EA043367B1 - MODULATION OF G PROTEIN COUPLED RECEPTOR (GPCR) WITH IMIPRIDONES - Google Patents

Description

State of the art

There are many receptors on the surfaces of human cells. G protein-coupled receptors (GPCRs or GPCRs) form one of the largest families of transmembrane receptor proteins. There are approximately 30,000 genes in the human genome, and 1,000 of them encode GPCRs. GPCRs have been divided into five classes. The first class is the class A rhodopsin-like receptor or GPCR family, with 670 receptor proteins. The rhodopsin-like receptor family can interact with a variety of ligands, including amines (alpha group), peptides (beta group), lipid-like substances (gamma group), nucleotides and glycoproteins (delta group) and contains many receptors that are drug targets funds. The second class is the secretin family of receptors, which possess peptide hormone binding domains. Receptors in this family are associated with homeostasis and are increasingly becoming targets for drug development. The third class is the adhesion receptor family, characterized by a GPCR proteolytic site (GPS). The development of drugs targeting this family of GPCRs is not yet underway because they have different N-terminal fragments and little is known about their ligands. The fourth class is the glutamate receptor family, with 22 GPCR receptors identified to date. Relatively little is known about the specificity of each protein. The last class is the Frizzled/Taste2 family, which includes 10 Frizzled receptors, for which Wnt glycoproteins serve as ligands, 5 SMO (smoothened) receptors, which do not require ligands, and 25 Taste2 receptors, which are necessary for the perception of various tastes. Receptors, including GPCRs, are also classified based on the identification of endogenous ligands. Receptors bind to known endogenous compounds or are classified as orphan receptors whose endogenous ligands have not yet been identified.

GPCRs are found in a wide range of tissue and cell types and are associated with many different physiological mechanisms. They are activated by a wide range of ligands, for example, hormones such as thyroid stimulating hormone (TSH), adrenocorticotropic hormone, glucagon and vasopressin, amines such as 5-HT, acetylcholine (muscarinic AChR) and histamines, lipids such as LPA and S1P, and signal transmitters such as amino acids, Ca ²⁺ , nucleic acids, peptides and light. The wide distribution of GPCRs and the variety of roles they play indicate their importance in various pathological diseases. Indeed, GPCRs are involved in a variety of diseases, including bronchoconstriction, hypertension, diabetes, inflammation, cell death, hormonal disorders, cancer, neurotransmission, and behavioral disorders. For these reasons, GPCRs are an important area of pharmaceutical product development. Currently, approximately 360 GPCRs are considered available for drug development, of which 46 have already been used for this purpose. There are an estimated 150 orphan GPCRs (oGPCRs). In the field of drug development, cell membrane receptors act as selective sites for drug action and are responsible for 50% of all drug targets; drugs that modulate GPCR activity account for 30% of the most commonly used drugs in the top 100 drugs ($40 billion, 9% of the total drug market). Therefore, GPCRs are one of the most significant targets for the development of new drugs.

GPCRs share common structural features. They contain seven hydrophobic transmembrane domains, each 20–30 amino acids long, which are linked by hydrophilic amino acid sequences of varying lengths. The receptors have an extracellular N-terminus, while the C-terminus is located in the cytoplasm. GTP-binding proteins (G proteins) act as mediators that transmit signals to intracellular effectors that are generated by binding hormones or other chemical ligands that stimulate GPCRs. Upon binding to a ligand, the intracellular domain of the GPCR undergoes a conformational change to allow the receptor to interact with the G protein, which in turn activates intracellular signal transducers, such as adenylate cyclase, phospholipase C, or ion channels. This system generates a signaling cascade in which many secondary transmitters act in response to the binding of a single ligand to a GPCR. Cells use this mechanism to detect changes in the environment outside the cell and respond appropriately to these changes. In general, endogenous ligands activate receptors with a concomitant generation of a conformational change that allows the receptors to couple to G proteins. Recent protein interaction studies have shown that GPCRs associate with various proteins, such as GRK or SH2 (Src homology 2) domain-containing proteins and the adapter Grb2, as well as G protein to participate in signal transduction.

Under normal conditions, signaling produces the final outcome, which is cell activation or suppression. In a physiological environment, GPCRs are in equilibrium between their inactive and active states in the cell membrane. Inactive receptors cannot carry out a biological response in conjunction with cellular signaling pathways. Receptors carry out biological responses through cellular signaling pathways (via G proteins) only when they change structurally and enter an active form. The receptor can be stabilized in its active form by compounds such as endogenous ligands or drugs. Therefore, functional studies such as cloning of such gene families and identification of new ligands are of the same importance as the development of new drug candidates, i.e., siRNA, antibodies, polypeptides, effectors, inhibitors, agonists, antagonists.

