides and / or polypeptides obtained from such cells (e.g., cell lysates or other cell extracts containing polynucleotides and / or polypeptides). A biological sample containing patient-derived infected cells may contain non-infected cells. In one embodiment, the biological sample is blood or a derivative thereof, such as plasma, serum, etc.

[0033] Sample Acquisition and Assay. As used herein, the term "assay" is used to include the physical process of manipulating a biological sample to generate data related to the sample. As will be readily understood by one of ordinary skill in the art, a biological sample must be "acquired" before the sample is assayed. Thus, the term "assay" implies that the sample has been acquired. As used herein, the terms "obtained" or "obtaining" include the act of receiving an extract or an isolated biological sample. For example, a testing facility can "obtain" a biological sample by mail (or delivery, etc.) before assaying the sample. In some cases, the biological sample is "extracted" or "isolated" (i.e., delivered, transferred, etc.) from an individual by another party prior to mailing and then "obtained" by the testing facility upon arrival of the sample. Thus, the testing facility can acquire the sample and then assay the sample, thereby generating data related to the sample.

[0034] As used herein, the terms "obtained" or "obtaining" can also include the physical extraction or isolation of a biological sample from a subject. Thus, a biological sample can be isolated (and thus "obtained") from a subject by the same person or entity that will subsequently assay the sample. If a biological sample is "extracted" or "isolated" from a first party or entity and then mailed (e.g., delivered, transported, etc.) to a second party, the sample was obtained (and also "isolated") by the first party and then "obtained" (not "isolated") by the second party. Thus, in some embodiments, the acquisition step does not include the step of isolating the biological sample.

[0035] In some embodiments, the acquisition step includes the step of isolating a biological sample (e.g., a biological sample before treatment, a biological sample after treatment, etc.). Methods and protocols for isolating various biological samples (e.g., blood samples, serum samples, plasma samples, biopsy samples, aspirates, etc.) are known to those of ordinary skill in the art, and any convenient method can be used to isolate the biological sample.

[0036] In some cases, it will be appreciated by those skilled in the art that it may be convenient for multiple samples (e.g., a biological sample before treatment and a biological sample after treatment) to wait to be obtained before assaying the samples. Thus, in some cases, isolated biological samples (e.g., a biological sample before treatment, a biological sample after treatment, etc.) are stored until all appropriate samples are obtained. Those skilled in the art understand how to appropriately store various different types of biological samples and can use any convenient storage method (e.g., refrigeration) suitable for a particular biological sample. In one aspect, the biological sample before treatment and the sample after treatment are assayed in parallel. In some cases, multiple different biological samples after treatment and / or biological samples before treatment are assayed in parallel. In some cases, biological samples are processed immediately or as soon as possible after they are obtained.

[0037] In the method of the present invention, the concentration (i.e., "level") of a gene product, such as RNA, protein, etc., that can be in a biological sample, or the expression level (referred to herein as a biomarker) is measured (i.e., "determined"). The "expression level" (or "level") means the level of a gene product (e.g., the RNA expression level of a biomarker, the expression level of the encoded polypeptide, or the absolute value and / or normalized value determined for the concentration of a protein in a biological sample). The terms "gene product" or "expression product" are used herein to refer to the RNA transcript of a gene (RNA transcript, e.g., mRNA, unspliced RNA, splice variant mRNA, and / or fragmented RNA) including mRNA, and the polypeptide translation product of such an RNA transcript. Gene products can be, for example, unspliced RNA, mRNA, splice variant mRNA, microRNA, fragmented RNA, polypeptide, post-translationally modified polypeptide, splice variant polypeptide, etc.

[0038] The terms "determine", "measure", "evaluate", "assess", "assay", and "analyze" are used interchangeably herein to refer to any form of measurement and include determining whether an element is present or not. These terms include quantitative and / or qualitative determinations. An assay can be relative or absolute. For example, an "assay" can be determining whether an expression level is less than a particular threshold, or "greater than, or equal to" (the threshold can be predetermined or determined by assaying a control sample). On the other hand, an "assay for determining an expression level" can mean determining (using any convenient metric) a quantitative value representing the expression level of a particular biomarker (i.e., the expression level, e.g., the amount of protein and / or RNA, e.g., mRNA). The level of expression can be represented in any unit associated with a particular assay (e.g., fluorescence units, e.g., mean fluorescence intensity (MFI)), or as an absolute value having a defined unit (e.g., the number of mRNA transcripts, the number of protein molecules, the concentration of protein, etc.). Further, a normalized value representing a normalized expression level can be derived by comparing the expression level of a biomarker to the expression levels of one or more additional genes (e.g., nucleic acids and / or the proteins encoded thereby). When evaluating multiple biological samples from the same individual (e.g., biological samples taken from the same individual at different time points), the particular metric criterion (or unit) selected is not important as long as the same unit is used (or a conversion to the same unit is made). This is because the units cancel out when calculating (i.e., determining the ratio) the fold change in the expression level from one biological sample to the next (e.g., biological samples taken from the same individual at different time points).

[0039] To measure the RNA level, the amount or level of RNA in a sample, such as the level of mRNA, is determined. In some examples, the expression levels of one or more additional RNAs can also be measured, and the level of biomarker expression compared to the level of one or more additional RNAs provides a normalized value of the biomarker expression level. Any convenient protocol for assessing the RNA level can be used, where the levels of one or more RNAs in the assayed sample are determined.

[0040] Multiple exemplary methods for measuring the RNA (e.g., mRNA) expression level (e.g., the expression level of a nucleic acid biomarker) in a sample are known to those skilled in the art, and any convenient method can be used. Exemplary methods include hybridization-based methods (e.g., Northern blot, array hybridization (e.g., microarray), in situ hybridization, in situ hybridization after FACS, etc.) (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNase protection assay (Hod et al, Biotechniques, 1992, 13 852-854 and references cited therein), PCR-based methods (e.g., reverse transcription PCR (RT-PCR), quantitative RT-PCR (qRT-PCR), real-time RT-PCR, etc.) (Weis et al, Trends in Genetics 1992 8 263-264 and references cited therein), nucleic acid sequencing methods (e.g., Sanger sequencing, next-generation sequencing (i.e., Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (SOLiD platform), Life Technologies' Ion Torrent platform, single molecule sequencing, etc., which is large-scale parallel high-throughput sequencing), etc., but are not limited thereto.

[0041] In some embodiments, the biological sample can be assayed directly. In some embodiments, the nucleic acid of the biological sample is amplified (e.g., by PCR) prior to the assay. Thus, techniques such as PCR (polymerase chain reaction), RT-PCR (reverse transcriptase PCR), qRT-PCR (quantitative RT-PCR) can be used prior to the hybridization methods and / or sequencing methods described above.

[0042] The starting material for measuring mRNA levels is generally total RNA or poly A+RNA isolated from a biological sample (e.g., a cell suspension from a peripheral blood sample, bone marrow sample, etc., or a homogenized tissue, e.g., a homogenized biopsy sample, aspirate, homogenized paraffin-embedded or OCT-embedded sample, etc.). General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). RNA isolation can also be performed using a manufacturer's purification kit, buffer set, and protease according to the manufacturer's instructions. For example, RNA from a cell suspension can be isolated using a Qiagen RNeasy mini column, and RNA from a cell suspension or homogenized tissue sample can be isolated using a TRIzol reagent-based kit (Invitrogen), MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE™, Madison, Wis.), paraffin block RNA isolation kit (Ambion, Inc.), or RNA Stat-60 kit (Tel-Test).

[0043] Various different methods for measuring mRNA levels are known in the art and are used, for example, in the field of differential gene expression analysis. One representative and convenient type of protocol for measuring mRNA levels is array-based gene expression profiling. Such protocols are hybridization assays, in which nucleic acids are used that display "probes" for each of the genes assayed / profiled in the generated profile. In these assays, a sample of the target nucleic acid is first prepared from the first nucleic acid sample to be assayed, where the preparation can include labeling the target nucleic acid with a label, e.g., a member of a signal generation system. After preparation of the target nucleic acid sample, the sample is contacted with the array under hybridization conditions, whereby a complex is formed between the target nucleic acids complementary to the probe sequences bound to the array surface. The presence of the hybridized complex is then detected either qualitatively or quantitatively.

[0044] Specific hybridization techniques that can be carried out to generate the expression profiles used in the method of the present invention include, as well as WO95 / 21265, WO96 / 31622, WO97 / 10365, WO97 / 27317, EP373203, EP785280, U.S. Patent Nos. 5,143,854, 5,288,644, 5,324,633, 5,432,049, 5,470,710, 5,492,806, 5,503,980, 5,510,270, 5,525,464, 5,547,839, 5,580,732, 5,661,028, 5,800,992, the disclosures of which are incorporated herein by reference. In these methods, an array of "probe" nucleic acids containing probes for each of the phenotypic determining genes whose expression is being assayed is contacted with the target nucleic acid as described above. The contacting is carried out under hybridization conditions, for example, under stringent hybridization conditions, and then unbound nucleic acids are removed. As used herein, the term "stringent assay conditions" refers to conditions that are compatible with generating binding pairs of nucleic acids having sufficient complementarity to provide the desired level of specificity in the assay, e.g., surface-bound and solution-phase nucleic acids, while being of low compatibility for forming binding pairs between binding members having insufficient complementarity to provide the desired specificity. Stringent assay conditions are the sum or combination (overall) of both hybridization conditions and wash conditions.

[0045] The pattern obtained from the hybridized nucleic acids provides information regarding the expression for each of the genes probed, where the expression information relates to whether the gene is expressed or not and typically at what level it is expressed, where the expression data, i.e., the expression profile (e.g., in the form of a transcriptosome), can be both qualitative and quantitative.

[0046] Alternatively, a non-array-based method for quantifying the level of one or more nucleic acids in a sample may be used. These include those based on amplification protocols, such as polymerase chain reaction (PCR)-based assays such as quantitative PCR, reverse transcription PCR (RT-PCR), real-time PCR, for example, TaqMan® RT-PCR, MassARRAY® System, BeadArray® technology, and Luminex® technology, those that rely on hybridization of a probe to a filter, such as Northern blot and some in situ examples of the nucleic acid sequencing methods listed above are described in the following references: Margulies et al., Nature 2005, 437, 376-80 and references cited therein, Ronaghi et al., Analytical Biochemistry 1996, 242, 84-89 and references cited therein, Shendure et al., Science 2005 309 1728 and references cited therein, Imelfort et al., Brief Bioinform. 2009, 10, 609-618 and references cited therein, Fox et al., Methods Mol Biol. 2009, 553, 79-108 and references cited therein, Appleby et al., Methods Mol Biol. 2009, 513, 19-39 and references cited therein, and Morozova et al., Genomics 2008, 92, 255-264 and references cited therein are incorporated by reference for a general description of the methods and for reference to specific steps of the methods, including all starting products, reagents, and final products for each step.

[0047] To measure protein levels, the amount or level of polypeptide in a biological sample is measured. In certain embodiments, extracellular protein levels are measured. For example, in some cases, the protein (i.e., polypeptide) being measured is a secreted protein (e.g., a cytokine or chemokine), and thus the concentration can be measured in the extracellular fluid of the biological sample (e.g., the protein concentration can be measured in serum). In certain embodiments, the concentration is a relative value measured by comparing the level of one protein to another protein. In other embodiments, the concentration is an absolute measurement of weight / volume or weight / weight hybridization.

[0048] In some cases, prior to measuring the concentration, cells are removed from the biological sample (e.g., via centrifugation, or via adhesion of the cells to a dish or plastic). In some cases, to measure the protein level in the cell contents, the intracellular protein level is measured by lysing the cells of the biological sample that have been removed. In some cases, the cell and fluid portions of the biological sample are separated (e.g., via centrifugation), and the extracellular level of the protein is measured by measuring the level of the protein in the fluid portion of the biological sample, and the intracellular level of the protein is measured by measuring the level of the protein in the cell portion of the biological sample (e.g., after lysing the cells), whereby both the extracellular and intracellular levels of the protein are measured. In some cases, the total level of the protein (i.e., a combination of extracellular and intracellular protein) is measured by lysing the cells of the biological sample and including the intracellular contents as part of the sample.

[0049] In some examples, the concentration of one or more additional proteins can also be measured, and the biomarker concentration is compared to the level of one or more additional proteins to provide a normalized value of the biomarker concentration. Any convenient protocol for assessing protein levels can be used, where the level of one or more proteins in the assayed sample is determined.

[0050] Various different methods for measuring protein levels are known to those skilled in the art, and any convenient method can be used. However, one representative and convenient type of protocol for measuring protein levels is the antibody-based method ELISA. In ELISA and ELISA-based assays, one or more antibodies specific to the protein of interest can be immobilized on a selected solid surface, preferably a surface showing protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed substances, the assay plate wells are coated with a non-specific "blocking" protein known to be antigenically neutral with respect to the test sample, such as a solution of bovine serum albumin (BSA), casein, or nonfat dry milk. This allows blocking of non-specific adsorption sites on the immobilized surface, thereby reducing the background caused by non-specific binding of antigens to the surface. After washing to remove unbound blocking protein, the immobilized surface is contacted with the sample to be tested under conditions that induce immune complex (antigen / antibody) formation. After incubation, the antiserum contact surface is washed to remove non-immune complex material. The bound immune complex can then be subjected to a second antibody having specificity for a target different from the first antibody, and by detecting the binding of the second antibody, the occurrence and amount of immune complex formation can be determined. In certain embodiments, the second antibody has an associated enzyme, such as urease, peroxidase, or alkaline phosphatase, which generates a color precipitate when incubated with an appropriate chromogenic substrate. After incubation with such a second antibody and washing to remove unbound substances, for example, in the case of a peroxidase label, incubation with a chromogenic substrate such as urea and bromocresol purple, or in the case of a peroxidase label, incubation with 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H2O2 quantifies the amount of the label. Quantification is then achieved, for example, by measuring the degree of color development using a visible spectrum spectrophotometer.

[0051] The aforementioned format can be changed by first binding the sample to the assay plate. The primary antibody is then incubated with the assay plate, and then the bound primary antibody is detected using a labeled secondary antibody having specificity for the primary antibody. The solid substrate to which the antibody is immobilized can be made of a wide variety of materials and in a wide variety of 30 shapes, such as microtiter plates, microbeads, dipsticks, resin particles, etc., and the substrate can be selected to maximize the signal-to-noise ratio, minimize background binding, and facilitate separation and cost. Washing can be affected in the most appropriate way for the substrate being used, for example, by removing beads or dipsticks from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing beads, particles, chromatography columns or filters with a washing solution or solvent.

[0052] Alternatively, a non-ELISA-based method for measuring the level of one or more proteins in a sample may be used. Representative exemplary methods include, but are not limited to, antibody-based methods (e.g., Western blotting, proteomics arrays, xMAP (trademark) microsphere technology (e.g., Luminex (registered trademark) technology), immunohistochemistry, flow cytometry, etc.) as well as non-antibody-based methods (e.g., mass spectrometry).

[0053] Biomarker. As used herein, the term "biomarker" refers to a gene product, i.e., a protein or RNA, the concentration (i.e., "level") and enzymatic activity (function) of which report the activity (level and / or both function) of the administered ClpP modulator. This ClpP modulator is also known as a ClpP agent. Since some individuals may not respond to treatment with a ClpP agent, biomarkers can be used to determine whether a ClpP agent has the desired effect in an individual (e.g., determining whether an individual responds to a ClpP agent, determining whether an individual maintains responsiveness to a ClpP agent, and whether an individual is a candidate for treatment with a ClpP agent, etc.). For example, a biomarker whose level increases upon administration of a ClpP agent when an individual responds to the ClpP agent is a "positive biomarker". A biomarker whose level decreases upon administration of a ClpP agent when an individual responds to the ClpP agent is a "negative biomarker". Also, a biomarker whose level does not change upon administration of a ClpP agent when an individual responds to the ClpP agent is a "neutral biomarker".

[0054] In some embodiments, the concentration or level of the biomarker is determined before and after administration of the ClpP agent, and the degree of change or lack of change thereof is interpreted as an indicator of whether the administered ClpP agent actually affects the function and / or level of ClpP and / or whether this blockade has the desired effect (i.e., whether the immune system is activated in response to contact with or administration of the ClpP agent). In summary, the concentration or level of the biomarker is determined before and after administration of the ClpP agent to an individual, and the degree of change or lack of change in level and / or enzymatic function (considered in relation to the exposure time to the ClpP agent) is interpreted as an indicator of whether an individual is likely to "respond" to a ClpP agent, whether an individual "responds" to a ClpP agent, and / or whether an individual "maintains responsiveness" to a ClpP agent.

[0055] A "positive biomarker" is a biomarker whose level increases in response to contact with and / or treatment with a ClpP agent when an individual and / or cell responds to the ClpP agent. Thus, a biological sample isolated from an individual administered a ClpP agent will show an increase in the level of the positive biomarker (compared to the level of the same biomarker measured from the same type of biological sample from the same individual prior to administration of the ClpP agent) if the ClpP agent has the desired effect. In some embodiments, the level of the positive biomarker increases by at least about 1.5-fold (e.g., at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold) in response to contact with and / or treatment with a ClpP agent when an individual and / or cell responds to the ClpP agent.

[0056] Positive biomarkers include, but are not necessarily limited to, ClpP, ClpX, ClpXP, H3K27M, LONP, and malic enzyme 1 (ME1). Further positive biomarkers (>2-fold increase) established by treating cancer cells with a compound of Formula I include the following.

[0057] JPEG0007709379000001.jpg225149

[0058] The levels of any combination of the above positive biomarkers can be measured and used in the methods of the present invention.

[0059] A "negative biomarker" is a biomarker whose level decreases in response to contact and / or treatment with a ClpP agent when an individual and / or cell responds to the ClpP agent. Thus, a biological sample isolated from an individual to whom a ClpP agent has been administered will show a decrease in the level of the negative biomarker (compared to the level of the same biomarker measured from the same type of biological sample from the same individual before administration of the ClpP agent) if the ClpP agent has the desired effect. In some embodiments, the level of the negative biomarker decreases by about 1.5-fold or more (e.g., 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 8-fold or more, 10-fold or more, 15-fold or more) in response to contact and / or treatment with a ClpP agent when an individual and / or cell responds to an anti-CD47 agent. Negative biomarkers include, but are not necessarily limited to, ClpP, ClpX, ClpXP, H3K27M, LONP, and malic enzyme 1 (ME1). Other negative biomarkers (>2-fold decrease) established by treating cancer cells with a compound of Formula I include the following.

[0060] JPEG0007709379000002.jpg225123

[0061] A "neutral biomarker" is a biomarker whose level does not significantly increase or decrease in response to contact and / or treatment with a ClpP agent when an individual and / or cell responds to the ClpP agent. The term "neutral biomarker" is used to refer to a protein or RNA that is expected to change in level (for example, because gene levels change during other situations that alter an individual's immune status, such as during an inflammatory response), but has been experimentally shown not to change in the situation where a ClpP agent is used to modulate ClpP level and / or function. Thus, a biological sample isolated from an individual administered a ClpP agent will exhibit similar levels of neutral biomarkers (relative to the levels of the same biomarkers measured from the same type of biological sample from the same individual prior to administration of the ClpP agent, or relative to a standardized control) when the ClpP agent has the desired effect. In some embodiments, the level of a neutral biomarker changes less than about 5-fold (for example, less than about 4.5-fold, less than about 4-fold, less than about 3.5-fold, less than about 3-fold, less than about 2.5-fold, less than about 2-fold, or less than about 1.5-fold) in response to contact with and / or treatment with a ClpP agent when an individual and / or cell responds to the ClpP agent. Neutral biomarkers include, but are not necessarily limited to, ClpP, ClpXP, ClpX, H3K27M, LONP, and malic enzyme 1 (ME1). Additionally, for neurodegenerative diseases, α-synuclein and α-synuclein A53T (mutant) may be used. The levels of any combination of the above neutral biomarkers can be measured and used in the methods of the present invention.

[0062] Chemical-related definitions

[0063] The chemical name, common name, and chemical structure can be used interchangeably to describe the structure. In the case of ambiguity between the structure and the name in the chemical structure and chemical name, the structure takes precedence. These definitions apply regardless of whether the term is used alone or in combination with other terms, unless otherwise specified. Thus, the definition of "alkyl" applies to the "alkyl" moieties such as "hydroxyalkyl", "fluoroalkyl", "-O-alkyl", etc.

[0064] As used throughout this specification and the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise specified.

[0065] As used herein, the term "therapeutically effective amount" refers to the amount of the composition of the compound of formula (I) and / or additional therapeutic agent or agents that, when administered to a patient suffering from cancer or other disease or disorder of unwanted cell proliferation, is effective to produce the desired therapeutic, ameliorating, inhibitory, or prophylactic effect. In the combination therapies of the present invention, the therapeutically effective amount can refer to each individual agent or the combination as a whole, where the amounts of all the agents administered are effective together, but the component agents of the combination may not individually be present in effective amounts. With respect to the treatment of cancer, a therapeutically effective amount means an amount having the effect of (1) reducing the size of the tumor, (2) inhibiting (to some extent decelerating and preferably stopping) tumor metastasis, (3) inhibiting (preferably stopping) tumor growth or tumor invasiveness to some extent, and / or (4) alleviating (or preferably eliminating) one or more symptoms or signs associated with the cancer.

[0066] As used herein with respect to a disease or disorder of cancer or unwanted cell proliferation, the term "prevent" means reducing the likelihood or rate of progression of the disease or disorder.

[0067] The use of a dashed or dotted line means a single bond between the said molecular fragment and another defined molecular fragment. For example, when Q1 is selected for Q in formula (I), the following structure results. JPEG0007709379000003.jpg38150

[0068] In another example, when Q2 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000004.jpg35150

[0069] In another example, when Q3 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000005.jpg32150

[0070] In another example, when Q4 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000006.jpg32150

[0071] In another example, when Q5 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000007.jpg36150

[0072] In another example, when Q6 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000008.jpg32150

[0073] In another example, when Q7 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000009.jpg34150

[0074] In another example, when Q8 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000010.jpg30150

[0075] In another example, when Q9 is selected for Q in formula (I), the following structure is obtained. JPEG0007709379000011.jpg35150

[0076] In another example, when Q10 is selected for Q in formula (I), the following structure results. JPEG0007709379000012.jpg29150

[0077] In another example, when Q11 is selected for Q in formula (I), the following structure results. JPEG0007709379000013.jpg30150

[0078] In another example, when Q12 is selected for Q in formula (I), the following structure results. JPEG0007709379000014.jpg30150

[0079] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group in which one of its hydrogen atoms is replaced by a bond having a specific number of carbon atoms. The alkyl group may be linear or branched. In addition to the term "alkyl", the alkyl group can be further defined by the number of carbon atoms. The alkyl substituent generally includes 1 to 20 carbon atoms "(C1-C20)alkyl", preferably 1 to 12 carbon atoms "(C1-C12)alkyl", more preferably 1 to 8 carbon atoms "(C1-C8)alkyl", or 1 to 6 carbon atoms "(C1-C6)alkyl", or 1 to 4 carbon atoms "(C1-C4)alkyl". In different embodiments, the alkyl group includes 7 to 12 carbon atoms "(C7-C12)alkyl" or 7 to 20 carbon atoms "(C7-C20)alkyl". Non-limiting examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl. All alkyl groups described herein may be optionally substituted by one or more substituents independently selected, unless otherwise expressly stated. The alkyl group described herein as substituted alkyl ("substituted alkyl") is substituted by one or more substituents independently selected, unless otherwise expressly stated. The total number of substituents can be equal to the total number of hydrogen atoms on the alkyl moiety, provided that such substitution is chemically meaningful. An optionally substituted alkyl group ("optionally substituted alkyl") generally includes 1 to 6 optional substituents, preferably 1 to 4 optional substituents, more preferably 1 to 3 optional substituents. For example, an optionally substituted ethyl group is "optionally substituted (C2)alkyl" or "(C2)optionally substituted alkyl", and a substituted ethyl group is "substituted (C2)alkyl" or "(C2)substituted alkyl".

[0080] Suitable substituents for alkyl, "alkyl", "optionally substituted alkyl" and "substituted alkyl" include (C3-C8) cycloalkyl, 3- to 12-membered heterocyclyl, (C6-C12) aryl, 5- to 12-membered heteroaryl, halo, =O (oxo), =S (thiono), =N-CN, =N-OR X , =NR X , -CN, -C(O)R X , -CO2R X , -C(O)NR X R Y , -SR X , -SOR X , -SO2R X , -SO2NR X R Y , -NO2, -NR X R Y , -NR X C(O)R y , -NR X C(O)NR X R Y , -NR X C(O)OR X , -NR X SO2R Y , -NR X SO2NR X R Y , -OR X , -OC(O)R X , and -OC(O)NR X R Y are included, but not limited to. Here, each R X and R Y are each independently hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C3-C6) cycloalkyl, 3- to 12-membered heterocyclyl, (C6-C12) aryl, or 5- to 12-membered heteroaryl, or R X and R Y can together with the nitrogen atom to which they are attached form a 3- to 12-membered heterocyclyl or a 5- to 12-membered heteroaryl system, each of which can optionally contain 0, 1, or 2 additional heteroatoms, and each R X and R Yis optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, =O, -CN, -C(O)R', -CO2R', -C(O)NR'2, -SO2R', -NR'2, or 3- to 12-membered heterocyclyl, where each R' is independently hydrogen, (C1-C6)alkyl, (C3-C6)cycloalkyl, or 3- to 12-membered heterocyclyl. However, suitable substituents for "substituted alkyl" do not include hydrogen.

[0081] "Alkenyl", as defined herein, refers to an alkyl group consisting of at least two carbon atoms and at least one carbon-carbon double bond. Generally, an alkenyl group has 2 to 20 carbon atoms "(C2-C20)alkenyl", preferably 2 to 12 carbon atoms "(C2-C12)alkenyl", more preferably 2 to 8 carbon atoms "(C2-C8)alkenyl", or 2 to 6 carbon atoms "(C2-C6)alkenyl", or 2 to 4 carbon atoms "(C2-C4)alkenyl". Representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, etc. The alkenyl group can be optionally substituted ("optionally substituted alkenyl"). Suitable substituents for alkenyl are as described herein for "optionally substituted alkyl", "substituted alkyl", and alkyl.

[0082] "Alkynyl", as defined herein, refers to an alkyl group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl group has 2 to 20 carbon atoms "(C2-C20) alkynyl", preferably 2 to 12 carbon atoms "(C2-C12) alkynyl", more preferably 2 to 8 carbon atoms "(C2-C8) alkynyl", or 2 to 6 carbon atoms "(C2-C6) alkynyl", or 2 to 4 carbon atoms "(C2-C4) alkynyl". Representative examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2- or 3-butynyl. Any alkynyl group can be optionally substituted. Suitable substituents for alkynyl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0083] As used herein, the term "fluoroalkyl" refers to an alkyl group as defined above in which one or more of the hydrogen atoms of the alkyl group are substituted with fluorine. In one embodiment, the fluoroalkyl group has 1 to 6 carbon atoms. In another embodiment, the fluoroalkyl group has 1 to 3 carbon atoms. In another embodiment, the fluoroalkyl group is substituted with 1 to 3 fluorine atoms. Non-limiting examples of fluoroalkyl groups include -CH2F, -CHF2 and CF3. The term "(C1-C3) fluoroalkyl" refers to a fluoroalkyl group having 1 to 3 carbon atoms. The term "(C1) fluoroalkyl" refers to -CH2F, -CHF2 and CF3.

[0084] As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic ring system containing 6 to about 14 carbon atoms. In one embodiment, the aryl group contains about 6 to 10 carbon atoms (C6-C10) aryl. In another embodiment, the aryl group is phenyl. Non-limiting examples of aryl groups include phenyl and naphthyl. The aryl group can be optionally substituted. Suitable substituents for aryl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0085] As used herein, the term "cycloalkyl" refers to a saturated ring containing a specific number of ring carbon atoms and no heteroatoms. Cycloalkyl substituents generally contain from 3 to 8 carbon atoms, "(C3-C8) cycloalkyl", preferably from 3 to 7 carbon atoms, "(C3-C7) cycloalkyl", more preferably from 3 to 6 carbon atoms, "(C3-C6) cycloalkyl", or from 3 to 5 carbon atoms, "(C3-C5) cycloalkyl". Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. All cycloalkyl groups described herein may be optionally substituted by one or more independently selected substituents, unless otherwise indicated. Cycloalkyl groups described herein as optionally substituted ("optionally substituted cycloalkyl") may be substituted by one or more independently selected substituents, unless otherwise indicated. Cycloalkyl groups described herein as substituted cycloalkyl ("substituted cycloalkyl") are substituted by one or more independently selected substituents, unless otherwise indicated. The total number of substituents can be equal to the total number of hydrogen atoms on the cycloalkyl moiety, to the extent that such substitution makes chemical sense. Optionally substituted cycloalkyl groups generally contain from 1 to 6 optional substituents, preferably from 1 to 4 optional substituents, more preferably from 1 to 3 optional substituents. For example, an optionally substituted cyclopropyl group is "optionally substituted (C3) cycloalkyl", and a substituted cyclopropyl group is "substituted (C2) cycloalkyl". In one embodiment, the cycloalkyl group is "(C3-C9) cycloalkyl" containing from 3 to 9 carbon atoms. In another embodiment, the substituted cycloalkyl group is "substituted (C3-C9) cycloalkyl" containing from 3 to 9 carbon atoms. Suitable substituents for cycloalkyl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0086] As used herein, the term "cycloalkenyl" refers to a partially unsaturated carbocyclic system containing a specific number of carbon atoms. A cycloalkenyl substituent generally contains 4 to 8 carbon atoms, "(C4-C8) cycloalkenyl", preferably 5 to 6 carbon atoms, "(C5-C6) cycloalkenyl". Non-limiting examples of monocyclic cycloalkenyl include cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl. The cycloalkenyl groups described herein may be optionally substituted with one or more substituents, which are independently selected unless otherwise indicated. The total number of substituents can be equal to the total number of hydrogen atoms on the cycloalkenyl moiety, to the extent that such substitution is chemically meaningful. An optionally substituted cycloalkenyl group generally contains 1 to 6 optional substituents, preferably 1 to 4 optional substituents, more preferably 1 to 3 optional substituents. For example, a cyclopentenyl group is "(C5) cycloalkenyl", and an optionally substituted cyclopentenyl group is "optionally substituted (C5) cycloalkenyl". In one embodiment, the cycloalkenyl group is "(C4-C8) cycloalkenyl" containing 4 to 8 carbon atoms. Suitable substituents for cycloalkenyl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0087] As used herein, the term "cycloalkylalkyl" refers to a cycloalkyl ring that is attached to a base molecule via an alkylene linker of 1 to 6 carbon atoms ("(C1-C6) alkylene") and is generally (C3-C9) cycloalkyl. The cycloalkylalkyl group is described by the number of carbon atoms in the carbocyclic ring and the number of carbon atoms in the linker. The cycloalkylalkyl groups described herein may be optionally substituted with one or more substituents, which are independently selected unless otherwise indicated. The cycloalkylalkyl groups described herein as optionally substituted ("optionally substituted cycloalkylalkyl") may be substituted with one or more independently selected substituents unless otherwise indicated. The cycloalkylalkyl groups described herein as substituted cycloalkylalkyl ("substituted cycloalkylalkyl") are substituted with one or more independently selected substituents unless otherwise indicated. The total number of substituents can be equal to the total number of hydrogen atoms on the cycloalkylalkyl moiety, to the extent such substitution is chemically meaningful. Optionally substituted cycloalkylalkyl groups generally contain from 1 to 6 optional substituents, preferably from 1 to 4 optional substituents, more preferably from 1 to 3 optional substituents. In one embodiment, the cycloalkyl group contains 3 to 9 carbon atoms and the linker alkyl group contains 1 to 6 carbon atoms, and is "(C3-C9) cycloalkyl (C1-C6) alkyl". For example, a cyclopropylethyl group is "(C3) cycloalkyl (C2) alkyl", and an optionally substituted cyclopropylethyl group is "optionally substituted (C3) cycloalkyl (C2) alkyl". Also, a substituted cyclopropylethyl group is "substituted (C3) cycloalkyl (C2) alkyl". Suitable substituents for cycloalkylalkyl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0088] As used herein, the term "cycloalkenylalkyl" refers to a cycloalkenyl ring that is generally (C4-C8) cycloalkenyl and is attached to a base molecule via an alkylene linker of 1 to 6 carbon atoms "(C1-C6) alkylene". The cycloalkenylalkyl group is represented by the number of carbon atoms in the carbon ring and the number of carbon atoms in the linker. Thus, a "(C5) cycloalkenyl (C1) alkyl" group is a cyclopentenyl group attached to the base molecule via a methylene group (-CH2-). Unless otherwise indicated, the cycloalkenylalkyl groups described herein may be optionally substituted with one or more substituents independently selected. The total number of substituents can be equal to the total number of hydrogen atoms on the cycloalkenylalkyl moiety, to the extent that such substitution is chemically meaningful. An optionally substituted cycloalkenylalkyl group generally contains from 1 to 6 optional substituents, preferably from 1 to 4 optional substituents, more preferably from 1 to 3 optional substituents. In one embodiment, the cycloalkenyl group contains from 4 to 8 carbon atoms and the linker alkyl group contains from 1 to 6 carbon atoms, and is "(C4-C8) cycloalkenyl (C1-C6) alkyl". For example, a cyclopentenylethyl group is "(C5) cycloalkenyl (C2) alkyl", and an optionally substituted cyclopentenylethyl group is "optionally substituted (C5) cycloalkenyl (C2) alkyl". Suitable substituents for cycloalkenylalkyl are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0089] In some cases, the substituted alkyl group can be specifically named with reference to the substituent. For example, "haloalkyl" is substituted by one or more halo substituents and generally refers to an alkyl group having a specific number of carbon atoms, containing 1 to 6 carbon atoms and 1, 2 or 3 halo atoms (i.e., "(C1-C6) haloalkyl"). Thus, (C1-C4) haloalkyl groups include trifluoromethyl (-CF3) and difluoromethyl (-CF2H). Unless otherwise indicated, the haloalkyl groups described herein can be optionally substituted with one or more independently selected substituents. The total number of substituents (the total number of halo and any other substituents as defined herein) can be made equal to the total number of hydrogen atoms on the unsubstituted parent alkyl moiety, to the extent that such substitution is chemically meaningful. For example, for -CH2CH2CH(OH)CH2CF3, the parent alkyl moiety is N-pentyl (-(CH2)4CH3) which has 11 possible positions for substitution. This example is not intended to be limiting. The haloalkyl groups described herein as optionally substituted ( "optionally substituted haloalkyl") can be substituted by one or more independently selected substituents, unless otherwise indicated. The haloalkyl groups described herein as substituted haloalkyl ( "substituted haloalkyl") are substituted by one or more independently selected substituents, unless otherwise indicated. The total number of substituents can be made equal to the total number of hydrogen atoms on the haloalkyl moiety, to the extent that such substitution is chemically meaningful. Optionally substituted haloalkyl groups generally include 1 to 6 arbitrary substituents, preferably 1 to 4 arbitrary substituents, more preferably 1 to 3 arbitrary substituents. For example, an optionally substituted halopropyl group is "optionally substituted (C3) haloalkyl", and a substituted halopropyl group is "substituted (C3) haloalkyl". In one embodiment, the cycloalkyl group is "(C1-C6) haloalkyl" containing 1 to 6 carbon atoms. In another embodiment, the substituted haloalkyl group is "substituted (C1-C6) haloalkyl" containing 1 to 6 carbon atoms.Suitable substituents for haloalkyl are as described herein for "optionally substituted alkyl" and "substituted alkyl".

[0090] "Alkoxy" refers to a monovalent -O-alkyl group, where the alkyl moiety has a specific number of carbon atoms. The alkyl moiety of the alkoxy group may be a straight-chain or branched-chain group. Alkoxy groups generally include 1 to 8 carbon atoms "(C1-C8) alkoxy", or 1 to 6 carbon atoms "(C1-C6) alkoxy" or 1 to 4 carbon atoms "(C1-C4) alkoxy". Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and t-butoxy. All alkoxy groups described herein may be optionally substituted with one or more independently selected substituents, unless otherwise indicated. An alkoxy group described herein as optionally substituted ("optionally substituted alkoxy") may be substituted with one or more independently selected substituents, unless otherwise indicated. An alkoxy group described herein as substituted ("substituted alkoxy") is substituted with one or more independently selected substituents, unless otherwise indicated. The total number of substituents can be equal to the total number of hydrogen atoms on the alkoxy moiety, to the extent that such substitution makes chemical sense. An optionally substituted alkoxy group generally includes 1 to 6 arbitrary substituents, preferably 1 to 4 arbitrary substituents, more preferably 1 to 3 arbitrary substituents. For example, an optionally substituted ethoxy group is "optionally substituted (C2) alkoxy", and a substituted butoxy group is "substituted (C4) alkoxy". In one embodiment, the alkoxy group is "(C1-C6) alkoxy" containing 1 to 6 carbon atoms. In another embodiment, the substituted alkoxy group is "substituted (C1-C6) alkoxy" containing 1 to 6 carbon atoms. Suitable substituents for alkoxy are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0091] "Cycloalkoxy" refers to a monovalent -O-cycloalkyl group, where the cycloalkyl moiety has a specific number of carbon atoms. The cycloalkyl moiety of the alkoxy group generally contains 3 to 9 carbon atoms "(C3-C9) cycloalkoxy" or 3 to 6 carbon atoms "(C3-C6) cycloalkoxy". Non-limiting examples of cycloalkoxy groups include cyclopropoxy, cyclobutoxy, and cyclopentoxy. All cycloalkoxy groups described herein may be optionally substituted with one or more substituents independently selected, unless otherwise indicated. The total number of substituents can be equal to the total number of hydrogen atoms on the cycloalkoxy moiety, to the extent that such substitution makes chemical sense. An optionally substituted cycloalkoxy group generally contains 1 to 6 optional substituents, preferably 1 to 4 optional substituents, more preferably 1 to 3 optional substituents. Suitable substituents for cycloalkoxy are "optionally substituted alkyl", "substituted alkyl", and as described herein for alkyl.

[0092] The term "haloalkoxy" refers to a monovalent -O-haloalkyl group, where the alkyl moiety is substituted by one or more halo substituents and generally has a specific number of carbon atoms containing from 1 to 6 carbon atoms and 1, 2, or 3 halo atoms (i.e., "(C1-C6) haloalkoxy"). In some cases, the substituted alkyl group can be specifically named with reference to the substituent. For example, "haloalkoxy" refers to an alkyl group having a specific number of carbon atoms. Thus, a (C1-C4) haloalkoxy group includes trifluoromethoxy (-OCF3). The haloalkoxy groups described herein can be substituted by one or more substituents independently selected, unless otherwise indicated. The total number of substituents can be made equal to the total number of hydrogen atoms on the haloalkyl moiety, to the extent such substitution is chemically meaningful. Optionally substituted haloalkoxy groups generally include from 1 to 3 optional substituents, preferably from 1 to 2 optional substituents. In one embodiment, the haloalkoxy group is "(C1-C6) haloalkoxy" containing from 1 to 6 carbon atoms. An example of a substituted haloalkoxy group is "(C1-C6) haloalkoxy" containing from 1 to 6 carbon atoms. Suitable substituents for haloalkyloxy are as described herein for "optionally substituted alkyl" and "substituted alkyl".

[0093] As used herein, the term "halo" means -F, -Cl, -Br, or -I. In one embodiment, the halo group is -Cl. In another embodiment, the halo group is -Br.

[0094] As used herein, the term "halogen" means -F, -Cl, -Br, or -I. In one embodiment, the halogen group is -Cl. In another embodiment, the halogen group is -Br.

[0095] As used herein, the term "acyl" means -C(O)alkyl or C(O)cycloalkyl. The alkyl group may be straight-chain or branched-chain. The alkyl substituent of the acyl group generally contains from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably from 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. The cycloalkyl substituent of the acyl group generally contains from 3 to 8 carbon atoms, preferably from 3 to 7 carbon atoms, more preferably from 3 to 6 carbon atoms, or 3 to 5 carbon atoms. The alkyl and cycloalkyl moieties of the acyl group may be substituted. Suitable substituents are as described herein for "optionally substituted alkyl", "substituted alkyl" and alkyl.

[0096] The term "aryl" or "aromatic" refers to an optionally substituted monocyclic biaryl or fused bicyclic ring system, having the well-known characteristics of aromaticity and containing a π-electron system in which at least one ring is completely conjugated. Generally, an aryl group contains from 6 to 20 carbon atoms, "(C6-20)aryl" as a ring member, preferably from 6 to 14 carbon atoms "(C6-C14)aryl", or more preferably from 6 to 12 carbon atoms "(C6-C12)aryl". A fused aryl group may include an aryl ring (e.g., a phenyl ring) fused to another aryl ring or to a saturated or partially unsaturated carbocyclic or heterocyclic ring. The point of attachment of a base molecule to such a fused aryl ring system may be a carbon atom of the aromatic portion of the ring system or a carbon atom or nitrogen atom of the non-aromatic portion. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl. The aryl groups described herein may be optionally substituted with one or more substituents independently selected, unless otherwise indicated. Suitable substituents for the aryl group are further described herein.

[0097] The terms "heteroaryl" or "heteroaromatic" are used interchangeably herein and refer to an aromatic monocyclic or polycyclic ring system containing from about 5 to about 14 ring atoms, where 1 to 4 of the ring atoms are independently N, O, or S and the remaining ring atoms are carbon atoms. These systems have well-known properties of aromaticity. The heteroaryl ring is attached to the base molecule through the ring atoms of the heteroaromatic ring so that aromaticity is maintained. By including heteroatoms, aromaticity can be obtained not only in 6-membered rings but also in 5-membered rings. In one embodiment, the heteroaryl group has 5 to 10 ring atoms. In another embodiment, the heteroaryl group is a monocyclic system and has 5 to 6 ring atoms. In another embodiment, the heteroaryl group is a bicyclic ring system. The term "heteroaryl" also includes the heteroaryl defined above and is fused to the heterocyclyl defined below. The term "heteroaryl" also encompasses the heteroaryl group defined above that is fused to a benzene, cyclohexadiene or cyclohexane ring. Non-limiting examples of heteroaryl include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridine (including N-substituted pyridine), isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiazolyl, pyrazolonyl, furyl, pyrrolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, indolyl, quinoxalinyl, phthalazinyl, oxyindolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, and the like. The heteroaryl or heteroaromatic groups described herein may be optionally substituted with one or more substituents independently selected, unless otherwise indicated. Suitable substituents for the heteroaryl or heteroaromatic group are further described herein.