Development, differentiation, homeostasis, responses to stimuli, cell cycle control, as well as aging and apoptosis of living organisms are mainly the result of the selective expression of certain genes in cells. This is also true for cellular mechanisms associated with diseases. In particular, pathological phenomena such as tumorigenesis are induced by gene mutations, which ultimately lead to changes in gene expression.

ONC201 (7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin5(1H)-one) is the founder of a class of antitumor compounds called imipridones, which is in phase II clinical trials against multiple advanced cancers. Since the discovery that ONC201 is a p53-independent inducer of TRAIL transcription, preclinical studies have shown that ONC201 has antiproliferative and proapoptotic effects on a wide range of tumor cells, but not normal cells. The mechanism of action of ONC201 involves PERK-independent activation of the integrated stress response, leading to up-regulation of DR5 and dual inactivation of Akt/ERK in the tumor, and subsequent activation of Foxo3a, leading to up-regulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animal models, produces sustained pharmacodynamic effects, and is not genotoxic. A first-in-human clinical study of ONC201 in advanced, aggressive, refractory solid tumors confirmed that the compound was well tolerated. Thus, the imipridone family, which includes ONC201 and its chemical analogues, represents a new class of therapeutic agents.

Brief description of the invention

In one aspect of the present invention there are provided compounds of formula (10) o

Ru R.·

Ν' Ν''

where R ₁ and R ₂ are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio and acyl radicals.

In one embodiment of the present invention, when R1 is CH ₂ Ph, R2 is not CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(2-CH ₃ -Ph) (ie ONC201). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH2-(2,4-di F-Ph) (ie ONC206). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(4-CF ₃ -Ph) (ie ONC212). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(3,4-diF-Ph) (ie ONC213). In one embodiment of the present invention, R1 is CH ₂ (3,4-di-Cl-Ph and R2 is CH ₂ -(4-CF ₃ -Ph) (i.e., ONC234). In one embodiment of the present invention R1 is CH ₂ -3-thienyl and R2 is CH ₂ -(4-CF ₃ -Ph) (ie ONC236).

In another aspect provided herein, methods are provided for treating or preventing a disease, disorder or condition in a subject in need thereof, comprising: administering to the subject in need of such treatment a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (10) or an analogue or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, said compound is selected from the group consisting of ONC201, ONC206, ONC212, ONC213, ONC234 and ONC236. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, said cancer is selected from a central nervous system tumor, a brain tumor, a peripheral nervous system tumor, pheochromocytoma, paraganglioma, neuroendocrine tumor, Ewing's sarcoma, pancreatic cancer, prostate cancer, endometrial cancer, hematologic malignancy, cancer bones and tumors of the lymphatic system. In one embodiment, said cancer is selected from meningioma, ependymoma, glioma, neuroblastoma, or diffuse brainstem glioma. In one embodiment of the present invention, said cancer is selected from acute leukemia, selected from acute lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome or myeloproliferative disease. In one embodiment of the present invention, said cancer has a histone H3 mutation (eg, the H3.3 K27M mutation) or an epigenetically silenced unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) gene. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment, said mental disorder is selected from psychosis, schizophrenia, bipolar disorder, or major depressive disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection).

In another aspect provided herein, methods are provided for treating or preventing a disease, disorder, or condition in a subject in need of selective modulation of the activity of a G protein-coupled receptor (GPCR) or G protein-coupled receptor (GPCR) signaling pathway. . Modulation includes, but is not limited to, agonism, partial agonism, reverse agonism, partial antagonism, antagonism, bivalent modulation, or bitopic modulation. In one embodiment of the present invention, these methods comprise administering to a subject in need of such treatment a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (10) or an analogue thereof or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment, said mental disorder is psychosis. In one embodiment, said mental disorder is schizophrenia. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the species Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection). In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, or combinations thereof. In one embodiment of the present invention, said GPCR is GPR132 (which is also called G2A). In one embodiment of the present invention, said GPCR is GPR91. In one embodiment of the present invention, said GPCR is MTNR1A. In one embodiment of the present invention, said GPCR is CXCR7.

In another aspect provided herein, methods are provided for treating or preventing a disease, disorder, or condition in a subject in need of selective modulation of the activity of a dopamine receptor or receptor of the dopamine receptor signaling pathway. In one embodiment of the present invention, these methods comprise administering to a subject in need of such treatment a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (10) or an analogue thereof or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment, said mental disorder is psychosis. In one embodiment, said mental disorder is schizophrenia. Cook in one

- 3 043367 ante implementation of the present invention, the specified subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection). In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family.