[0098] The terms "heterocyclic", "heterocyclic", or "heteroalicyclic" are used interchangeably herein and refer to a non-aromatic saturated or partially saturated monocyclic or polycyclic ring system containing 3 to 11 ring atoms, where 1 to 4 of the ring atoms are independently O, S, or N and the remainder of the ring atoms are carbon atoms. In one embodiment, the heterocyclic group is monocyclic and is a "6-membered heterocycle" having 6 ring atoms. In another embodiment, the heterocyclic group is monocyclic, has 6 ring atoms, and is a "6-membered heterocycle containing one or two heteroatoms", where either one or two of the ring atoms are heteroatoms. In another embodiment, the heterocyclic group is monocyclic and is a "4- or 5-membered heterocycle" having either 4 or 5 ring atoms. In another embodiment, the heterocyclic group is a "7-, 8-, or 9-membered heterocycle" having 7, 8, or 9 ring atoms. In another embodiment, the heterocyclic group is bicyclic. The heterocyclic group can be attached to the remainder of the molecule via a ring carbon or ring nitrogen atom. The nitrogen or sulfur atom of the heterocyclyl can optionally be oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. A carbon atom having two hydrogens can be oxidized to the corresponding carbonyl. Non-limiting examples of monocyclic heterocycles include oxetanyl, piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, dihydropyranyl, pyran, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, delta-lactam, delta-lactone, and the like. The heterocyclic groups described herein can optionally be substituted with one or more substituents independently selected, unless otherwise indicated. Suitable substituents for the heterocyclic groups are further described herein. The heterocyclic group may be unsubstituted or may be substituted with the same groups suitable for alkyl, aryl, or heteroaryl. In one embodiment, the heterocycle as defined herein contains 6 atoms and is a "6-membered heterocycle substituted with 1 to 4 groups" substituted with 1 to 4 groups. Further, when specified, the ring nitrogen atom can optionally be substituted with a group suitable for an amine, such as an alkyl, acyl, carbamoyl, sulfonyl substituent, etc., and the ring S atom can optionally be substituted with one or two oxo groups (i.e., S(O) qwhere q is 0, 1, or 2). In one embodiment, the 4- or 5-membered heterocyclic ring is optionally substituted as described above and is an "optionally substituted 4- or 5-membered heterocyclic ring". In another embodiment, the 7-, 8-, or 9-membered heterocyclic ring is optionally substituted as described above and is an "optionally substituted 7-, 8-, or 9-membered heterocyclic ring".

[0099] Aryl, heteroaryl, and heterocyclic moieties described herein as being optionally substituted (the "optionally substituted") may be substituted by one or more substituents, which are independently selected unless otherwise indicated. Aryl, heteroaryl, and heterocyclic moieties described herein as being substituted (the "substituted") are substituted by one or more substituents that are independently selected unless otherwise indicated. Optionally substituted aryl, heteroaryl, or heterocyclic groups generally include from 1 to 5 optional substituents, sometimes from 1 to 4 optional substituents, preferably from 1 to 3 optional substituents, or more preferably from 1 to 2 optional substituents. Substituted aryl groups, heteroaryl groups, or heterocyclic groups contain at least one substituent and may optionally contain up to 5 substituents each independently selected, as described herein. The substituents used are substituents suitable for the uses described herein.

[0100] Suitable substituents for aryl, heteroaryl, and heterocyclic rings include, but are not limited to: (C1-C8)alkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, (C3-C8)cycloalkyl, 3- to 12-membered heterocyclyl, (C6-C12)aryl, 5- to 12-membered heteroaryl, halo, =O (oxo), =S (thiono), =N-CN, =N-OR X 、=NR X 、-CN, -C(O)R X 、-CO2R X 、-C(O)NR X R Y 、-SR X 、-SOR X 、-SO2R X 、-SO2NR X R Y, -NO2, -NR X R Y , -NR X C(O)R y , -NR X C(O)NR X R Y , -NR X C(O)OR X , -NR X C(O)OR X , -NR X SO2R Y , -NR X SO2NR X R Y , -OR X , -OC(O)R X and -OC(O)NR X R Y ; wherein each R X and R Y is independently hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C3-C6)cycloalkyl, 3- to 12-membered heterocyclyl, (C6-C12)aryl, or 5- to 12-membered heteroaryl, or R X and R Y may together with the nitrogen atom to which they are attached form a 3- to 12-membered heterocyclyl, or 5- to 12-membered heteroaryl system, each optionally containing 0, 1, or 2 additional heteroatoms; each R X and R Y may be optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, =O, -CN, -C(O)R', -CO2R', -C(O)NR'2, -SO2R', -NR'2, -OR', wherein R' is independently hydrogen, (C1-C6)alkyl, (C3-C6)cycloalkyl, or 3- to 12-membered heterocyclyl. However, suitable substituents for "substituted alkyl" do not include hydrogen.

[0101] "Unsubstituted amino" refers to the -NH2 group. When amino is described as substituted or optionally substituted, the term includes groups of the form -NR X R Y , wherein each R X and R Yis independently selected from hydrogen, (C1-C8)alkyl, (C3-C9)cycloalkyl, alkynyl, heterocyclyl, acyl, aryl, heteroaryl, thioacyl, cycloalkylalkyl, arylalkyl, or heteroalkylalkyl, each having a specified number of atoms and optionally substituted as described herein. Generally, alkyl substituents on the amine contain from 1 to 8 carbon atoms, preferably from 1 to 6 carbon atoms, more preferably from 1 to 4 carbon atoms. This term also includes R X and R Y which, together with the nitrogen to which they are attached, form a 3- to 12-membered heterocyclyl or 5- to 12-membered heteroaryl ring, each of which may be optionally substituted for the heterocyclyl or heteroaryl ring as described herein and may contain 1 to 3 additional heteroatoms selected from N, O, and S as ring members, provided that such a ring does not contain adjacent oxygen atoms or adjacent sulfur atoms. The above terms also cover amino residues of other functional groups (e.g., -C(O)NR X R Y , -S(O)2NR X R Y , etc.). In one embodiment, the R X R Y of -NR X R Y and the R X and R Y of -C(O)NR X R Y may, together with the nitrogen to which they are attached (''together with the nitrogen to which they are attached''), form a ring (a 3- to 12-membered heterocyclyl or 5- to 12-membered heteroaryl ring, each of which may be optionally substituted for the heterocyclyl or heteroaryl ring as described herein and may contain 1 to 3 additional heteroatoms selected from N, O, and S as ring members, provided that such a ring does not contain consecutive oxygen atoms or consecutive sulfur atoms). In another embodiment, the R X R Y of -NR X and R Y(which can form a ring together with the nitrogen to which they are attached, being a 3- to 12-membered heterocyclyl or 5- to 12-membered heteroaryl ring, each of which may be optionally substituted with respect to the heterocyclyl or heteroaryl ring as described herein, and such a ring may contain 1 to 3 additional heteroatoms selected from N, O, and S as ring members, provided that such a ring does not contain adjacent oxygen atoms or adjacent sulfur atoms).

[0102] Two adjacent substituents on the ring can form a ring together with the atoms to which they are attached. The term "can form a ring together with the carbon atom to which they are attached" is defined herein to mean that two adjacent residues present on the ring can form a 4- to 6-membered heterocyclyl, 4- to 6-membered carbocyclyl, or 4- to 6-membered heteroaryl ring together with the carbon atom to which they are attached, each of which may be optionally substituted with respect to the heterocyclyl ring or heteroaryl ring as described herein. The heterocyclyl and heteroaryl rings thus formed may contain 1 to 3 additional heteroatoms selected from N, O, and S as ring members (when such a ring does not contain adjacent oxygen atoms or adjacent sulfur atoms). Representative examples derived from the phenyl moiety include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzimidazolyl, indazolyl, benzotriazolyl, indazolyl, quinolinyl, isoquinolinyl, cinnolinyl, azakinazoline, quinoxalinyl, 2,3-dihydro-1H-indenyl, phthalanyl, 2,3-dihydrobenzofuryl, benzodioxolyl, benzodioxanyl, etc. Representative examples of the heterocyclyl rings thus formed include, but are not limited to, the following. JPEG0007709379000015.jpg26150 etc. Representative examples of the carbocyclyl rings thus formed include, but are not limited to, the following. JPEG0007709379000016.jpg41150 etc.

[0103] Two substituents bonded to a common carbon can form a ring together with the carbon to which they are bonded. The term "capable of forming a non-aromatic ring having two oxygen atoms together with the carbon to which they are bonded" is defined herein to mean that two alkoxy or two oxygen-substituted alkyl groups can combine with the carbon atom to which they are bonded to form a ring of 4 to 7 atoms containing two oxygen atoms. Representative examples of the heterocyclic ring thus formed include, but are not limited to, the following. JPEG0007709379000017.jpg38150 etc.

[0104] Two substituents bonded to a common nitrogen atom can form a ring together with the nitrogen to which they are bonded. The term "capable of forming a ring together with the nitrogen atom to which they are bonded" is defined herein to mean that two residues present on the nitrogen atom can be joined together to form a 3- to 12-membered heterocyclyl, 3- to 7-membered carbocyclyl, or 5- to 12-membered heteroaryl ring, each of which can be optionally substituted on the heterocyclyl or heteroaryl ring as described herein. The heterocyclyl and heteroaryl rings thus formed can contain 1 to 3 additional heteroatoms selected from N, O, and S as ring members (when such rings do not contain adjacent oxygen or adjacent sulfur atoms). Non-limiting examples derived from the nitrogen atom include the following moieties: azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, 1,4-azathianyl, 1,3,4-triazolyl, tetrazolyl, imidazolyl, and the like.

[0105] Two substituents can combine to form an oxo residue (=O). "Capable of forming =O together with R5 and R6" means that an oxygen atom is double-bonded to the carbon atom having the residues of both R5 and R6. Refer to 1AA for A1 which can have the following sub-structure. Further, "capable of forming =O together with R7 and R8" means that an oxygen atom is double-bonded to the carbon atom having the residues of both R7 and R8. Refer to 1AB. JPEG0007709379000018.jpg29150

[0106] The term "replaced" means that one or more specified hydrogen atoms are replaced by a selection from the specified group, provided that the replacement does not exceed the normal valence of the atom in the existing situation and results in a stable compound. "Stable compound" or "stable structure" means a compound that is robust enough to withstand isolation from the reaction mixture to useful purity and formulation into an effective therapeutic agent.

[0107] If any substituent or variable occurs one or more times in any component or compound of formula (I), unless otherwise indicated, its definition at each occurrence is independent of its definition at all other occurrences.

[0108] As used herein, the term "in purified form" refers to the physical state of a compound after it has been isolated from a synthetic process (e.g., from a reaction mixture), a natural source, or a combination thereof. The term "in purified form" also refers to the physical state of a compound after it has been obtained from a purification process described herein or well known to those of skill in the art (e.g., chromatography, recrystallization, etc.).

[0109] The term optionally substituted alkyl with dye ("optionally substituted alkyl with dye") means that the alkyl residue can be substituted with the substituents defined for the optionally substituted alkyl residues defined herein, and either the carbon of the alkyl residue or the appropriate substituent can be modified with the dye. There can be a linker moiety such as an alkyl chain or a polyether chain as part of the dye residue. The compounds described by Q being either Q2 or Q3 can be coupled with various infrared, fluorescent, phosphorescent, radioactive or infrared fluorescent as shown in the synthesis scheme 3. The compound shown as SS10 is a useful intermediate for making the compounds of the present invention into other diagnostic agents. The length of the carbon linker determined by n can be 1-30, but n=1-5 is more optimal. These analogs are made as described above with the appropriate protecting group for the terminal functionality. The amine end of the alkyl chain has special value as a reactive species, and many common functional groups such as amides, carbamates, secondary amines, etc. can be easily formed using acid chlorides, ketenes, carboxylic acids (including coupling agents), etc. In addition to amines, other terminal residues can be used to form linkers such as -SH, -OH, -Cl, -Br and -I. These terminal residues can be linked to a variety of dyes and imaging agents. Fluorescent dyes containing a wide variety of functional groups to facilitate coupling and different lengths of PEG spacers to increase water solubility are commercially available (BroadPharm, Inc., 6625 Top Gun Street, Suite 103, San Diego, CA 92121). They allow efficient biolabeling in imaging and diagnostic research and development. The types of agents sold by BroadPharm, Inc. include BDP, Cyanine 3, Cyanine 5, Cyanine 5.5, Cyanine 7, Fluorescein and Pyrene. This example is not intended to be limiting.

[0110] It should be noted that in this specification, schemes, examples and tables, any carbon and heteroatom with unsatisfied valences is assumed to have sufficient hydrogen atoms to satisfy the valences.

[0111] Compounds are known by one or more names. For example, ONC201 is also TIC10. Other compounds can be referred to by names starting with "TR". For these agents, the following examples show the nomenclature referring to the same compound. For example, TR57, TR-57, Tr57, Tr-57, tr-57 and tr57 refer to the same compound.

[0112] One or more compounds of the present invention can exist in an unsolvated form and in a solvated form with a pharmaceutically acceptable solvent such as water, ethanol, etc., and the present invention is intended to encompass both the solvated and unsolvated forms.

[0113] The compounds of formula (I) can contain one or more stereocenters and can thus exist as racemates, racemic mixtures, single enantiomers, diastereomer mixtures and individual diastereomers. Each such asymmetric center independently gives rise to two optical isomers, and all possible optical isomers and diastereomers in mixtures and as purified or partially purified compounds are intended to be included within the scope of the present invention.

[0114] As used herein, the term "composition" is intended to encompass a product containing a specific amount of a specific ingredient and any product directly or indirectly obtained from a combination of specific amounts of specific ingredients.

[0115] In the compounds of general formula (I) and the compounds of general formulas 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A, the atoms may represent their natural isotope abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number but a different atomic mass or mass number than the atomic mass or mass number predominantly found in nature. The present invention is meant to include all appropriate isotope variations of the compounds of general formula (I) and the compounds of general formulas 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A. Enriching a particular isotope can provide advantageous properties, for example, enriching deuterium can provide certain therapeutic advantages such as an increase in in vivo half-life or a decrease in dosage. Additionally, isotope enrichment may also enhance the utility of the compounds in the characterization of biological samples. Compounds enriched in a particular isotope can be prepared by methods known to those skilled in the art by using the synthetic methods described herein and reagents and starting materials enriched in the particular isotope.

[0116] Prodrugs of the compounds of the present invention are contemplated herein. The term "prodrug" as used herein refers to a compound that, when administered to a subject, undergoes a chemical transformation by a metabolic or chemical process to yield a compound of formula (I). Prodrugs can have beneficial properties such as, but not limited to, enhanced absorption and / or oral bioavailability.

[0117] The compounds of formula (I) may optionally form salts which are also included within the scope of the present invention. References to the compounds of formula (I) herein are to be understood to include references to their salts, unless otherwise indicated. The term "salt" as used herein means acidic and / or basic salts formed with inorganic and / or organic acids and bases. Zwitterions (internal or inner salts) are included within the term "salt" as used herein (and can be formed, for example, when the R substituent contains an acidic moiety such as a carboxyl group). Quaternary salt ammonium salts such as alkylammonium salts are also included herein. Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, but other salts may be useful, for example, in the isolation or purification steps used during preparation. The salts of the compounds of formula (I) can be formed, for example, by reacting a compound of formula (I) with an equivalent amount of an acid or base in a medium (e.g., ether) that allows precipitation of the salt, or in an aqueous medium, followed by lyophilization.

[0118] Examples of acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates), and the like. Further, acids generally considered suitable for forming pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH. This disclosure is incorporated herein by reference.

[0119] Examples of basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (e.g., organic amines) such as dicyclohexylamine and t-butylamine, and salts with amino acids such as arginine and lysine. The basic nitrogen-containing group can be quaternized with lower alkyl halides (e.g., methyl chloride, ethyl chloride, butyl chloride, bromide, iodide), dialkyl sulfates (e.g., dimethyl sulfate, diethyl and dibutyl), long-chain halides (e.g., decyl chloride, lauryl, and stearyl, bromide and iodide), aralkyl halides (e.g., benzyl bromide and phenethyl), and other such agents.

[0120] The present invention further includes the compounds of formula (I) in all of their isolated forms.

[0121] The present invention provides a method for determining whether a mammal responds to a compound of formula I, the method comprising the following.

[0122] Administering a compound of formula I to an individual after isolating a pre-treatment biological sample and before isolating a post-treatment biological sample of the same sample type as the biological sample, wherein the biological sample is selected from a blood sample, a serum sample, a plasma sample, a bone sample, a biopsy sample, a fine needle aspirate, a lymph node aspirate, a cyst aspirate, a puncture sample, a thoracentesis sample, and the administering step; Assaying the pre-treatment and post-treatment biological samples to determine the level of biomarker ClpP; Determining that the individual is a candidate for treatment with a compound of formula I if the level of the pre-treatment biomarker is 1.5 times or more higher than the normal level, or determining whether the individual responds to treatment with a compound of formula I if the level of any of the biomarker ClpP is decreased by more than 50% of the pre-treatment biomarker.

[0123] Z1-Q Formula I

[0124] Z1 is JPEG0007709379000019.jpg20150

[0125] Z2 is JPEG0007709379000020.jpg19150

[0126] Q is independently selected from the group consisting of JPEG0007709379000021.jpg61150JPEG0007709379000022.jpg67150

[0127] Ar1 and Ar2 are independently selected from aryl, heteroaryl, thiophenyl and phenyl.

[0128] Ar1 may be optionally substituted with 1 to 5 J groups.

[0129] Ar2 may be substituted with 1 to 5 JJ groups.

[0130] J is independently selected from halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6) optionally substituted alkyl, (C1-C6) haloalkyl, -CF3, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6) alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl(C1-C6) alkyl, aryl, heteroaryl, heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15 and NR15C(O)R16.

[0131] JJ is independently selected from hydrogen, halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6) alkyl, (C1-C6) haloalkyl, -CF3, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6) alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl(C1-C6) alkyl, aryl, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkyloxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15 and -NR15C(O)R16.

[0132] R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from hydrogen, halogen and optionally substituted (C1-C3) alkyl.

[0133] R9, R10, R11 and R12 are each independently selected from the group consisting of hydrogen, halogen, (C3-C6) cycloalkyl and optionally substituted (C1-C6) alkyl.

[0134] R10 and R11, together with the carbon atom to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0135] R13 is independently selected from the group consisting of hydrogen, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C6) cycloalkyl, (C1-C6) haloalkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, -CN, -S(O)2R15, -NR17R18, -S(O)2R15, -C(NH)NH2, -C(O)R15, ZW, and C(O)OR15.

[0136] R14 is independently selected from hydrogen, optionally substituted (C1-C6) alkyl, (C3-C6) cycloalkyl, (C1-C6) haloalkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, -CN, -S(O)2R15, -NR17R18, -S(O)2R15, -C(NH)NH2, -C(O)R15, and -C(O)OR15.

[0137] R15, R16, R17, R18, R19, R28 and R29 are independently selected from hydrogen and optionally substituted (C1-C6) alkyl.

[0138] R17 and R18, together with the nitrogen to which they are attached, may form a ring of 3 to 6 atoms.

[0139] ZW is optionally substituted (C1-C6) alkyl with a dye.

[0140] W1 and W2 are independently selected from the following.

[0141] nitrogen and JPEG0007709379000023.jpg18150

[0142] W3 is independently selected from oxygen, -N(R15)-, and sulfur.

[0143] W4 is independently selected from the group consisting of =C(R14)- and nitrogen.

[0144] W5 is a single bond, SS and It is independently selected from the group consisting of JPEG0007709379000024.jpg19150.

[0145] W6 is oxygen, sulfur, and -NR 14 It is independently selected from the group consisting of.

[0146] A is SS and It is independently selected from the group consisting of JPEG0007709379000025.jpg19150.

[0147] G is SS and It is independently selected from the group consisting of JPEG0007709379000026.jpg17150.

[0148] M is SS and It is independently selected from the group consisting of JPEG0007709379000027.jpg19150.

[0149] E is a single bond, SS, and It is independently selected from the group consisting of JPEG0007709379000028.jpg19150.

[0150] SS is independently selected from the group consisting of the following. JPEG0007709379000029.jpg40150

[0151] R20, R21, R26 and R27 are each independently selected from the group consisting of hydrogen, halogen and optionally substituted (C1 - C6) alkyl.

[0152] R22, R23, R24, and R25 are each independently selected from the group consisting of hydrogen, halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl (C1-C6) alkyl, (C1-C6) haloalkyl, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl (C1-C6) alkyl, (C3-C9) cycloalkyl (C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl (C1-C6) alkyl, aryl, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15, and NR15C(O)R16.

[0153] R22 and R23, together with the carbon to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0154] R22 and R23, together with the carbon to which they are attached, may form a non-aromatic ring having 1 to 2 oxygen atoms.

[0155] R24 and R25, together with the carbon to which they are attached, may form a non-aromatic ring having 1 to 2 oxygen atoms.

[0156] R24 and R25, together with the carbon to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0157] R30 and R31 are each independently selected from the group consisting of hydrogen and optionally substituted (C1-C6) alkyl.

[0158] Embodiments of the present invention include testing the level of ClpP ex vivo in a sample taken from a mammal.

[0159] Embodiments of the present invention include that the sample to be tested is derived from normal tissue, tumor tissue, circulating tumor cells, plasma or whole blood.

[0160] Embodiments of the present invention include that the sample to be tested is derived from tumor tissue or circulating tumor cells.

[0161] Embodiments of the present invention include predicting an effective response to the disease of a higher level of ClpP in an untreated sample compared to a standard value or a set of standard values, to treatment with a compound of formula I or a pharmaceutically acceptable formulation thereof.

[0162] Embodiments of the present invention include predicting an effective response to a lower level of ClpP in a sample relative to a standard value or a set of standard values after treatment with a compound of formula I or a pharmaceutically acceptable formulation thereof.

[0163] Embodiments of the present invention include other biomarkers described in the present invention. These include the use of the positive biomarkers described herein. Further, the present invention can use negative biomarkers. Also, the negative biomarkers described herein can be used.

[0164] The present invention provides a compound of formula 1A: JPEG0007709379000030.jpg37150 or a pharmaceutically acceptable salt thereof.

[0165] The present invention provides a compound of formula 2A: JPEG0007709379000031.jpg29150 or a pharmaceutically acceptable salt thereof.

[0166] The present invention provides a compound of formula 3A: JPEG0007709379000032.jpg39150 or a pharmaceutically acceptable salt thereof.

[0167] The present invention provides a compound of formula 4A: Provide 34150 of JPEG0007709379000033.jpg or a pharmaceutically acceptable salt thereof.

[0168] The present invention relates to Formula 5A: Provide 39150 of JPEG0007709379000034.jpg or a pharmaceutically acceptable salt thereof.

[0169] The present invention relates to Formula 6A: Provide 37150 of JPEG0007709379000035.jpg or a pharmaceutically acceptable salt thereof.

[0170] The present invention relates to Formula 7A: Provide 40150 of JPEG0007709379000036.jpg or a pharmaceutically acceptable salt thereof.

[0171] The present invention relates to Formula 8A: Provide 41150 of JPEG0007709379000037.jpg or a pharmaceutically acceptable salt thereof.

[0172] The present invention relates to Formula 9A: Provide 34150 of JPEG0007709379000038.jpg or a pharmaceutically acceptable salt thereof.

[0173] The present invention relates to Formula 10A: Provide 33150 of JPEG0007709379000039.jpg or a pharmaceutically acceptable salt thereof.

[0174] The present invention relates to Formula 11A: Provide 35150 of JPEG0007709379000040.jpg or a pharmaceutically acceptable salt thereof.

[0175] The present invention relates to Formula 12A: Provide 35150 of JPEG0007709379000041.jpg or a pharmaceutically acceptable salt thereof.

[0176] The present invention relates to Formula 13A: JPEG0007709379000042.jpg provides 33150 or a pharmaceutically acceptable salt thereof.

[0177] The present invention relates to formula 14A: JPEG0007709379000043.jpg provides 36150 or a pharmaceutically acceptable salt thereof.

[0178] The present invention relates to formula 15A: JPEG0007709379000044.jpg provides 42150 or a pharmaceutically acceptable salt thereof.

[0179] The present invention relates to formula 16A: JPEG0007709379000045.jpg provides 37150 or a pharmaceutically acceptable salt thereof.

[0180] Various radicals and / or variants for A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 are defined herein with respect to formula (I).

[0181] In another embodiment, the present invention provides compounds of formula A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts thereof.

[0182] In another embodiment, the present invention provides compounds of formula A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts thereof, wherein

[0183] Z1 is JPEG0007709379000046.jpg is 17150,

[0184] Z2 is JPEG0007709379000047.jpg is 17150.

[0185] Ar1 and Ar2 are independently selected from aryl, heteroaryl, thiophenyl and phenyl.

[0186] Ar1 may be optionally substituted with 1 to 5 J groups.

[0187] Ar2 may be substituted with 1 to 5 JJ groups.

[0188] J is independently selected from halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6) alkyl, (C1-C6) haloalkyl, -CF3, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6) alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl(C1-C6) alkyl, aryl, heteroaryl, heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15 and -NR15C(O)R16.

[0189] JJ is independently selected from hydrogen, halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6) alkyl, (C1-C6) haloalkyl, -CF3, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6) alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl(C1-C6) alkyl, aryl, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkyloxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15 and -NR15C(O)R16.

[0190] R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from hydrogen, halogen and optionally substituted (C1-C3) alkyl.

[0191] R9, R10, R11 and R12 are each independently selected from the group consisting of hydrogen, halogen, (C3-C6) cycloalkyl and optionally substituted (C1-C6) alkyl.

[0192] R10 and R11, together with the carbon atom to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0193] R13 is independently selected from the group consisting of hydrogen, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C6) cycloalkyl, (C1-C6) haloalkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, -CN, -S(O)2R15, -NR17R18, -S(O)2R15, -C(NH)NH2, -C(O)R15, ZW, and -C(O)OR15.

[0194] R14 is independently selected from hydrogen, optionally substituted (C1-C6) alkyl, (C3-C6) cycloalkyl, (C1-C6) haloalkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, -CN, -S(O)2R15, -NR17R18, -S(O)2R15, -C(NH)NH2, -C(O)R15, and -C(O)OR15.

[0195] R15, R16, R17, R18, R19, R28 and R29 are independently selected from hydrogen and optionally substituted (C1-C6) alkyl.

[0196] R17 and R18, together with the nitrogen to which they are attached, may form a ring of 3 to 6 atoms.

[0197] ZW is optionally substituted (C1-C6) alkyl with a dye.

[0198] W1 and W2 are independently selected from the following.

[0199] nitrogen and JPEG0007709379000048.jpg18150

[0200] W3 is independently selected from oxygen, -N(R15)-, and sulfur.

[0201] W4 is independently selected from the group consisting of =C(R14)- and nitrogen.

[0202] W5 is independently selected from the group consisting of a single bond, SS and JPEG0007709379000049.jpg19150.

[0203] W6 is independently selected from the group consisting of oxygen, sulfur, and -NR 14 and.

[0204] A is SS and It is independently selected from the group consisting of JPEG0007709379000050.jpg19150.

[0205] G is SS and It is independently selected from the group consisting of JPEG0007709379000051.jpg17150.

[0206] M is SS and It is independently selected from the group consisting of JPEG0007709379000052.jpg19150.

[0207] E is a single bond, SS, and It is independently selected from the group consisting of JPEG0007709379000053.jpg19150.

[0208] SS is independently selected from the group consisting of the following.

[0209] JPEG0007709379000054.jpg39150

[0210] R20, R21, R26 and R27 are each independently selected from the group consisting of hydrogen, halogen and alkyl optionally substituted with (C1-C6).

[0211] R22, R23, R24, and R25 are each independently selected from the group consisting of hydrogen, halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6) alkyl, (C1-C6) haloalkyl, -NH2, -NO2, -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6) alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl(C1-C6) alkyl, aryl, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O)2OR15, -SO2NR17R18, -S(O)2R15, -NR15S(O)2R16, -C(O)NR17R18, -C(O)R15, and -NR15C(O)R16.

[0212] R22 and R23, together with the carbon to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0213] R22 and R23, together with the carbon to which they are attached, may form a non-aromatic ring having 1 to 2 oxygen atoms.

[0214] R24 and R25, together with the carbon to which they are attached, may form a non-aromatic ring having 1 to 2 oxygen atoms.

[0215] R24 and R25, together with the carbon to which they are attached, may form a non-aromatic ring having 3 to 6 carbon atoms.

[0216] R30 and R31 are each independently selected from the group consisting of hydrogen and optionally substituted (C1-C6) alkyl.

[0217] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, where as follows.

[0218] Z1 is substituted with 0 to 5 J groups JPEG0007709379000055.jpg21150.

[0219] Z2 is substituted with 1 to 5 JJ groups JPEG0007709379000056.jpg20150.

[0220] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, where as follows.

[0221] Z1 is substituted with 1 J group JPEG0007709379000057.jpg17150.

[0222] Z2 is substituted with 1 to 5 JJ groups JPEG0007709379000058.jpg15150.

[0223] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, where as follows.

[0224] Z1 is substituted with 1 J group JPEG0007709379000059.jpg18150.

[0225] Z2 is substituted with 1 JJ group JPEG0007709379000060.jpg17150.

[0226] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, wherein as follows.

[0227] Z1 is JPEG0007709379000061.jpg17150.

[0228] Z2 is substituted with 1 to 5 JJ groups JPEG0007709379000062.jpg17150.

[0229] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, wherein as follows.

[0230] Z1 is JPEG0007709379000063.jpg18150.

[0231] Z2 is substituted with 1 JJ group JPEG0007709379000064.jpg17150.

[0232] In another embodiment, the present invention provides compounds of formulas A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15 and A16 and pharmaceutically acceptable salts, wherein as follows.

[0233] Z1 is substituted with 1 J group JPEG0007709379000065.jpg17150.

[0234] Z2 is substituted with 1 JJ group It is JPEG0007709379000066.jpg18150.

[0235] R5, R6, R7, R8, R9, R10, R11 and R12 are hydrogen.

[0236] R14 is independently selected from hydrogen, (C1-C6) alkyl and -NH2.

[0237] W1 and W2 are nitrogen.

[0238] W3 is independently selected from oxygen and sulfur.

[0239] W4 is independently selected from nitrogen and carbon.

[0240] W5 is a single bond, JPEG0007709379000067.jpg20150, and is independently selected from the group consisting of.

[0241] W6 is independently selected from oxygen, sulfur and NH2.

[0242] R13 is independently selected from hydrogen and (C1-C6) alkyl.

[0243] A is It is JPEG0007709379000068.jpg18150.

[0244] G is JPEG0007709379000069.jpg19150, and is independently selected from.

[0245] M is JPEG0007709379000070.jpg18150, JPEG0007709379000071.jpg18150, and is independently selected from the group consisting of.

[0246] E is a single bond, It is independently selected from the group consisting of JPEG0007709379000072.jpg18150 and JPEG0007709379000073.jpg18150.

[0247] R14 is independently selected from hydrogen, (C1-C6) alkyl, and NH2.

[0248] R19 is independently selected from hydrogen and (C1-C6) alkyl.

[0249] The method for treating cancer described herein includes a method for treating cancer in a subject, the method comprising administering an effective amount of a compound of formula 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, and 16A or a pharmaceutically acceptable salt thereof.

[0250] The pharmaceutical composition described herein includes a compound of formula 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, and 16A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0251] The present invention also provides a treatment for diseases in which the activation of ClpP is effective. The methods described herein for the treatment of such diseases include the administration of a compound of formula: 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A or a pharmaceutically acceptable salt thereof. Further, various neurodegenerative diseases can be treated with the compounds described herein. The methods described herein for the treatment of various neurodegenerative diseases include the administration of a compound of formula: 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A or a pharmaceutically acceptable salt thereof. Also, the methods described herein for the treatment of Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and Alzheimer's disease include the administration of a compound of formula: 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A or a pharmaceutically acceptable salt thereof.

[0252] The present invention also provides a treatment for diseases in which a decrease in the concentration and / or activity of ClpX is effective. The methods described herein for the treatment of such diseases include the administration of a compound of formula: 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A or a pharmaceutically acceptable salt thereof. The present invention also provides a treatment for diseases in which a decrease in the concentration and / or activity of TUFM is effective. The methods described herein for the treatment of such diseases include the administration of a compound of formula: 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A and 16A or a pharmaceutically acceptable salt thereof.

[0253] In one aspect of the present invention, the following compounds are expected to be activators of the protein ClpP. These compounds are formed by the selection of the FA2 fragment and the independent selection of the fragments: FA1 and FA3, and form a single molecule. For FA1, Ar1 is phenyl which may be substituted with 1 to 5 J groups.

[0254] FA1: JPEG0007709379000074.jpg26150

[0255] FA3: JPEG0007709379000075.jpg27150

[0256] FA2: JPEG0007709379000076.jpg72150

[0257] In another embodiment, the compound is FA1 - FA2 - FA3.

[0258] In another embodiment, the preferred compounds of the present invention are Examples 66, 76 and 77.

[0259] The present invention provides the following compounds or pharmaceutically acceptable salts thereof.

[0260] JPEG0007709379000077.jpg80150JPEG0007709379000078.jpg100150JPEG0007709379000079.jpg48150

[0261] The method for treating cancer described herein includes a method for treating cancer in a subject, which comprises administering an effective amount of the following compound or a pharmaceutically acceptable salt thereof.

[0262] JPEG0007709379000080.jpg79150JPEG0007709379000081.jpg53150JPEG0007709379000082.jpg91150

[0263] The present invention contemplates the following compounds or pharmaceutically acceptable salts thereof.

[0264] JPEG0007709379000083.jpg83150JPEG0007709379000084.jpg113150JPEG0007709379000085.jpg92150

[0265] The contemplated method for treating cancer described herein includes a method for treating cancer in a subject, which comprises administering an effective amount of the following compound or a pharmaceutically acceptable salt thereof. JPEG0007709379000086.jpg85150JPEG0007709379000087.jpg98150JPEG0007709379000088.jpg107150

[0266] Another embodiment is a method for determining whether a mammal responds to the following compound or a pharmaceutically acceptable salt thereof.

[0267] JPEG0007709379000089.jpg23150

[0268] Another embodiment is a method for determining whether a mammal responds to the following compound or a pharmaceutically acceptable salt thereof.

[0269] JPEG0007709379000090.jpg25150

[0270] Another embodiment is a method for treating a bacterial infection in a subject, which comprises administering an effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof.

[0271] Another embodiment is a method for treating a bacterial infection in a subject, which comprises administering an effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof, wherein Q is independently selected from Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q12.

[0272] The compounds of the present invention

[0273] Dosage form and regimen Administration of the compounds of the present invention can be affected by any method that enables delivery of the compound to the site of action. These methods include oral route, intraduodenal route, parenteral injection (including intravenous, subcutaneous, intramuscular, or infusion), topical and rectal administration.

[0274] The dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, it may be administered in divided doses over time, or the dosage may be proportionally decreased or increased as indicated by the exigencies of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as a single dosage for the mammalian subject to be treated, each unit containing a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by, and may directly depend on, (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of sensitivity in individuals.

[0275] Accordingly, one of ordinary skill in the art will understand that, based on the disclosure provided herein, the dosage and administration regimen will be adjusted according to methods well known in the art of therapy. That is, the maximum allowable dosage can be readily established, as can the effective amount to provide a detectable therapeutic benefit to a patient, similar to the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient. Accordingly, while specific dosages and administration regimens are exemplified herein, these examples in no way limit the dosages and administration regimens that can be provided to a patient in practicing the present invention. It should be noted that the dosage varies depending on the type and severity of the condition to be alleviated and can be administered in a single or multiple doses. Further, for any particular subject, the specific dosage regimen should be adjusted over time according to the individual needs of the individual administering or supervising the administration of the composition and the judgment of the expert, and it should be understood that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed composition. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters that can include clinical effects such as toxic effects and / or clinical test values. Accordingly, the present invention includes an increase in the dosage within a patient determined by one of ordinary skill in the art. Determining the appropriate dosage and regimen for the administration of chemotherapeutic agents is well known in the relevant art and will be understood to be encompassed by one of ordinary skill in the art when the teachings disclosed herein are provided.

[0276] The amount of the compound of the invention administered depends on the subject being treated, the severity of the disorder or condition, the rate of administration, and the discretion of the physician treating and prescribing the compound. However, effective dosages are in the range of about 0.001 to about 100 mg / kg body weight per day, preferably about 1 to about 35 mg / kg / day, administered in a single or divided dose. For a 70 kg human, this is about 0.05 to about 7 g / day, preferably about 0.1 to about 2.5 g / day. In some cases, dosage levels below the lower limit of the above range may be sufficient, while in other cases, more dosage may be used without causing harmful side effects, provided that such more dosage is first divided into several smaller dosages for administration throughout the day.

[0277] Formulations and routes of administration

[0278] As used herein, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not inhibit the biological activity and properties of the active compound.

[0279] Pharmaceutically acceptable carriers can include any conventional pharmaceutical carrier or excipient. The selection of the carrier and / or excipient depends largely on factors such as the particular method of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0280] Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents (such as hydrates and solvates). The pharmaceutical composition may contain additional ingredients such as flavorings, binders, excipients, etc., if desired. Thus, for oral administration, tablets containing various excipients such as citric acid can be used with various disintegrants such as starch, alginic acid, and certain complex silicates, as well as binders such as sucrose, gelatin, and acacia. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol. Furthermore, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often useful for tableting purposes. Solid compositions of the same type can also be used in soft and hard filled gelatin capsules. Thus, non-limiting examples of materials include lactose or milk sugar and high molecular weight polyethylene glycol. When an aqueous suspension or elixir is desired for oral administration, the active compound therein can be combined with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof, along with various sweetening or flavoring agents, coloring agents or dyes, and, if desired, emulsifying or suspending agents.

[0281] The pharmaceutical composition may be in a form suitable for oral administration, such as tablets, capsules, pills, powders, sustained-release formulations, solutions, suspensions or emulsions, topical administration as ointments or wrinkles, or rectal administration as suppositories.

[0282] Examples of parenteral administration forms include sterile aqueous solutions, such as solutions or suspensions of the active compound in aqueous propylene glycol or aqueous dextrose. Such administration forms can be appropriately buffered if desired.

[0283] The pharmaceutical composition may be in unit dosage form suitable for precise single-dose administration.

[0284] Pharmaceutical compositions suitable for delivery of the active agent and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in "Remington’s Pharmaceutical Sciences", 19th Edition (Mack Publishing Company, 1995), the disclosure of which is hereby incorporated by reference in its entirety.

[0285] The compounds of the present invention can be administered orally. Oral administration can include swallowing such that the compound enters the gastrointestinal tract, or the compound can enter the bloodstream directly from the mouth using oral or sublingual administration.

[0286] Formulations suitable for oral administration include solid formulations such as tablets, capsules containing microparticles, liquids or powders. Troches (including liquid fillings), gums, multiparticulates and nanoparticles, gel solid solutions, liposomes, films, ovules, sprays and liquid formulations.

[0287] Liquid formulations include suspensions, solutions, syrups, and elixirs. Such formulations can be used as fillers for soft or hard capsules and generally contain a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying and / or suspending agents. Liquid formulations can also be prepared, for example, by adding water to a solid from a sachet and then restoring it.

[0288] The compounds of the present invention can also be used in dosage forms that dissolve rapidly and disintegrate rapidly, such as the dosage forms described in Liang and Chen (2001), Expert Opinion in Therapeutic Patents, 11(6), 981-986, the disclosure of which is hereby incorporated by reference in its entirety.

[0289] For tablet dosage forms, the active agent can constitute from 1% to 80% by weight, more generally from 5% to 60% by weight of the dosage form. In addition to the active agent, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch, and sodium alginate. Generally, the disintegrant can constitute from 1% to 25% by weight, preferably from 5% to 20% by weight of the dosage form.

[0290] Binders are generally used to impart cohesiveness to tablets. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic rubbers, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose, and hydroxypropyl methylcellulose. Tablets can also contain diluents such as lactose, mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate.

[0291] The tablets may optionally also contain surfactants such as sodium lauryl sulfate and polysorbate 80, and lubricants such as silicon dioxide and talc. When present, the surfactant is generally in an amount of from 0.2% to 5% by weight of the tablets, and the lubricant is generally in an amount of from 0.2% to 1% by weight of the tablets.

[0292] Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate and sodium lauryl sulfate. The lubricant is generally present in an amount of from 0.25% to 10% by weight of the tablets, preferably from 0.5% to 3% by weight.

[0293] Exemplary tablets may contain up to about 80% by weight of the active agent relative to about 10% to about 90% by weight of binder, about 0% to about 85% by weight of diluent, about 2% to about 10% by weight of disintegrant, and about 0.25% to about 10% by weight of lubricant.

[0294] Details of tablet formulations are described in "pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X), the disclosure of which is incorporated herein by reference in its entirety.

[0295] Suitable modified release formulations are described in U.S. Patent No. 6,106,864. Details of other suitable release techniques such as high energy dispersion and osmotic and coated particles can be found in Verma et al, Pharmaceutical Technology On-line 25(2), 1-14(2001). The disclosure of this document is incorporated herein by reference in its entirety.

[0296] It is understood that the compounds of formula (I) can be formulated as the di-salts.

[0297] Parenteral administration

[0298] The compounds of the present invention can also be administered directly into the bloodstream, muscle, or viscera. Suitable means for parenteral administration include intravenous administration, intra-arterial administration, intraperitoneal administration, intrathecal administration, intraventricular administration, intraurethral administration, intracranial administration, intramuscular administration, and subcutaneous administration. Devices suitable for parenteral administration include syringe injectors, syringe-free injectors, and injection techniques.

[0299] Parenteral formulations are generally aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9), but for some applications they may be more suitably formulated as sterile non-aqueous solutions or in dry form in combination with a suitable vehicle such as sterile pyrogen-free distilled water.

[0300] The preparation of parenteral formulations under aseptic conditions, for example by freeze-drying, can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art.

[0301] The solubility of the compounds of the present invention used in the preparation of parenteral solutions can be increased by using suitable formulation techniques such as the incorporation of solubility enhancers.

[0302] Formulations for parenteral administration can be formulated to be immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release, and programmed release. Thus, the compounds of the present invention can be formulated as solids, semi-solids, or thixotropic liquids for administration as implanted depots that provide modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.

[0303] The compounds of the present invention can also be administered topically to the skin or mucosa, i.e., dermally or transdermally. General formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, wafers, implants, sponges, fiber bandages, and microemulsions.

[0304] Dosage and administration

[0305] The amount of the active compound to be administered depends on the subject to be treated, the severity of the disorder or condition, the rate of administration, the treatment with the compound, and the discretion of the prescribing physician. However, the effective dose generally ranges from about 0.001 to about 100 mg / kg body weight / day, preferably 0.01 to about 35 mg / kg / day, in single or divided doses. In the case of humans, this amounts to about 0.07 to about 700 mg / day, preferably about 0.7 to about 2500 mg / day. In some cases, dose levels below the lower limit of the above range may be sufficient, while in other cases, higher doses can be used without causing harmful side effects, and such higher doses are generally divided into several smaller doses for administration throughout the day.

[0306] Combination therapy

[0307] As used herein, the term "combination therapy" refers to administering a compound of the present invention, either sequentially or simultaneously, with at least one additional pharmaceutical or medicinal agent (e.g., an anti-cancer agent).

[0308] As described above, the compounds of the present invention can be used in combination with one or more additional anti-cancer agents described below. When combination therapy is used, the one or more additional anti-cancer agents can be administered continuously or simultaneously with the compounds of the present invention. In one embodiment, the additional anti-cancer agent is administered to a mammal (subject, patient) prior to administration of the compound of the present invention. In another embodiment, the additional anti-cancer agent is administered to the mammal after administration of the compound of the present invention. In another embodiment, the additional anti-cancer agent is administered to the mammal simultaneously with the administration of the compound of the present invention.

[0309] The present invention also relates to a pharmaceutical composition for the treatment of abnormal cell growth in mammals, including humans, comprising an amount of a compound of the present invention as defined herein in combination with one or more (preferably 1 to 3) anti-cancer agents selected from the group consisting of anti-angiogenic agents and signal transduction inhibitors and a pharmaceutically acceptable carrier, wherein the amounts of the active agent and the combined anti-cancer agents are therapeutically effective for treating the abnormal cell growth when taken as a whole.

[0310] In one embodiment of the present invention, the anti-cancer agent used together with the compound of the present invention and the pharmaceutical composition described herein is an anti-angiogenic agent (for example, an agent that stops a tumor from developing new blood vessels). Examples of anti-angiogenic agents include, for example, VEGF inhibitors, VEGFR inhibitors, TIE-2 inhibitors, PDGFR inhibitors, angiopoietin inhibitors, PKCβ inhibitors, COX-2 inhibitors, integrins, MMP-2 (matrix-metalloprotease 2) inhibitors, and MMP-9 (matrix-metalloprotease 9) inhibitors.

[0311] Preferred anti-angiogenic agents include sunitinib (Sutent®), bevacizumab (Avastin®), axitinib (AG 13736), SU 14813 (Pfizer), and AG 13958 (Pfizer).

[0312] Additional anti-angiogenic agents include batatinib (CGP 79787), sorafenib (Nexavar®), pegaptanib octasodium (Macugen®), vandetanib (Zactima®), PF-0337210 (Pfizer), SU 14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis®), Neovastat® (AE 941), tetrathiomolybdate (Coprexa®), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), teratinib (BAY 57-9352), and CP-868,596 (Pfizer).

[0313] Other anti-angiogenic agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex®), and UCN 01 (Kyowa Hakko).

[0314] Other examples of anti-angiogenic agents that can be used in combination with the compounds of the present invention and the pharmaceutical compositions described herein include celecoxib (Celebrex®), parecoxib (Dynastat®), deracoxib (SC 59046), lumiracoxib (Preige™), valdecoxib (Bextra™), rofecoxib (Viox™), iguratimod (Careram®), IP 751 (Invedus), SC-58125 (Pharmacia), and etoricoxib (Arcoxia®).

[0315] Other anti-angiogenic agents include Aptosyn®, Amigesic®, Dolobid®, Motrin®, Orudis®, Relafen®, Feldene®, Aleve®, Naprosyn®, Voltarn®, Indocin®, Clinoril®, Tolectin®, Lodine®, Toradol®, and Day-pro®.

[0316] Other anti-angiogenic agents include ABT 510 (abbott), aplastatin (TMI 005), AZD 8955 (AstraZeneca), incerinide (Metastat®), and PCK 3145 (Procyon).

[0317] Other anti-angiogenic agents include acitretin (Neotigason®), plitidepsin (Aplidine®), thalidomide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4 HPR), halofuginone (Tempostatin®), Panzem®, rebimastat (BMS 275291), catumaxomab (Removab®), lenalidomide (Revlimid®), squalamine (EVIZON®), thalidomide (Thalomid®), Ukrain® (NSC 631570), Vitaxin® (MEDI 522), and zoledronic acid (Zomata®).

[0318] In another embodiment, the anti-cancer agent is a so-called signal transduction inhibitor (e.g., inhibiting by means of regulatory molecules transmitted intracellularly that govern the basic processes of cell proliferation, differentiation, and survival). Signal transduction inhibitors include small molecules, antibodies, antisense molecules, etc. Signal transduction inhibitors include, for example, kinase inhibitors (e.g., tyrosine kinase inhibitors or serine / threonine kinase inhibitors) and cell cycle inhibitors. More specifically, signal transduction inhibitors include, for example, farnesyl protein transferase inhibitors, EgF inhibitors, ErbB-1 (EGFR) inhibitors, ErbB-2 inhibitors, pan-erb inhibitors, IGF1R inhibitors, MEK (1,2) inhibitors, c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, Akt inhibitors, mTOR inhibitors, P70S6 kinase inhibitors, CDK inhibitors, CDK4 / 6 inhibitors, BTK inhibitors of the WNT pathway, and so-called multi-target kinase inhibitors.

[0319] Preferred signal transduction inhibitors include gefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), trastuzumab (Herceptin®), sunitinib (Sutent®), imatinib (Gleevec®), Trametinib® (GSK1120212), abemaciclib (Verzenio®), palbociclib (Ibrance®), ribociclib (Kisqali®), ibrutinib (IMBRUVICA®), acalabrutinib (CALQUENCE®), LOXO-305, and Cobimetinib® (XL518).

[0320] Further examples of signal transduction inhibitors that can be used together with the compounds of the present invention and the pharmaceutical compositions described in the present specification include BMS214662, lonafarnib (Sarasar®), peritolaxol (AG2037), matuzumab (EMD 7200), nimotuzumab (TheraCIM h-R3®), panitumumab (Vectibix®), vandetanib (Zactima®), pazopanib (SB786034), BIBW2992 (Boehringer Ingelheim), and Cervene® (TP 38).

[0321] Other examples of signal transduction inhibitors include canertinib (CI 1033), pertuzumab (Omnitarg®), lapatinib (Tycerb®), pelitinib (EKB 569), miltefosine (Miltefosin®), BMS 599626, lapuleucel-T (Neuvenge®), NeuVax®, Osidem® (IDM 1), mubritinib (TAK-165), panitumumab (Vectibix®), lapatinib (Tycerb®), pelitinib (EKB 569), eribafatinib (Balversa), pertuzumab (Omnitarg®).

[0322] Other examples of signal transduction inhibitors include ARRY 142886, everolimus (Certican®), zotarolimus (Endeavor®), temsirolimus (Torisel®), and VX 680 (Vertex).

[0323] The present invention contemplates the use of the compounds of the present invention together with an anti-tumor agent. Examples of the anti-tumor agent include, but are not limited to, hormonal agents, anti-estrogen therapeutic agents, histone deacetylase (HDAC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteomics, topoisomerase I inhibitors, camptothecin derivatives, topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors, microtubulin inhibitors, antibiotics, spindle inhibitors, platinum coordination compounds, gene therapy agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins.

[0324] Examples of antitumor agents used in combination therapy with the compounds of the present invention include glucocorticoids such as dexamethasone, prednisone, prednisolone, methylprednisolone, hydrocortisone, and progestins such as medroxyprogesterone, megestrol acetate (Megace), mifepristone (RU-486), selective estrogen receptor modulators (SERMs such as tamoxifen, raloxifene, lasofoxifene, afimoxifene, arzoxifene, bazedoxifene, fispemifene, ormeloxifene, ospemifene, temoxifen, toremifene, trianthene, and CHF 4227 (Cheisi)), selective estrogen receptor downregulators (SERDs such as fulvestrant), exemestane (Aromasin (registered trademark)), astrozole (Arimidex (registered trademark)), atamestane, fadrozole, letrozole (Femara), buserelin (Suprefact), goserelin (Zoladex), leuprolide (Lupron), and gonadotropin-releasing hormones (generally also called luteinizing hormone-releasing hormone [LHRH] GnRH) agonists such as triptorelin (Trelstar (registered trademark)), abarelix (Plenaxis (registered trademark)), bicalutamide (Casodex (registered trademark)), cyproterone, flutamide (Eulexin (registered trademark)), megestrol, nilutamide (Nilandron), and osaterone, dutasteride, epristeride, finasteride, abarelix, goserelin, leuprorelin, triptorelin, bicalutamide, tamoxifen, exemestane, anastrozole, fadrozole, formestane, letrozole, and combinations thereof, but are not limited thereto.

[0325] Other examples of anti-tumor agents used in combination with the compounds of the present invention include suberoylanilide hydroxamic acid (SAHA®, Merck), depsipeptide (FR901228), G2M-777, MS-275, pivaloyloxymethyl butyrate, and PXD-101 / Onconase® (ranpimase), PS-341, Valcade® (bortezomib), 9-aminocamptothecin, velotecan, BN-80915, camptothecin, difluorocamptothecin, edotecarin, exatecan, gimotcan, 10-hydroxycamptothecin, irinotecan HCl (Camptosar®), lutetium, Orathecin® (rubitecan, Supergen®), SN-38, topotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, irinotecan, aclarubicin, adriamycin, amonafide, amrubicin, anamycin, daunorubicin, doxorubicin, elsamitrucin, epirubicin, etoposide, idarubicin, gallarubicin, hydroxycarbamide, nembutal, Novantrone (mitoxantrone), pirarubicin, pisanthrone, procarbazine, levomycetin, sobuzoxane, tafurposide, valrubicin, Zinecard® (dexrazoxane), nitrogen mustard N-oxide, cyclophosphamide, AMD-473, altretamine, Ap-5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, chlorambucil, dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, KW-2170, lomustine, mafosfamide, mechlorethamine, melphalan, mitobronitol, mitractol, mitomycin C, mitoxantrone, nimustine, ranimustine, temozolomide, thiotepa, platinum coordination alkylating agents such as cisplatin, but not limited to these. Paraplatin (carboplatin), heptaplatin, lobaplatin, nedaplatin, Eloxatin® (oxaliplatin), streptozocin, satraplatin, and combinations thereof.

[0326] The present invention also contemplates the use of the compounds of the present invention in combination with dihydrofolate reductase inhibitors (e.g., methotrexate and NeuTrexin® (trimetrexate glucuronate)), purine antagonists (e.g., 6-mercaptopurine riboside, mercaptopurine, 6-thioguanine, cladribine, clofarabine (Clolar®), fludarabine, nelarabine, and larotrexed), pyrimidine antagonists (e.g., 5-fluorouracil (5-FU), Alimta® (pemetrexed disodium), capecitabine (Xeloda®), cytosine arabinoside, Gemzar® (gemcitabine), Tegafur® (UFT Orzel® or UForal® and combinations of tegafur, gimeracil and oteracil of TS-1), doxifluridine, carmofur, cytarabine (including the octaphosphate, phosphoric acid stearate, sustained release and liposomal forms), enocitabine, 5-azacytidine (Vidaza®), decitabine and ethynyl-cytidine), and other antimetabolites such as efomithine, hydroxyurea, leucovorin, nolatrexed, triapine, trimetrexate, ABT-472, Ino-1001, KU-0687 and GPI 18180, and combinations thereof.

[0327] Further examples of anti-tumor agents used in combination therapy with the compounds of the present invention include, in combination therapy with optionally one or more other agents, Advexin®, Genasense (oblimersen, Genta®), combretastatin A4P (CA4P), Oxi4503, AVE-8062, ZD-6126, TZT 1027, atorvastatin (Lipitor®), pravastatin (Pravachol®), lovastatin (Mvacor®), simvastatin (Zocor®), fluvastatin (Lescol®), cerivastatin (Baycol®), rosuvastatin (Crestor®), niacin (Advicor®), Caduet, and combinations thereof, but are not limited thereto.

[0328] The present invention also contemplates using the compounds of the present invention together with agents that modulate the immune system, including, but not limited to, pembrolizumab (Keytruda®), nivolumab (Opdivo®), cemiplimab (Liptayo®), atezolizumab (Tecentrig®), avelumab (Bavencio®), durvalumab (Imfinzi®), ipilimumab (Yervoy®), rituximab (RITUXAN®, Thor-707, and dexamethasone.

[0329] The present invention also contemplates using the compounds of the present invention together with agents that modulate proteins of the BCL-2 family, including, but not limited to, venetoclax (Venelexta®, ABT-199) and AMG 176.

[0330] The present invention also contemplates using the compounds of the present invention together with agents that inhibit the androgen receptor, including, but not limited to, apalutamide (Erleada®), flutamide (Eulexin®), nilutamide (Nilandron®), bicalutamide (Casodex®), and enzalutamide (Xtandi®).

[0331] The present invention also contemplates using the compounds of the present invention together with agents that modulate proteins of the PARP family, including, but not limited to, niraparib (Zejula®), olaparib (Lynparza®), rucaparib (Rubraca®), and talazoparib (Talzenna®).

[0332] Another aspect of the present invention of particular interest relates to a method for treating breast cancer in a human in need of treatment, comprising administering to the human an amount of a compound of the present invention in combination with one or more (preferably 1 to 3) anti-cancer agents selected from the group consisting of trastuzumab, tamoxifen, docetaxel, paclitaxel, capecitabine, gemcitabine, vinorelbine, exemestane, letrozole, and anastrozole.

[0333] Another embodiment of the present invention relates to a method for treating neurodegenerative diseases in a human in need of treatment, comprising administering to the human an amount of a compound of the present invention in combination with one or more agents selected from the group consisting of anti-tau mAb, anti-β amyloid mAb, BIIB067 (tofersen), BAN2401, BIIB054 (anti-α-synuclein), BIIB074, BIIB092, BIIB092 (gosuranemab), BIIB104, natalizumab, BIIB076 (anti-tau mAb), BIIB078 (IONIS-C9RX), BIIB080 (IONIS-MAPTRX), BIIB095 (NAV 1.7), BIIB (XPO1 inhibitor), BIB110, cholinesterase inhibitors (Aricept®, Exelon®, Razadyne®), memantine (Namenda®), levodopa, rosin, dopamine agonists (pramipexole, ropinirole, rotigotine and apomorphine), MAO B inhibitors (selegiline, rasagiline, safinamide), catechol O-methyltransferase (COMT) inhibitors (entacapone and tolcapone), anti-cholinergic drugs (benztropine and trihexyphenidyl), amantadine, riluzole, edavarone, xedazine, antipsychotics and benzodiazepines.

[0334] Methods of treatment and uses

[0335] The present invention further provides methods of treatment and uses comprising administering a compound of the invention or a pharmaceutically acceptable salt thereof, alone or in combination with one or more other therapeutic or palliative agents. The compositions and methods described herein are useful for treating many disease states including cancer.

[0336] Cancers treated using the methods, compositions and / or agents described herein are characterized by abnormal cell growth and include, but are not limited to, pre-neoplastic overgrowth, intraepithelial carcinoma, tumors and metastases. The methods and compositions described herein can be used for the prevention and amelioration of cancer signs and / or symptoms.

[0337] In one aspect, the compositions and methods described herein are used for treating diseases such as melanoma, desmoplastic small round cell tumor, chondrosarcoma, leptomeningeal disease, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal or rectal cancer, appendiceal cancer, astrocytoma, and atypical teratoid / rhabdoid tumor.

[0338] In one aspect, the compositions and methods described herein are used for treating diseases such as basal cell carcinoma, basal cell nevus syndrome, Gorlin- nevus syndrome, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma, and malignant fibrous histiocytoma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, and spinal cord tumor.

[0339] In one aspect, the compositions and methods described herein are used for treating diseases such as carinoid tumors, cancers of unknown primary origin, central nervous system atypical teratoid / rhabdoid tumors, leptomeningeal diseases, central nervous system germ cell tumors, central nervous system lymphomas, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, central nervous system germ cell tumors, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer, Ewing sarcoma, extracranial primitive neuroectodermal tumors, extragonadal primitive neuroectodermal tumors, extrahepatic bile duct cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumors, germ cell tumors, gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lung cancer, non-Hodgkin lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer of unknown primary origin, multiple neoplasia syndrome, oral cancer, multiple / plasma cell neoplasms, fungating polyps, myelodysplastic syndromes, neoplasms, multiple myeloma and myeloproliferative disorders.

[0340] In one aspect, the compositions and methods described herein are used for treating cancer.

[0341] The present invention further provides methods of treatment and uses comprising administering a compound of the invention or a pharmaceutically acceptable salt thereof, alone or in combination with one or more therapeutic or palliative agents.

[0342] In one aspect, the present invention provides a method of treating a disease in a subject in which a protein that is a substrate of ClpP is present at an abnormally high concentration in the subject, comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

[0343] In one aspect, the present invention provides a method of treatment comprising administering to a subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof for a disease state including cancer in which a decrease in the concentration of a protein that is a substrate of ClpP in the subject leads to improvement of the disease.

[0344] In one aspect, the present invention provides a method of treatment comprising administering to a subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof for a disease state comprising a cancer in which an abnormally high concentration of a protein, ClpP, is present in the subject.

[0345] In one aspect, the present invention provides a method of treatment of a disease in which an abnormally low concentration of a protein, ClpP, is present in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0346] In one aspect, the present invention provides a method of treatment of abnormal cell proliferation in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0347] In another aspect, the present invention provides a method for treating abnormal cell proliferation in a subject, comprising administering to the subject a quantity of a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a quantity of an anti-tumor agent, the quantity of the anti-tumor agent being effective, together with the compound, to treat the abnormal proliferation. In some aspects, the anti-tumor agent is selected from the group consisting of a mitotic inhibitor, an alkylating agent, an antimetabolite, an intercalating antibiotic, a growth factor inhibitor, radiation, a cell cycle inhibitor, an enzyme, a topoisomerase inhibitor, a biological response modifier, an antibody, a cytotoxic agent, an anti-hormone and an anti-androgen.

[0348] In another aspect, the present invention provides a method of inhibiting cancer cell proliferation in a subject, comprising administering to the subject an amount of a compound of the present invention or a pharmaceutically acceptable salt thereof effective to inhibit cell proliferation.

[0349] In another aspect, the present invention provides a method for treating cancer selected from the group consisting of solid tumors, liquid tumors, lymphomas, leukemias or myelomas. In some aspects, the treatment of cancer includes preventing tumor growth in a cancer subject, which includes administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof that is effective to inhibit cell growth.

[0350] In another aspect, the present invention provides a method for inhibiting cancer cell invasiveness in a subject, which includes administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof that is effective to inhibit cell growth.

[0351] In another aspect, the present invention provides a method for inducing apoptosis in cancer cells in a subject, which includes administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof that is effective to inhibit cell growth.

[0352] In another aspect, the present invention provides a method for inducing apoptosis in a subject, which includes administering to the subject an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof that is effective to inhibit cell growth.

[0353] In a frequent embodiment of the methods provided herein, the abnormal cell growth is cancer, where the cancer is selected from the group consisting of basal cell carcinoma, medulloblastoma, liver cancer, rhabdomyosarcoma, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal area cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft leukemia, urethral cancer, penile cancer, prostate cancer, chronic or acute lymphocytic, lymphoma, lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell cancer, renal pelvic cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, spinal cord axis tumor, brain stem glioma, pituitary adenoma, or a combination of one or more of these cancers. In some aspects, the cells are in a tissue or tumor, and the tissue or tumor can be in a subject including a human.

[0354] The cancers treated using the methods and compositions described herein are characterized by abnormal cell proliferation including, but not limited to, metastasis, pre-neoplastic hyperplasia, carcinoma in situ, and tumors. The compounds of the invention can be for prevention in addition to improvement of cancer signs and / or symptoms. Examples of cancers treated by the compounds of the invention include, but are not limited to, breast cancer, CNS cancer, colon cancer, prostate cancer, leukemia, lung cancer, and lymphoma.

[0355] In another aspect, the invention provides a method of treating leukemia selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloproliferative disorders, hairy cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and Langerhans cell histiocytosis.

[0356] In another aspect, the invention provides a method of treating lymphoma selected from the group consisting of diffuse large B-cell lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, Sézary syndrome, mycosis fungoides (MF), histiocytosis, Burkitt lymphoma, central nervous system lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Hodgkin lymphoma, Waldenström macroglobulinemia, mycosis fungoides, and lymphoplasmacytic lymphoma.

[0357] In another aspect, the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

[0358] In another aspect, the invention provides a method of treating cancer selected from the group consisting of vaginal cancer, vulvar cancer, endometrial cancer, carcinoma of unknown primary, and cancer of unknown primary.

[0359] In another aspect, the invention provides a method of treating a bacterial infection in a subject comprising administering to the subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

[0360] In another aspect, the present invention provides a method for treating Staphylococcus aureus infection in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0361] In another aspect, the present invention provides a method for treating neurodegenerative diseases in a subject, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, spinal muscular atrophy, and motor neuron diseases, the method comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0362] In another aspect, the present invention provides a method for treating erythropoietic protoporphyria (EPP) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0363] In another aspect, the present invention provides a method for treating erythropoietic protoporphyria (EPP) in a subject having a dominant mutation (ClpX:p.Gly298Asp), the method comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0364] In another aspect, other conditions that may be suitable for the methods described herein include, but are not limited to, attention deficit disorder, addiction, epilepsy, viral infection, inflammation, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, cardiovascular diseases such as coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, arrhythmia, inflammatory heart disease, endocarditis, inflammatory cardiac hypertrophy, myocarditis, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, rheumatic heart disease, diabetes, and light chain amyloidosis.

[0365] In another aspect, the present invention provides a method for treating cystic fibrosis.

[0366] In another aspect, the present invention provides a method for treating Pelizaeus-Merzbacher disease.

[0367] In another aspect, the present invention provides a method for treating type 3 Peyronie's syndrome.

[0368] In another aspect, the present invention provides a method for treating an autoimmune disease. Autoimmune diseases include, but are not limited to, alopecia areata, antiphospholipid antibody, autoimmune hepatitis, celiac disease, type 1 diabetes, Graves' disease, Guillain - Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, psoriatic arthritis, Crohn's disease, vitiligo.

[0369] In another aspect, the present invention provides a method for treating allograft rejection. In another aspect, the present invention provides a method for treating hereditary spastic paraplegia.

[0370] In another aspect, the present invention provides a method for treating the condition of acquired immunodeficiency syndrome (AIDS).

[0371] In another aspect, the present invention provides a method for treating the condition of HIV and acquired immunodeficiency syndrome (AIDS).

[0372] In another aspect, the present invention provides a method for treating the condition of pneumonia.

[0373] In another aspect, the present invention provides a method for treating the condition of sepsis.

[0374] In another aspect, the present invention provides a method for treating the condition of viral infection.

[0375] In another aspect, the present invention provides a method for treating hepatitis in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0376] In another aspect, the present invention provides a method for treating idiopathic cirrhosis in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0377] In another aspect, the present invention provides a method for treating hepatocyte senescence in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0378] In another aspect, the present invention provides a method for treating non-alcoholic fatty liver disease (NAFLD) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0379] In another aspect, the present invention provides a method for treating non-alcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0380] Preparation method, chemical compound

[0381] The compounds of the present invention can be prepared by various methods including standard chemistry. The variables defined previously retain their previously defined meanings unless otherwise noted. Exemplary general synthetic methods are described below, specific compounds of formula (I) are prepared in the examples, and additional information regarding the synthesis of these compounds is described in the following cited references: Sun H. et al ACS Med.Chem.Lett. 2019, 10, 191-195 and the references cited therein, WO2018 031990 and the references cited therein, WO2018 031987 and the references cited therein, CN1048600948 and the references cited therein, and US8,318,751 and the references cited therein.

[0382] Currently, there are many suppliers of chemical reagents. Examples of chemical suppliers include, but are not limited to, Sigma Aldrich, Saint Louis, MO; Alfa Aesar, Tewksbury, MA; TCI America, Portland, OR; BroadPharm, San Diego, CA and Cambridge BioSciences, Cambridge, UK. BroadPharm also offers a custom service providing reagents for the synthesis of the compounds of the present invention. ONC201 (CAS 1616632-77-9) is commercially available from multiple suppliers such as MEDCHEM Express, 1 Deer Park Drive, Suite Q, Monmouth Junction, NJ, 08852. 2-(3-Iodopropyl)isoindoline-1,3-dione is available from multiple vendors including Sigma-Aldrich (Aldrich CPR-R465674). Further, 2-(4-Iodobutyl)isoindoline-1,3-dione is also available from multiple vendors including Sigma-Aldrich (Aldrich CPR-R260312). Both ONC201 and ONC206 are available from commercial suppliers including SelleckChem, Houston, TX 77014, MedKoo BioSciences, Inc and Matrix Scientific (Columbia, SC 29224).

[0383] The compounds of formula (I) can be prepared by methods known in the art of organic synthesis as described in part by the following synthetic schemes. In all the schemes described below, it is well understood that, in accordance with the general principles of chemistry, protecting groups for sensitive or reactive groups are often used as necessary. Protecting groups are manipulated according to standard organic synthetic methods (T.W. Green and P.G.M. Wuts (1991) Protecting Groups in Organic Synthesis, John Wiley & Sons). Those skilled in the art will recognize whether there are chiral centers in the compounds of formula (I). Thus, the present invention includes all possible stereoisomers, including mixtures of stereoisomers (such as racemic compounds) as well as individual stereoisomers. If a compound is desired as a single isomer, it can be obtained by various separation methods of the final product or key intermediates, or by stereospecific synthesis using methods that impart enantiomeric purity or enantiomerically pure intermediates. These are known to those skilled in the art.

[0384] The compounds were analyzed by general methods known to those skilled in the art. NMR, HPLC, and LCMS were used for the evaluation of isolated compounds and reaction mixtures. Under LCMS conditions, a Symmetry C18, 5um, 4.6X50mm column was used, with water and MeCN as the two solvents. A linear gradient was used from time 0 (90% H20, 10% MeCN, 0.1% TFA) to 4.5 minutes (5% H20, 95% MeCN, 0.1% TFA). The flow rate was 1.7 ml / min. The evaluation was performed at 254 nm.

[0385] The following solvents, reagents, protecting groups, moieties, and other names can be referred to by their abbreviations. Me: Methyl; Et: Ethyl; Pr: Propyl; i-Pr: Isopropyl; Bu: Butyl; t-Bu: tert-Butyl; Ac: Acetyl ACN: Acetonitrile AcOH: Acetic acid Aq.: Aqueous AUC: Area under the curve BOC or Boc: tert-Butyloxycarbonyl Conc.: Concentrated DMF: Dimethylformamide DMSO: Dimethyl sulfoxide EDCI or EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: Ethyl acetate EtOH: Ethanol Ex.: Example g: Gram h: Hour HPLC: High performance liquid chromatography LCMS: Liquid chromatography mass spectrometry MeOH: Methanol MeI: Methyl iodide MS: Mass spectrometry NA: Not applicable ND: No data NMR: Nuclear magnetic resonance spectroscopy NT: Not tested Ph: Phenyl Ret Time: Retention time RT or rt: Room temperature Satd, Sat’d, sat’d and satd.: Saturated TFA: Trifluoroacetic acid THF: Tetrahydrofuran

[0386] Notable with respect to particulate matter is the use of toluene analogs as reagents and synthetic intermediates. There are numerous commercial sources of toluene analogs that are used directly or can be converted to useful reagents or intermediates in the synthesis of the compounds of the present invention. Many methods for the interconversion of toluene analogs to provide reagents and intermediates useful in the synthesis of the compounds of the present invention are known to those of skill in the art. The examples described herein include bromination of methyl residues (Example 64) and conversion of functionalized benzyl alcohols to the corresponding bromides (Example 79). Further, benzyl alcohols can be converted to the corresponding benzylamines via oxidation to aldehydes followed by a reductive amination process. These examples are not limiting.

[0387] An aromatic residue having a single J substituent means various J residue(s) as described herein and means various positions on the aromatic residue to which it is shown to be attached.

[0388] The compounds described when Q is Q3 can be prepared as shown in Scheme 1. Further, the scheme used to prepare Example 61 can be used to prepare the compounds of the present invention. Those of skill in the art can extrapolate this method of preparation to form the agents using the information contained in the references cited herein and general synthetic chemical knowledge. Of particular note is the information regarding the synthesis of chemically related substances described in U.S. Patent No. 8,318,751 and the references cited therein. Further, additional synthetic details for the preparation of the compounds when Q is Q3 are found in WO2008 / 130584 and the references contained therein. Similarly, the compounds of the present invention where Q is Q10 can be prepared in the same manner as the compounds where Q is Q3. The chemistry described for the synthesis of the compounds where Q is Q3 uses various functionalized piperidine compounds as synthetic intermediates, and the compounds where Q is Q10 can use various functionalized piperidine compounds as synthetic intermediates and use similar or analogous synthetic routes.

[0389] Scheme 1 JPEG0007709379000091.jpg74150

[0390] When Q is Q4, the compounds described can be prepared as shown in Scheme 2. Those skilled in the art can extrapolate this preparation method using the information contained in the references cited herein (Stahl M., et al, Angew. Chem. Int. Ed. 2018, 57, 14, 602 - 14, 607 and the references cited therein) and general synthetic chemical knowledge to form the agents.

[0391] Scheme 2 JPEG0007709379000092.jpg61150

[0392] When Q is Q2, the compounds described can be prepared as described in WO2018 031990 and the references cited therein. Furthermore, the synthetic methods and schemes described by Ma, Z (Ma, Z. et al, ACS Med. Chem. Lett. 2019, 10, 191 - 195 and the references cited therein) and Furrer (US5,556,854 and the references cited therein) are applicable to the manufacture of the agents of the present invention. Those skilled in the art can extrapolate this preparation method using the information contained in the references cited herein and general synthetic chemical knowledge to form the agents.

[0393] When Q is Q1, the compounds described can be prepared as described in WO2018 031987 and the references cited therein. There are many other publications, such as El-Deiry, W.S. et al, Cell Cycle 2017, 16, 1790 - 1799 and the references cited therein, which describe the synthesis of these agents. Those skilled in the art can extrapolate this preparation method using the information contained in the references cited herein and general synthetic chemical knowledge to form the agents.

[0394] When Q is Q2, the compounds described may be conjugated to various infrared, fluorescent, phosphorescent, radioactive or infrared fluorescent as shown in Scheme 3. Compounds designated as SS10 are useful intermediates for forming the compounds of the invention into other diagnostic agents. The length of the carbon linker determined by n can be from 1 to 30, although n = 1 to 5 is more optimal. These analogs are prepared as described above using appropriate protecting groups for terminal functionality. The amine terminus of the alkyl chain has particular value as a reactive species and can readily form many common functional groups such as amides, carbamates, secondary amines, etc. using acid chlorides, ketenes, carboxylic acids (including coupling agents), etc. In addition to amines, other terminal residues can be used to form linkers such as -SH, -OH, -Cl, -Br and -I. These terminal residues can be conjugated to various dyes and contrast agents. Fluorescent dyes containing a wide variety of functional groups to facilitate coupling and PEG spacers of different lengths to increase water solubility are commercially available (BroadPharm, Inc., 6625 Top Gun Street, Suite 103, San Diego, CA 92121). Efficient bio-labeling in imaging and diagnostic research and development is made possible. The types of agents sold by BroadPharm, Inc. include BDP, Cy3, Cy5, Cy5.5, Cy7, fluorescein and pyrene. This example is not intended to be limiting.

[0395] Further experimental information on the synthesis of the coupled dyes can be found in the following references: Wang L. et al, Angew Chem Int Ed. 2019 Mar 7. Doi: 10.1002 / anie.201901061 and the references cited therein, Gomes da Costa, S. et al, Morphologie 2019, Mar; 103(341): 11-16 and the references cited therein, Wei H. et al, Future Med Chem 2018, Dec 6. doi:10.4155 / fmc-2018-0198 and the references cited therein, Alamudi, S.H. et al, Chem Commun 2018 Dec 4; 54(97): 13641-13653 and the references cited therein, Iliopoulos-Tsoutsouvas C. et al, Expert Opin Drug Discov 2018 Oct; 13(10): 933-947 and the references cited therein, Vernall A.J. et al, Br J Pharmacol 2014 Mar; 171(5): 1073-84 and the references cited therein, and Broyles C.N. et al, Cells 2018 May 31; 7(6) and the references cited therein.

[0396] Scheme 3 JPEG0007709379000093.jpg37150

[0397] The general synthetic scheme shown as Scheme 4 is a series of reactions that can be used by those skilled in the art to prepare the compounds of the present invention. The substituents X and Y represent various substituents that can be used in this series of reactions, and their positions on their respective aromatic residues are not limited. Further, one or more substituents may be present on one aromatic residue. The center of this chemical synthesis route is the use of the isocyanate shown here as SS15. When J is a single chlorine atom and the remaining positions that can be substituted are hydrogen, the required isocyanate has the chemical formula C8H6ClNO. Further, the final step (d) is envisioned to allow the attachment of various residues identified here by R. Alternative methods for N-alkylation are known to those skilled in the art. For example, SS13 can be prepared from SS11 using the corresponding benzaldehyde and a reducing agent. This example is not limiting with respect to the number and type of substituents that can be used therein. Other reaction conditions known to those skilled in the art can be used for the various transformations of Scheme 4.

[0398] Scheme 4 JPEG0007709379000094.jpg105150Synthesis of the compound according to Scheme 4: (a) DMF, Et3N; (b) sodium carbonate, NH3, ethanol, 70 o C for 5 hours; (c) Et3N, toluene, reflux, 80 o C for 8 hours; (d) RBr, K2CO3, DMF, 100 o C, 12 hours

[0399] The general synthetic scheme shown as Scheme 5A is a series of reactions that can be used by those skilled in the art to prepare the compounds of the present invention. The substituent J is independently selected Y, and their positions in the aromatic system are not restricted. The center of this chemical synthesis route is to form a ring using a two-step series of syntheses. A carbon-nitrogen bond is formed on SS16 to produce SS19. Of great importance is the SS18 reagent with a protected nucleophile (nitrogen). This was once an unprotected nucleophile, and the resulting SS21 is now attempting to condense onto SS21 to form the ring of SS23. An example where Q is Q5 is SS23. These examples are not restrictive with respect to the number and type of substituents that can be used therein. Other reaction conditions known to those skilled in the art can be used for the various transformations in Scheme 5A.

[0400] Scheme 5A JPEG0007709379000095.jpg119150Synthesis of the compound according to Scheme 5a: (a) Sodium carbonate, DMF, 85 o C for 5 hours; (b) CH3NH2, EtOH, reflux, 80 o C for 4 hours; (c) pTSA, DMF, iPrOH, 80 o C for 12 hours.

[0401] Scheme 5B shows the preparation of the compounds of the present invention as an alternative to the synthetic scheme shown in Scheme 5A.

[0402] Scheme 5B JPEG0007709379000096.jpg55150Synthesis of the compound according to Scheme 5b: (a) 4-Cl-benzylamine, DMF, 85 o C for 5 hours; (b) SS25, cat p-TSA, EtOH, reflux, 80 o C for 4 hours.

[0403] Scheme 6 shows the preparation of amine-protected alkylating agents. Other reaction conditions known to those skilled in the art can be used for the various transformations in Scheme 6.

[0404] Scheme 6 JPEG0007709379000097.jpg84150Synthesis of compounds according to Scheme 6: (a) SS29, K2CO3, DMF.

[0405] Scheme 7 shows the preparation of various compounds of the present invention using the following important reagents: SS33, SS35, SS37, and SS39. Paying attention to the chemistry disclosed herein, particularly the transformations shown as Schemes 5A, 5B, and 6, indicates a series of reactions that a person skilled in the art can use to prepare the compounds of the present invention. Of particular note are the reaction conditions that promote the alkylation reaction, for example, sodium carbonate, DMF, 85 o C for 12 hours. The J substituent represents various substituents that can be used in this series of reactions, and their positions on the molecule are not limited. This example is not limiting with respect to the number and type of substituents that can be used therein. Other reaction conditions known to those skilled in the art can be used for the various transformations for preparing the compounds in Scheme 7.

[0406] Scheme 7 JPEG0007709379000098.jpg132150

[0407] Scheme 8 shows the preparation of various compounds of the present invention using the following important reagents: SS41, SS42, SS43, and SS44. Using the chemistry disclosed herein, particularly paying attention to the series of reactions shown in Schemes 5a and 5b, indicates a series of reactions that a person skilled in the art can use to prepare the compounds of the present invention. The J substituent represents various substituents that can be used in this series of reactions, and their positions on the aromatic system are not limited. This example is not limiting with respect to the number and type of substituents that can be used therein. Other reaction conditions known to those skilled in the art can be used for the various transformations for preparing the compounds in Scheme 8.

[0408] Scheme 8 JPEG0007709379000099.jpg113135

[0409] Scheme 9 shows the preparation of various compounds of the invention and shows the use of SS40 and SS45, which are particularly important synthetic intermediates. The terminal olefin of SS40 and the ketone residue of SS45 are converted to many novel analogs under reaction conditions known to those skilled in the art.

[0410] Scheme 9 JPEG0007709379000100.jpg113149Synthesis of compounds according to Scheme 9: (a) Iodomethylzinc iodide, Et2O (Simmons-Smith reaction) or (CH3)2S(O)CH2, DMSO, THF 50 o °C; (b) O3, CH2Cl2, -78 o °C followed by Me2S, and (c) pTSA, DMF, ROH (or HOCH2CH2OH), 80 o °C for 12 hours.

[0411] Scheme 10 is a general synthetic scheme for producing the compounds of the invention. This scheme can be used to prepare compounds in which Q is Q6, together with other chemistries disclosed herein and chemistries known to those skilled in the art. In particular, the chemistries of Schemes 4, 5a and 5b can be applied to this synthetic route.

[0412] Scheme 10 JPEG0007709379000101.jpg33150Synthesis of compounds according to Scheme 10: (a) Z2-N=C=O, Et3N, toluene, reflux, 80 o °C, 8 hours and (b) RBr, K2CO3, DMF, 100 o °C, 12 hours.

[0413] Scheme 11 is a general synthetic scheme for producing the compounds of the invention. This scheme can be used to prepare compounds in which Q is Q6, together with other chemistries disclosed herein and chemistries known to those skilled in the art. Note that SS51 is prepared as shown in Scheme 10 using the chemistries particularly described in Schemes 5a, 5b, 6, 7 and 8 herein.

[0414] Scheme 11 JPEG0007709379000102.jpg37150Synthesis of the compound according to Scheme 11: (a) pTSA, DMF, iPrOH, 80 o C for 12 hours.

[0415] Schemes 12A and 12B are general synthetic schemes for preparing the compounds of the present invention. These schemes can be used to prepare compounds where Q is Q8, together with the other chemistries disclosed herein and chemistries known to those skilled in the art. In particular, the chemistry of Scheme 4 can be applied to this synthetic route. Additionally, the chemistries described in CN104860948 and WO2016 / 184437 can be used. SS53 can be prepared from the corresponding secondary amine by a reductive amination process that forms the Z1 residue using the corresponding aldehyde and a reducing agent.

[0416] Scheme 12A JPEG0007709379000103.jpg28150Synthesis of the compound according to Scheme 12A: (a) Z2-NH(CO)Cl, Et3N, toluene, reflux, 80 o °C, 8 hours and (b) RBr, K2CO3, DMF, 100 o °C, 12 hours.

[0417] Scheme 12B JPEG0007709379000104.jpg70150Synthesis of the compound according to Scheme 12B: (a) NH3, t-BuOH, (b) O=N=CH(Ph-JJ), TNF, (c) toluene, Et3N, 100 o °C or toluene, cat p-TSA, 100 o °C, (d) R13-Br, K2CO3, DMF, 100 o °C, (e) TFA, CH2Cl2 and (f) CH3CN, BrCH2Ph-J, Et3N.

[0418] Schemes 13A and 13B are general synthetic schemes for preparing the compounds of the present invention. These schemes can be used, together with the other chemistries disclosed herein and chemistries known to those skilled in the art, to prepare compounds where Q is Q9. It should be noted that SS56 is prepared as shown in Scheme 12 using the chemistries specifically described in Schemes 5a, 5b, 6, 7 and 8 herein. Alternatively, SS57 can be prepared using the series of chemistries shown in Scheme 5b.

[0419] Scheme 13A JPEG0007709379000105.jpg36150 Synthesis of the compound according to Scheme 13A: (a) pTSA, DMF, iPrOH, 80 o C for 12 hours.

[0420] Scheme 13B JPEG0007709379000106.jpg62150 Synthesis of the compound according to Scheme 13B: a) MeI, Et3N, THF, 50 o C; b) J-benzylamine, reflux in THF; c) Et3N, reflux in toluene; d) TFA, CH2Cl2; e) J-benzyl bromide, Cs2CO3.

[0421] Scheme 14 is a general synthetic scheme for preparing the compounds of the present invention. This scheme can be used, together with the other chemistries disclosed herein and chemistries known to those skilled in the art, to prepare the compounds of formula 8A.

[0422] Scheme 14 JPEG0007709379000107.jpg80150 Synthesis of the compound according to Scheme 14: (a) MeI, Et3N, THF, 50 o C; (b) 4-Cl-benzylamine, reflux in THF; (c) Cl(CO)OEt, NaOEt, EtOH, 60 o C; (d) SS60, Et3N, reflux in toluene.

[0423] Scheme 15 is a general synthetic scheme for preparing the compounds of the present invention. This scheme can be used to prepare the compounds of Formula 9A, along with other chemistry disclosed herein and chemistry known to those skilled in the art.

[0424] Scheme 15 JPEG0007709379000108.jpg63150Synthesis of the compound according to Scheme 15: (a) NH3, cat. NH4Cl, EtOH, reflux; (b) HN=C=O or equivalent, Et3N, toluene reflux; (c) 4-Cl-benzyl bromide, Et3N, DMF heat; (d) K2CO3, MeI, DMF heat.

[0425] Examples

[0426] Chemical examples

[0427] Examples of the compounds are shown below. This is not intended to be limiting in any way.

[0428] Example 1 JPEG0007709379000109.jpg29150

[0429] D9 was prepared as described in Sieber S.A. et al, Angew.Chem.Int.Ed. 2008, 57, 14, 602 - 14, 607.

[0430] Examples 2 - 27

[0431] Examples 2 - 27 were prepared as described in WO2018 031987.

[0432] JPEG0007709379000110.jpg29150

[0433] JPEG0007709379000111.jpg225135JPEG0007709379000112.jpg210125JPEG0007709379000113.jpg22399

[0434] JPEG0007709379000114.jpg31150

[0435] Examples 28 to 58

[0436] Examples 28 to 58 were prepared as described in WO2018 031990 and the references cited therein.

[0437] JPEG0007709379000115.jpg29150

[0438] JPEG0007709379000116.jpg223113JPEG0007709379000117.jpg223108JPEG0007709379000118.jpg223106JPEG0007709379000119.jpg223102

[0439] Example 57

[0440] 3-((1-(3-aminopropyl)-2,4-dioxo-3-(4-(trifluoromethyl)benzyl)-1,2,3,4,7,8-hexahydropyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzonitrile

[0441] JPEG0007709379000120.jpg27150JPEG0007709379000121.jpg36150

[0442] Step 1: A mixture of methyl 1-(3-cyanobenzyl)-4-oxopiperidine-3-carboxylate SS26 (8.55 g, 31.4 mmol) and ammonia solution (7 ml, 25%) in ethanol (110 ml) was heated at 70 °C for 5 h. The solution was concentrated, extracted with DCM (2 × 300 ml), and washed with borane. The extract was dried over Na2SO4 and evaporated under reduced pressure to give 8 g of 2-((4-amino-3-(methoxycarbonyl)-5,6-dihydropyridin-1(2H)-yl)methyl)-4-cyanobenzene-1-ide INT2 (oil), which was used directly in the next step.

[0443] Step 2: To a solution of INT2 (2 g, 7.4 mmol) in 20 mL of toluene was added 1-(isocyanatomethyl)-4-(trifluoromethyl)benzene (1.6 g, 7.5 mmol) and triethylamine (1.1 g, 10.4 mmol). The solution was heated at 80 °C for 8 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure. The formed white solid was filtered and dissolved in MeOH (20 ml). NaOMe (350 mg) was added and the mixture was refluxed overnight. Then about 10 - 15 ml of methanol was removed and the precipitate was filtered. The desired product 3-((2,4-dioxo-3-(4-(trifluoromethyl)benzyl)-1,2,3,4,7,8-hexahydropyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzonitrile, INT2 was obtained as a pale yellow solid (0.8 g, 25%).

[0444] Step 3: To a solution of INT2 (200 mg) in DMF (2 ml) were added potassium carbonate (150 mg) and 2-(3-iodopropyl)isoindoline-1,3-dione (150 mg). The mixture was heated at 100 °C for 12 h. Water (about 3 ml) was added and the solution was extracted with EtOAc (3 × 5 ml). The combined extracts were washed 3 times with borane (about 5 ml), dried over Na2SO4, filtered, concentrated under reduced pressure to give the crude product. The purified product, INT3 was obtained by preparative TLC, 100 mg, 35% yield.

[0445] Procedure 4: To a solution of INT3 (100 mg) in EtOH (3 ml) of the product, methylamine solution (0.25 ml, 30%) was added. The mixture was heated at 80 °C for 4 hours. Water was added and the solution was extracted with DCM (3 × 3 ml). The combined organic extracts were dried over Na2SO4, filtered, concentrated under reduced pressure to obtain the crude product, Example 57. The final product, Example 57, was obtained by preparative HPLC, 15 mg, yield 19%.

[0446] 1 1H NMR (400 MHz, CD3OD) δ 2.03 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 6.8 Hz, 2H), 3.18 (s, 2H), 3.67 (s, 2H), 4.01 (t, J = 6.8 Hz, 2H), 4.07 (s, 2H), 4.62 (s, 2H), 5.17 (s, 2H), 7.5 - 7.57 (m, 4H), 7.69 (t, J = 8 Hz, 1H), 7.86 - 7.93 (m, 2H), 7.99 (s, 1H); LC-MS: m / z = 498.1 (M + 1).

[0447] Example 58

[0448] 3 - ((1 - (4 - aminobutyl) - 2,4 - dioxo - 3 - (4 - (trifluoromethyl)benzyl) - 1,2,3,4,7,8 - hexahydropyrido[4,3 - d]pyrimidin - 6(5H) - yl)methyl)benzonitrile

[0449] JPEG0007709379000122.jpg35150

[0450] Example 58 was prepared in the same manner as Example 57.

[0451] 1HNMR(400MHz,CD3OD)δ 1.7(s,4H),2.95(s,2H),3.16(s,2H),3.64(s,2H),3.9(s,2H),4.03(s,2H),4.59(s,2H),5.15(s,2H),7.49-7.57(m,4H),7.67-7.7(m,1H),7.88 (t,J=8Hz,2H),7.98(s,1H);LC-MS:m / z=512.2(M+1).

[0452] Example 59

[0453] 3-((1-(4-Aminobutyl)-3-(4-chlorobenzyl)-2,4-dioxo-1,2,3,4,7,8-hexahydropyrido[4,3-d]pyrimidin-6(5H)-yl)methyl)benzonitrile

[0454] JPEG0007709379000123.jpg36150

[0455] Example 59 is prepared in the same manner as Example 57.

[0456] 1 HNMR(400MHz,CD3OD)δ 1.72(s,4H),2.98-2.99(d,2H),3.15-3.17(d,2H),3.61(t,J=5.6Hz,2H),3.91-3.93(d,2H),4.01(s,2H),4.57(s,2H),5.08(s,2H),7.28-7.3(d,2H),7.35-7.37(d,2H),7.71(t,J=7.6Hz,1H),7.9-7.92(d,2H),7.99(s,1H).

[0457] Example 60

[0458] 11-Benzyl-7-[(2,4-difluorophenyl)methyl]-2,5,7,11-tetraazatricyclo[7.4.0.0 2,6 trideca-1(9),5-dien-8-one

[0459] JPEG0007709379000124.jpg30150

[0460] Example 60 was prepared as described in WO2018 031987.

[0461] Example 61

[0462] 3-({3-[(4-chlorophenyl)methyl]-2-methyl-4-oxo-3H,4H,5H,6H,7H,8H-pyrimidin-6-yl}methyl)benzonitrile

[0463] JPEG0007709379000125.jpg27150

[0464] The synthesis of Example 61 was carried out according to the following scheme.

[0465] JPEG0007709379000126.jpg30150

[0466] To a 10 mL three-necked flask were charged SS26 (0.4 mmol), acetamidine hydrochloride (0.4 mmol), methanol (3 mL) and K2CO3 (1.2 mmol). The mixture was refluxed for 12 - 15 h. It was confirmed by LC-MS that the reaction was complete. The reaction mixture was cooled to room temperature and half of the solvent was removed under vacuum. Water (2 mL) was added dropwise. The precipitated white solid was filtered and washed with water. This solid was dried under vacuum to obtain INT4 (yield 72%).

[0467] To a 10 mL three-necked flask were charged INT4 (0.4 mmol), 1-(bromomethyl)-4-chlorobenzene (0.4 mmol), THF (3 mL) and Cs2CO3 (1.2 mmol). The mixture was refluxed for 12 - 15 h. It was confirmed by LC-MS that the reaction was complete. This solution was washed with water (100 mL × 2) and borane (100 mL × 1). The combined organic layers were dried over Na2SO4 and purified by silica gel column to obtain Example 61 (yield 30%).

[0468] 1HNMR (400 MHz, CDOD3) δ 7.78 (s, 1H), 7.72 - 7.74 (d, J = 8 Hz, 1H), 7.65 - 7.67 (d, J = 8 Hz, 1H), 7.54 (t, J = 8 Hz, 1H), 7.34 - 7.36 (d, J = 8 Hz, 2H), 7.17 - 7.19 (d, J = 8 Hz, 2H), 5.32 (s, 2H), 3.81 (s, 2H), 3.41 (s, 2H), 2.81 (t, J = 6 Hz, 2H), 2.74 (t, J = 5.2 Hz, 2H), 2.46 (s, 3H); LC-MS: m / z = 404.9 (M).

[0469] Example 62 (TR98)

[0470] 3 - [(8 - oxo - 9 - {methyl} - 1,5,9,11 - tetraazatricyclo[8.4.0.0 2,7 tetradeca - 2(7),10 - diene - 5 - yl)methyl]benzonitrile

[0471] JPEG0007709379000127.jpg25150

[0472] Example 62 was prepared using the following scheme.

[0473] JPEG0007709379000128.jpg26150JPEG0007709379000129.jpg29150

[0474] Imidazolidine - 2 - thione (59.8 mmol) INT5 was dissolved in methanol (70 mL), and CH3I (89.7 mmol) was added dropwise at 25 °C. After refluxing for 30 minutes, the solvent was removed under vacuum. The residue was suspended in MTBE (50 mL) and filtered. The solid was dried under vacuum to obtain INT6 (yield 83%) as a white solid.

[0475] Compound INT6 (2 mmol) and ((4-trifluoromethyl)phenyl)methylamine (4.2 mmol) were dissolved in dioxane (5 mL). The mixture was refluxed for 12 hours. It was confirmed by LC-MS that the reaction was complete. The solvent was removed, and the residue was suspended in toluene for 12 hours. The suspension was filtered, and the filtered cake was dried under vacuum to obtain Compound INT7.

[0476] Into a 10 mL three-necked flask, Compound INT7 (0.4 mmol), SS26 (0.4 mmol), methanol (3 mL) and MeONa (1.2 mmol) were charged. The mixture was refluxed for 12 - 15 hours. It was confirmed by LC-MS that the reaction was complete. The reaction was cooled to room temperature. Half of the solvent was removed under vacuum. Water (2 mL) was added dropwise. The precipitated white solid was filtered and washed with water. The solid was dried under vacuum to obtain Example 62 (yield 25%).

[0477] 1 1H-NMR (400 MHz, CD3OD): δ 7.64 - 7.77 (m, 4H), 7.52 - 7.57 (m, 2H), 7.38 - 7.45 (m, 2H), 5.25 (s, 1H), 5.20 (s, 1H), 3.72 - 3.88 (m, 4H), 3.42 (s, 2H), 3.26 (s, 2H), 2.57 - 2.76 (m, 4H), 1.86 - 1.91 (m, 2H).

[0478] LCMS [Mobile phase: From 20% water (0.05% NH3.H2O) and 80% CH3CN (0.05% NH3.H2O) to 5% water (0.05% NH3.H2O) and 95% CH3CN (0.05% NH3.H2O) in 6.0 minutes (linear gradient, C18 (50 mm, 5 micron, 1 micron injection) column), at 0.5 ml / min under these conditions.] The purity was 97.5%, Rt = 3.6 minutes; MS Calcd.: 479.5. MS Found: 480.1 [M + 1] + )。

[0479] Example 63

[0480] N-[(4-Chlorophenyl)methyl]-5-[(3-cyanophenyl)methyl]-1,3,4-oxadiazole-2-carboxamide

[0481] JPEG0007709379000130.jpg30150

[0482] Example 63 is prepared according to the following synthetic scheme.

[0483] JPEG0007709379000131.jpg51150

[0484] 1 HNMR(400MHz,DMSO_d6):δ 9.83(s,1H),7.72-7.87(m,3H),7.6(t,J=8Hz,1H),7.38(t,J=7.2Hz,4H),4.44(t,J=4.8Hz,4H);LC-MS:m / z=352.9(M+)

[0485] Example 64

[0486] 7-[(4-Chlorophenyl)methyl]-11-[(3-oxo-2,3-dihydro-1H-inden-5-yl)methyl]-2,5,7,11-tetraazatricyclo[7.4.0.0 2,6 trideca-1(9),5-dien-8-one

[0487] JPEG0007709379000132.jpg25150

[0488] Example 64 is prepared according to the following synthetic scheme.

[0489] JPEG0007709379000133.jpg57150

[0490] 1HNMR(400MHz,CDCl3)δ 2.46(s,3H),2.75 - 7.92(m,5H),3.05(s,1H),3.43 - 3.46(d,J = 12Hz,1H),3.62 - 3.66(d,J = 16Hz,1H),4.07(s,2H),4.21(s,2H),4.99(s,1H),5.21(s,2H),7.29(s,2H),7.33 - 7.35(d,J = 8Hz,2H),7.53 - 7.55(d,J = 8Hz,1H),7.63 - 7.64(d,J = 8Hz,2H);LC-MS:m / z = 460.9(M + 1).

[0491] Example 65

[0492] 3 - ({3 - [(4 - chlorophenyl)methyl]-4 - oxo - 3H,4H,5H,6H,7H,8H - pyrido[4,3 - d]pyrimidin - 6 - yl}methyl)benzonitrile

[0493] JPEG0007709379000134.jpg25150

[0494] Example 65 is prepared according to the following synthetic scheme.

[0495] JPEG0007709379000135.jpg29150

[0496] 1 HNMR(400MHz,CDCl3)δ 3.06(s,2H),3.42(s,2H),3.92(s,2H),4.35(s,2H),5.03(s,2H),7.24(s,2H),7.33 - 7.35(d,J = 8Hz,2H),7.6(t,J = 8Hz,1H),7.72 - 7.81(m,3H),8.14(s,1H);LC-MS:m / z = 390.9(M + 1)

[0497] Example 66(TR108)

[0498] 3 - ({8 - [(4 - chlorophenyl)methyl]-7 - oxo - 1,4,8,10 - tetraazatricyclo[7.3.0.0 2,6{Dodeca-2(6),9-dien-4-yl}methylbenzonitrile

[0499] JPEG0007709379000136.jpg28150

[0500] Example 66 is prepared according to the following synthetic scheme.

[0501] JPEG0007709379000137.jpg65150

[0502] 1 HNMR(400MHz,CDCl3)δ 3.72 - 3.98(m,10H),5.0(s,2H),7.24(s,1H),7.39 - 7.47(m,4H),7.57 - 7.59(d,J = 8Hz,2H),7.66(s,1H);LC-MS:m / z = 418(M + 1).

[0503] Example 67 (TR109)

[0504] 3 - [(5 - oxo - 4 - {[4 - (trifluoromethyl)phenyl]methyl}-1H,2H,4H,5H,6H,7H,8H,9H - imidazo[1,2 - a]quinazolin - 7 - yl)methyl]benzonitrile

[0505] JPEG0007709379000138.jpg28150

[0506] Example 67 is prepared according to the following synthetic scheme.

[0507] JPEG0007709379000139.jpg53150

[0508] 1 HNMR(400MHz,CDCl3)δ 1.36 - 1.4(m,1H),1.8 - 1.95(m,3H),2.37 - 2.75(m,5H),3.87 - 3.97(m,4H),5.1(s,2H),7.39 - 7.57(m,8H);LC-MS:m / z = 465(M + 1).

[0509] Example 68 (TR122)

[0510] 3-({4-[(4-chlorophenyl)methyl]-5-oxo-1H,2H,4H,5H,6H,7H,8H,9H-imidazo[1,2-a]quinazolin-7-yl}methyl)benzonitrile JPEG0007709379000140.jpg28150

[0511] Example 68 is prepared by the series of syntheses described in Example 67.

[0512] 1 HNMR (400 MHz, CDCl3) δ 1.32 - 1.42 (m, 1H), 1.81 - 1.94 (m, 3H), 2.31 - 2.74 (m, 5H), 3.86 - 3.96 (m, 4H), 5.01 (s, 2H), 7.25 (t, J = 5.6 Hz, 2H), 7.37 - 7.45 (m, 5H), 7.51 (t, J = 4 Hz, 1H); LC-MS: m / z = 431 (M+1).

[0513] Example 69

[0514] 3-({3-[(4-chlorophenyl)methyl]-2-methyl-4-oxo-3H,4H,5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-yl}methyl)benzonitrile

[0515] JPEG0007709379000141.jpg27150

[0516] Example 69 is prepared by the following synthetic scheme.

[0517] JPEG0007709379000142.jpg61150

[0518] 1HNMR (400 MHz, CDCl3) δ 2.51 (s, 3H), 4.46 - 4.48 (ss, 6H), 5.26 (s, 2H), 7.11 - 7.13 (d, J = 8 Hz, 2H), 7.33 - 7.35 (d, J = 8 Hz, 2H), 7.63 (t, J = 8 Hz, 1H), 7.74 - 7.79 (m, 2H), 7.85 - 7.87 (d, J = 8 Hz, 1H); LC-MS: m / z = 390.9 (M + 1).

[0519] Example 70

[0520] 3 - ({9 - [(4 - chlorophenyl)methyl] - 13,13 - dimethyl - 8 - oxo - 1,5,9,11 - tetraazatricyclo[8.4.0.0 2,7 tetradeca - 2(7),10 - diene - 5 - yl}methyl)benzonitrile

[0521] JPEG0007709379000143.jpg28150

[0522] Example 70 is prepared according to the following synthetic scheme.

[0523] JPEG0007709379000144.jpg49150

[0524] 1 HNMR (400 MHz, CDCl3) δ 1.03 (s, 6H), 2.98 (s, 2H), 3.1 - 3.17 (m, 4H), 3.59 - 3.68 (m, 4H), 3.75 (s, 2H), 4.15 (s, 2H), 5.25 (s, 2H), 7.28 - 7.3 (d, J = 8 Hz, 2H), 7.40 - 7.42 (d, J = 8 Hz, 2H), 7.65 (t, J = 8 Hz, 1H), 7.8 - 7.82 (d, J = 8 Hz, 1H), 7.86 - 7.88 (d, J = 8 Hz, 1H), 7.93 (s 1H); LC-MS: m / z = 473.9 (M + 1).

[0525] Example 71

[0526] 3-({9-[(4-chlorophenyl)methyl]-13,13-difluoro-8-oxo-1,5,9,11-tetraazatricyclo[8.4.0.0 2,7 tetradeca-2(7),10-dien-5-yl}methyl)benzonitrile

[0527] JPEG0007709379000145.jpg27147

[0528] Example 71 is prepared according to the following synthetic scheme.

[0529] JPEG0007709379000146.jpg50150

[0530] 1 HNMR(400MHz,CDCl3)δ 2.94(s,2H),3.58 - 3.75(m,8H),4.31(s,2H),5.17(s,2H),7.05 - 7.07(d,J = 8Hz,1H),7.26 - 7.33(m,3H),7.59 - 7.79(m,4H); LC-MS: m / z = 481.9(M+1).

[0531] Example 72

[0532] 3-({3-[(4-bromophenyl)methyl]-2-methyl-4-oxo-3H,4H,5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-6-yl}methyl)benzonitrile

[0533] JPEG0007709379000147.jpg24150

[0534] Example 72 is prepared according to the following synthetic scheme. JPEG0007709379000148.jpg25150

[0535] 11H NMR (400 MHz, CDCl3) δ 2.04 (s, 3H), 2.43 (s, 4H), 3.46 (s, 2H), 3.75 (s, 2H), 5.21 (s, 2H), 7.05 - 7.07 (d, J = 8 Hz, 2H), 7.42 - 7.47 (m, 3H), 7.56 - 7.61 (m, 2H), 7.7 (s, 1H); LC-MS: m / z = 450.9 (M+1).

[0536] Example 73

[0537] 3-[(2-Methyl-4-oxo-3-{[4-(trifluoromethyl)phenyl]methyl}-3H,4H,5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-6-yl)methyl]benzonitrile

[0538] JPEG0007709379000149.jpg24150

[0539] Example 73 is prepared by the same synthetic route as described in Example 72.

[0540] LC-MS: m / z = 439.0 (M+1) and retention time 1.743 min.

[0541] Example 74

[0542] 3-({3-[(4-Bromophenyl)methyl]-4-oxo-3H,4H,5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-6-yl}methyl)benzonitrile

[0543] JPEG0007709379000150.jpg25150

[0544] Example 74 is prepared according to the scheme described in Example 65.

[0545] 1HNMR(400MHz,CDCl3)δ 2.73 - 2.79(m,4H),3.46(s,2H),3.75(s,2H),5.03(s,2H),7.21 - 7.23(d,2H),7.43 - 7.5(m,3H),7.59(t,J = 8.8Hz,2H),7.7(s,1H),8.06(s,1H);LC - MS:m / z = 434.1(M + 2).

[0546] Example 75

[0547] 3 - [(4 - Oxo - 3 - {[4 - (trifluoromethyl)phenyl]methyl}-3H,4H,5H,6H,7H,8H - pyrido[4,3 - d]pyrimidin - 6 - yl)methyl]benzonitrile

[0548] JPEG0007709379000151.jpg23150

[0549] Example 75 is prepared according to the scheme described in Example 65.

[0550] 1 HNMR(400MHz,CDCl3)δ 3.09(s,2H),3.45(s,2H),3.95(s,2H),4.37(s,2H),5.13(s,2H),7.43 - 7.75(d,2H),7.59 - 7.65(m,3H),7.75 - 7.82(m,3H),8.18(s,1H);LC - MS:m / z = 424.2(M).

[0551] Example 76

[0552] 3 - ({8 - [(4 - Bromophenyl)methyl]-7 - oxo - 1,4,8,10 - tetraazatricyclo[7.3.0.0 2,6}dodeca - 2(6),9 - diene - 4 - yl}methyl)benzonitrile

[0553] JPEG0007709379000152.jpg26150

[0554] Example 76 is prepared according to the following synthetic scheme.

[0555] JPEG0007709379000153.jpg58150

[0556] 1 HNMR (400 MHz, CDCl3) δ 4.1 - 4.3 (m, 10H), 5.19 (s, 2H), 7.25 (s, 1H), 7.27 (s, 1H), 7.43 - 7.45 (d, 2H), 7.53 (t, J = 7.6 Hz, 1H), 7.66 - 7.72 (m, 3H); LC-MS: m / z = 463.8 (M + 2).

[0557] Example 77

[0558] 3 - [(7 - oxo - 8 - {[4 - (trifluoromethyl)phenyl]methyl}-1,4,8,10 - tetraazatricyclo[7.3.0.0 2,6 dodeca - 2(6),9 - diene - 4 - yl)methyl]benzonitrile

[0559] JPEG0007709379000154.jpg27150

[0560] Example 77 is prepared according to the synthetic scheme described in Example 76.

[0561] 1 HNMR (400 MHz, CDCl3) δ 4.06 - 4.15 (m, 8H), 4.28 (t, J = 8.4 Hz, 2H), 5.33 (s, 2H), 7.52 - 7.61 (m, 5H), 7.66 - 7.68 (d, 2H), 7.72 (s, 1H); LC-MS: m / z = 451.9 (M).

[0562] Example 78

[0563] 2 - [(4 - (bromophenyl)methyl]-7 - {[3 - (prop - 1 - yn - 1 - yl)phenyl]methyl}-1,2,5,6,7,8 - hexahydro - 2,7 - naphthyridin - 1 - one

[0564] JPEG0007709379000155.jpg25150

[0565] Example 78 is prepared according to the following synthetic scheme.

[0566] JPEG0007709379000156.jpg23150JPEG0007709379000157.jpg49150

[0567] 1 HNMR(400MHz,DMSO_d6)δ 1.97(s,3H),2.91(s,2H),3.32 - 3.36(m,1H),3.62 - 3.65(m,1H),3.91(s,2H),4.46(s,2H),5.08(s,2H),7.29 - 7.6(m,8H),8.71(s,1H);LC - MS:m / z = 449.8(M + 2).

[0568] Example 79

[0569] 7 - {[3 - (prop - 1 - yn - 1 - yl)phenyl]methyl}-2 - {[4 - (trifluoromethyl)phenyl]methyl}-1,2,5,6,7,8 - hexahydro - 2,7 - naphthyridin - 1 - one

[0570] JPEG0007709379000158.jpg22150

[0571] Example 79 is prepared using the synthetic scheme described in Example 78.

[0572] 1 HNMR(400MHz,DMSO_d6)δ 2.06(s,3H),2.92(s,2H),3.29 - 3.36(m,1H),3.62 - 3.65(m,1H),3.93(s,2H),4.46(s,2H),5.2(s,2H),7.44 - 7.81(m,8H),8.75(s,1H);LC - MS:m / z = 437.9(M).

[0573] Example 80

[0574] 4-Benzyl-8-[(4-chlorophenyl)methyl]1,4,8,10-tetraazatricyclo[7.3.0.0 2,6 dodeca-2(6),9-dien-7-one

[0575] JPEG0007709379000159.jpg27150

[0576] Example 80 is prepared according to the following synthetic scheme.

[0577] JPEG0007709379000160.jpg57150

[0578] LC-MS: Retention time: 1.546 min, m / z = 393.1 (M+1). For conditions, see Figure 8 and Example 62.

[0579] Example 81

[0580] 4-Benzyl-8-[(4-(bromophenyl)methyl]1,4,8,10-tetraazatricyclo[7.3.0.0 2,6 dodeca-2(6),9-dien-7-one

[0581] JPEG0007709379000161.jpg27150

[0582] Example 81 is prepared using the synthetic scheme described in Example 80.

[0583] 1 HNMR(400MHz,CDCl3)δ 4.0(s,2H),4.2 - 4.24(d,6H),4.4(s,2H),5.15(s,2H),7.23 - 7.24(d,2H),7.42(s,7H); LC-MS: m / z = 439.1 (M+2).

[0584] Example 82

[0585] 4-Benzyl-8-{[4-(trifluoromethyl)phenyl]methyl}-1,4,8,10-tetraazatricyclo[7.3.0.0 2,6 dodeca-2(6),9-dien-7-one

[0586] JPEG0007709379000162.jpg28150

[0587] Example 82 is prepared using the synthetic scheme described in Example 80.

[0588] 1 HNMR(400MHz,CDCl3)δ 4.01(s,2H),4.21-4.25(d,6H),4.41(s,2H),5.26(s,2H),7.37-7.46(m,7H),7.54-7.56(d,2H);LC-MS:m / z=426.9(M).

[0589] Example 83

[0590] 3-({9-[(4-chlorophenyl)methyl]-8-oxo-1,5,9,11-tetraazatricyclo[8.4.0.0 2,7 tetradeca-2(7),10-dien-5-yl}methyl)benzonitrile

[0591] JPEG0007709379000163.jpg29150

[0592] Example 83 is prepared using the synthetic scheme described in Example 62.

[0593] 1 HNMR(400MHz,DMSO&CDCl3)2.13(s,2H),2.86(s,4H),3.38(s,2H),3.5(s,2H),3.84(s,2H),4.05(s,2H),5.28(s,2H),7.27-7.34(m,3H),7.53(t,J=8Hz,1H),7.65-7.67(d,2H),7.74(s,1H),8.0(s,1H);LC-MS:m / z=446.1(M+1).

[0594] Example 84

[0595] 3-({9-[(4-(Bromophenyl)methyl]-8-oxo-1,5,9,11-tetraazatricyclo[8.4.0.0 2,7 tetradeca-2(7),10-dien-5-yl}methyl)benzonitrile

[0596] JPEG0007709379000164.jpg28150

[0597] Example 84 is prepared using the synthetic scheme described in Example 62.

[0598] 1 HNMR(400MHz,DMSO)2.05(s,2H),2.87(s,4H),3.36 - 3.43(m,4H),3.89(s,2H),3.99(t,J = 5.6Hz,2H),5.16(s,2H),7.22 - 7.24(d,2H),7.54 - 7.62(m,3H),7.71 - 7.73(d,1H),7.8 - 7.83(d,2H);LC-MS:m / z = 492.1(M + 2).

[0599] Biological examples and experiments

[0600] The following examples are set forth to provide those skilled in the art with a complete disclosure and description of the methods of manufacture and use of the invention and are not intended to limit the scope of the invention. Efforts have been made to ensure the accuracy of the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account.

[0601] a) Experimental procedures / materials and methods Measurement of human ClpP activityMeasurement of the in vitro activity of recombinant human caseinolytic peptidase hClpP (Cat#MBS204060, MyBioSource, Boston USA) based on monitoring the release of fluorescent coumarin from the fluorogenic substrate Ac-WLA-AMC (Cat#S330, Boston Biochem, Inc., Cambridge, MA), following with slight modifications the previous descriptions (Maurizi, M.R. et al, Methods Enzymol. 1994, 244, 314 - 331 and the references cited therein, and Woo, K.M. et al, Biol. Chem. 1989, 264, 2088 - 2091 and the references cited therein). Briefly, the activity of the recombinant hClpP proteolytic subunit (1 μg / mL) was measured using 10 μM of the fluorogenic Ac-WLA-AMC substrate in an assay buffer composed of 50 mM Tris, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 4 mM ATP, 0.02% Triton X-100 and 5% glycerol, pH 8.0 (HCl), as described in the above references. Two different protocols were used to examine the effects of ONC201 and the compounds of the present invention on ClpP activity. Using the first protocol (Protocol 1), the reaction was initiated by immediately mixing the enzyme and the substrate in the presence of the indicated doses of the compound. Applying the second protocol (Protocol 2), the enzyme and the compound were mixed and incubated in the assay buffer for 60 min before adding the Ac-WLA-ACM substrate to initiate the reaction. The kinetics of the free coumarin fluorescence was monitored using a black μ-CLEAR 96-well flat bottom plate (Cat#655090, Greiner Germany), and the fluorescence of the free coumarin was recorded at 350 nm excitation and 460 nm emission using a BMR PHERA star plate reader equipped with an appropriate FI module (BMG LABTECH, Durham NC). The time gradient of the linear portion of the fluorescence signal was a measure of the activity of hClpP. The measurements were performed in triplicate and shown as the rate of fluorescence change at a given concentration of hClpP and substrate in the presence or absence of ONC201 or the compounds of the present invention. The dose-dependence of hClpP activation by different compounds was determined for the substrate (relative IC 50) It was used for , and the activity of the sample treated with DMSO (vehicle) measured as background was subtracted from the experimental data, and the activity of ClpP was expressed as RFU / μg of ClpP / h. See also Greer, Y.E. et al, Oncotarget, 2018, 9, 18, 454 - 18479 and the references cited therein.

[0602] cancer Cell lines The cell data described in Tables 1 and 2 were determined as described in CN104860948 and US10,526,332. Further information for the cell tests is as follows: HCT116 (human colon cancer) or MDA - MB - 231 (MDA231, human breast adenocarcinoma) was dispensed into 100 μl of cell suspension in a 96 - well plate. The plate was incubated in a humidified incubator (37 °C, 5% CO2) for 24 hours. The compound of the present invention was added to the medium of the plate at an appropriate test concentration. The plate was incubated for 48 hours. CCK - 8 (10 μl, see below) was added to each well. The plate was incubated for 1 - 4 hours under the above conditions, and the absorbance at 450 nm and 650 nm was measured with a plate reader.

[0603] Cell Counting Kit - 8 (CCK - 8) enables a sensitive colorimetric assay for the determination of the number of viable cells in proliferation and cytotoxicity assays. Cell counting with CCK - 8 was performed using WST - 8 (2 - (2 - methoxy - 4 - nitrophenyl) - 3 - (4 - nitrophenyl) - 5 - (2,4 - disulfophenyl) - 2H - tetrazolium, monosodium salt), which produces a water - soluble formazan dye by bioreduction in the presence of the electron carrier 1 - methoxy PMS. The CCK - 8 solution was added directly to the cells. WST - 8 is bioreduced by cellular dehydrogenases to form an orange - colored formazan product soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of live cells.

[0604] Measurement of antibacterial activity. Several publications have described the testing of ClpP modulators for antibacterial activity (Kao, Y. T. et al, PNAS 2018, 115, 8003 - 8008 and the references included therein, and Quellette S. P. et al, J. Bacteriol 2018, 201(2) pii: e00635 - 18, doi: 10.1128 / JB.00635 - 18 and the references cited therein. The experimental conditions described by Kao, Y. T. et al and Quellette S. P. et al can be used to measure the antibacterial effect of the compounds of the present invention, including the activity against Staphylococcus aureus.)

[0605] b) Results

[0606] ONC201 and TR compounds activate CLPP peptidase activityTo examine the effects of ONC201 and the compounds of the present invention on ClpP activity, their effects on the enzymatic activity of isolated human hClpP were tested. Using purified recombinant human mitochondrial ClpP proteolytic subunit (Cat#MBS204060, MyBioSource, Cambridge, MA) and the selective fluorogenic 7-amino-methylcoumarin-linked tripeptide Ac-WLA-AMC (Cat#S330, MyBioSource, Cambridge, MA), hClpP peptidase activity was measured in the presence or absence of ONC201 and TR compounds. The enzymatic activity of hClpP was measured in assay buffer (described in the experimental procedures / materials and methods), and the fluorescence levels of the released coumarin were continuously monitored. As shown in Figure 1, we observed that when hClpP was incubated with ONC201 or a selected TR compound (TR-57), the fluorescence of coumarin AMC released due to hClpP peptidase activity increased in a time-dependent and exponential manner. However, when the recombinant hClpP proteolytic subunit was pre-incubated for 60 minutes in standard assay buffer with the selected compounds, the activity of the enzyme increased continuously, the coumarin release rate became linear over time, and examples of the kinetic and dose-dependent activity changes of hClpP for ONC201 and TR57 shown in Figure 2 were obtained. By plotting the dose-dependence of hClpP activity against the concentration of the compound on a semi-logarithmic scale, the concentration of the agent that increases the activity of pre-incubated hClpP by 50% is the IC 50 can be determined (Figure 3).

[0607] The biological activities against human cancer cells for the selected examples are shown in Tables 1 and 2.

[0608] Table 1: Biological activity data for selected analogs against human cancer cells JPEG0007709379000165.jpg150139

[0609] Table 2: Biological activity data for selected analogs against human cancer cells JPEG0007709379000166.jpg202159JPEG0007709379000167.jpg150162

[0610] List of abbreviations A549: Human non-small cell lung cancer cell line BSA: Bovine serum albumin ClpP: Caseinolytic protease P DMSO: Dimethyl sulfoxide DNA: Deoxyribonucleic acid EDTA: Ethylenediaminetetraacetic acid ELISA: Enzyme-linked immunosorbent assay FACS: Fluorescence-activated cell sorting HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid HsClpP: Human mitochondrial ClpP HsClpX: AAA+ protein unfoldase HsClpXP: ATP-dependent protease complex in the mitochondrial matrix IHC: Immunohistochemistry MAB: Monoclonal antibody mRNA: Messenger ribonucleic acid PBS: Phosphate-buffered saline RPMI-1640: Cell culture medium used for culturing transformed and non-transformed eukaryotic cells and cell lines siRNA: Small interfering ribonucleic acid TR compound or TR compounds: Any compound or set of compounds described herein using a nomenclature starting with TR. For example, TR57.

[0611] Amino acid sequence Protein: ClpP Organism: Homo sapiens (sp|Q16740|CLPP_HUMAN ATP-dependent Clp protease proteolytic subunit, mitochondrial OS=Homo sapiens OX=9606 GN=CLPP PE=1 SV=1) (SEQ ID NO: 1) MWPGILVGGARVASCRYPALGPRLAAHFPAQRPPQRTLQNGLALQRCLHATATRALPLIP IVVEQTGRGERAYDIYSRLLRERIVCVMGPIDDSVASLVIAQLLFLQSESNKKPIHMYIN SPGGVVTAGLAIYDTMQYILNPICTWCVGQAASMGSLLLAAGTPGMRHSLPNSRIMIHQP SGGARGQATDIAIQAEEIMKLKKQLYNIYAKHTKQSLQVIESAMERDRYMSPMEAQEFGI LDKVLVHPPQDGEDEPTLVQKEPVEAAPAAEPVPAST

Claims

1. A compound of formula I, Z1-Q Formula I or a pharmaceutically acceptable salt thereof, wherein Z1 is and Q is independently selected from the group consisting of Q3 and Q5 ; Z2 is and Ar1 is independently selected from aryl, heteroaryl, thiophenyl and phenyl, and Ar1 can be optionally substituted with 1 to 3 J groups; Ar1 can be optionally substituted with 1 to 3 J groups; Ar2 is independently selected from thiophenyl and phenyl and is substituted with 1 to 3 JJ groups; J is halogen, -CN, optionally substituted alkyl having 1 to 6 carbon atoms, optionally substituted cycloalkyl having 3 to 9 carbon atoms, cycloalkyl having 3 to 9 carbon atoms (alkyl having 1 to 6 carbon atoms), haloalkyl having 1 to 6 carbon atoms, -CF ３ , -NH ２ , -NO ２ , -SH, -SR15, -OH, optionally substituted alkoxy having 1 to 6 carbon atoms, -NR17R18, substituted cycloalkyl having 3 to 9 carbon atoms (alkyl having 1 to 6 carbon atoms), cycloalkyl having 3 to 9 carbon atoms (alkynyl having 2 to 6 carbon atoms), cycloalkenyl having 4 to 8 carbon atoms, cycloalkenyl having 4 to 8 carbon atoms (alkyl having 1 to 6 carbon atoms), aryl, heteroaryl, heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, -C(O)OH, -C(O)OR15, -OC(O)OR15, alkynyl having 2 to 6 carbon atoms, alkenyl having 2 to 8 carbon atoms, haloalkoxy having 1 to 6 carbon atoms, -S(O) ２ OR15, -SO ２ NR17R18, -S(O) ２ R15, -NR15S(O) ２ R16, -C(O)NR17R18, -C(O)R15 and -NR15C(O)R16, and is independently selected from JJ is selected independently from halogen, -CN, (C1-C6) haloalkyl, (C1-C6) optionally substituted alkyl, -CF ３ , -NH ２ , -NO ２ , -SH, -SR15, -OH, (C1-C6) optionally substituted alkoxy, -NR17R18, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O) ２ OR15, -SO ２ NR17R18, -S(O) ２ R15, -NR15S(O) ２ R16, -C(O)NR17R18, -C(O)R15 and -NR15C(O)R16, R1, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, halogen, -OH and (C1-C3) optionally substituted alkyl; R2 and R3 are hydrogen; R5 and R6 together can form =O; R7 and R8 together can form =O; R14 is independently selected from hydrogen, halogen, optionally substituted (C1-C6) alkyl, (C3-C6) cycloalkyl, (C1-C6) haloalkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, -CN, -S(O) ２ R15, -NR17R18, -S(O) ２ R15, -C(NH)NH ２ , -C(O)R15, and -C(O)OR15 R15, R16, R17, R18, and R28 are each independently selected from hydrogen and (C1-C6) optionally substituted alkyl; R17 and R18 together with the nitrogen to which they are attached can form a ring of 3 to 6 atoms; W4 is independently selected from the group consisting of =C(R14)- and nitrogen; A is independently selected from the group consisting of SS and ; G is independently selected from the group consisting of SS and ; M is independently selected from the group consisting of SS and ; E is independently selected from the group consisting of a single bond, SS, and ; SS is and is independently selected from the group consisting of; R20, R21, R26 and R27 are each independently selected from the group consisting of hydrogen, halogen and (C1-C6) optionally substituted alkyl; R22, R23, R24, and R25 are each independently selected from the group consisting of hydrogen, halogen, -CN, optionally substituted (C1-C6) alkyl, optionally substituted (C3-C9) cycloalkyl, (C3-C9) cycloalkyl(C1-C6)alkyl, (C1-C6) haloalkyl, -NH ２ , -NO ２ , -SH, -SR15, -OH, optionally substituted (C1-C6) alkoxy, -NR17R18, substituted (C3-C9) cycloalkyl(C1-C6)alkyl, (C3-C9) cycloalkyl(C2-C6) alkynyl, (C4-C8) cycloalkenyl, (C4-C8) cycloalkenyl(C1-C6)alkyl, aryl, heteroaryl, -C(O)OH, -C(O)OR15, -OC(O)OR15, (C2-C6) alkynyl, (C2-C8) alkenyl, (C1-C6) haloalkoxy, -S(O) ２ OR15, -SO ２ NR17R18, -S(O) ２ R15, -NR15S(O) ２ R16, -C(O)NR17R18, -C(O)R15, and -NR15C(O)R16 R22 and R23 together with the carbon to which they are attached can form a non-aromatic ring having 3 to 6 carbon atoms; R22 and R23 together with the carbon to which they are attached can form a non-aromatic ring having 1 to 2 oxygen atoms; R24 and R25 together with the carbon to which they are attached can form a non-aromatic ring having 1 to 2 oxygen atoms; R24 and R25 together with the carbon to which they are attached can form a non-aromatic ring having 3 to 6 carbon atoms; R30 and R31 are each independently selected from the group consisting of hydrogen and (C1-C6) optionally substituted alkyl, the compound or a pharmaceutically acceptable salt thereof.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein Q is Q3.

3. R1, R4, R5, R6, R7 and R8 are each hydrogen, The compound or a pharmaceutically acceptable salt thereof according to claim 2, wherein Ar1 and Ar2 are each phenyl.

4. W4 is nitrogen, J is independently selected from halogen, -CN, optionally substituted alkyl having 1 to 6 carbon atoms, optionally substituted cycloalkyl having 3 to 9 carbon atoms, haloalkyl having 1 to 6 carbon atoms, -CF ３ , optionally substituted alkoxy having 1 to 6 carbon atoms, -NR17R18, optionally substituted heterocyclyl, alkynyl having 2 to 6 carbon atoms, alkenyl having 2 to 8 carbon atoms, and haloalkyloxy having 1 to 6 carbon atoms; JJ is independently selected from halogen, -CN, optionally substituted alkyl having 1 to 6 carbon atoms, optionally substituted cycloalkyl having 1 to 6 carbon atoms, -CF ３ , haloalkyl having 1 to 6 carbon atoms, optionally substituted alkoxy having 1 to 6 carbon atoms, alkynyl having 2 to 6 carbon atoms, alkenyl having 2 to 8 carbon atoms, and haloalkyloxy having 1 to 6 carbon atoms, The compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein R14 is independently selected from hydrogen, halogen, optionally substituted (C1-C6) alkyl, and -CN.

5. J is independently selected from halogen, -CN, and (C2-C6) alkynyl, JJ is selected independently from halogen, -CF ３ and (C1-C6) haloalkyl, the compound according to claim 4 or a pharmaceutically acceptable salt thereof.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 5, wherein the compound is represented by the following formula.

7. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein Q is Q5.

8. R1, R4, R5, R6, R7, and R8 are hydrogen, A is where, M is where, The compound or a pharmaceutically acceptable salt thereof according to claim 7, wherein E is a single bond.

9. J is independently selected from halogen, -CN, optionally substituted alkyl having 1 to 6 carbon atoms, optionally substituted cycloalkyl having 3 to 9 carbon atoms, haloalkyl having 1 to 6 carbon atoms, -CF ３ , optionally substituted alkoxy having 1 to 6 carbon atoms, -NR17R18, optionally substituted heterocyclyl, alkynyl having 2 to 6 carbon atoms, alkenyl having 2 to 8 carbon atoms, and haloalkyloxy having 1 to 6 carbon atoms, JJ is independently selected from halogen, -CN, (C1-C6) haloalkyl, optionally substituted (C1-C6) alkyl, -CF ３ , optionally substituted (C1-C6) alkoxy, (C2-C6) alkynyl, (C2-C8) alkenyl, and (C1-C6) haloalkyloxy, the compound according to claim 8 or a pharmaceutically acceptable salt thereof.

10. J is independently selected from halogen, -CN, and (C2-C6) alkynyl, JJ is independently selected from halogen, -CF ３ , and (C1-C6) haloalkyl, the compound according to claim 9 or a pharmaceutically acceptable salt thereof.

11. The compound The compound or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of.

12. The compound The compound or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of.

13. The compound The compound or a pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of.

14. A compound according to any one of claims 1 to 11 for use in a method of treating cancer in a subject, said method comprising administering to the subject an effective amount of said compound or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier or excipient.

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