In another aspect provided herein, methods for treating or preventing liver fibrosis or liver tissue regeneration are described, comprising: administering to a subject in need of such treatment a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (10) or a compound of formula (100) ( for example, TIC-10), or an analogue thereof, or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, the compound is a CXCR7 agonist.

In another aspect provided herein, methods are provided for stimulating the immune system (e.g., activating natural killer cells, NK cells) in a subject in need thereof, comprising: administering to the subject a pharmaceutical composition containing a therapeutically effective amount of a compound of formula (10) or an analogue thereof or a pharmaceutically acceptable salt thereof. In one embodiment of the present invention, the compound is a GPR91 agonist. In one embodiment of the present invention, said compound is ONC213. In one embodiment of the present invention, said subject has cancer, and said method is a method of cancer immunotherapy. In one embodiment of the present invention, said subject has a viral infection (eg, HIV). In one embodiment of the present invention, said subject has systemic lupus erythematosus. In one embodiment of the present invention, said method further comprises administering a vaccine (eg, a cancer vaccine) to said subject, and said compound is administered as an adjuvant.

In another aspect provided herein, methods are described for determining whether a subject having a condition may respond to a treatment regimen described herein. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring expression levels of at least one dopamine receptor or G protein-coupled receptor (GPCR) in the sample; (iii) comparing the specified measured levels in the sample with the levels for a predetermined standard; and (iv) determining the likelihood of a subject's response to the treatment regimen based on the measured levels in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said treatment regimen further comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, or combinations thereof. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, CXCR7, or combinations thereof. In one embodiment of the present invention, said GPCR is GPR132.

In one aspect provided herein, methods are described for assessing the effectiveness of a treatment regimen described herein, monitoring, or determining a prognosis for a subject with a condition. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring expression levels of at least one dopamine receptor or G protein-coupled receptor (GPCR) in the sample; (iii) comparison of the specified measured levels in the sample with levels for a predetermined standard

- 4 043367 ta; and (iv) determining a prognosis, or determining the likelihood of whether a subject may respond to a treatment regimen, based on the measured levels in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring the number of gene copies or mutations found in at least one dopamine receptor in the sample; (iii) comparing the specified measured number of copies or mutations in the sample with levels for a predetermined standard; and (iv) determining the likelihood of a subject's response to a specified treatment regimen based on the measured number of copies or mutations found in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor is selected from DRD2, DRD2S, DRD2L and DRD3. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, or combinations thereof.

In one aspect provided herein, methods for screening a drug candidate for a specified condition are described. In one embodiment, the method comprises (i) contacting at least one G protein-coupled receptor (GPCR) with a test molecule suspected of being a drug for the condition; (ii) measuring the binding, interaction, or signaling affinity of the GPCR of the test compound to the GPCR; and (iii) comparing the binding, interaction or signaling affinity of the specified test molecule with a predetermined threshold value. In one embodiment, modulation of a GPCR or modulation of a GPCR signal by a test molecule that is comparable to or greater than a threshold value is indicative of a drug for the specified condition. In one embodiment of the present invention, said condition is cancer. In one embodiment of the present invention, said threshold value is GPCR modulation or GPCR signal modulation by a drug, such as a compound of formula (10), or a pharmaceutically acceptable salt or analog thereof. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, or combinations thereof. In one embodiment of the present invention, said GPCR is GPR132. In one embodiment of the present invention, said GPCR is GPR91. In one embodiment of the present invention, said GPCR is MTNR1A. In one embodiment of the present invention, said GPCR is CXCR7.

In one aspect, provided herein, methods are provided for screening a drug candidate for a specified condition. In one embodiment, the method comprises (i) contacting at least one dopamine receptor with a test molecule suspected of being a drug for the condition; (ii) measuring the binding, interaction, or signaling affinity of the test compound to said at least one dopamine receptor; and (iii) comparing the binding or interaction affinity of the specified test molecule with a predetermined threshold value. In one embodiment of the present invention, modulation of a dopamine receptor by a test molecule that is equal to or greater than a threshold value is indicative of a drug for the condition. In one embodiment of the present invention, said condition is cancer. In one embodiment of the present invention, said dopamine receptor is a receptor from the D2-like dopamine receptor family. In one embodiment of the present invention, said threshold value is dopamine receptor modulation or dopamine receptor signal modulation by a drug, such as a compound of formula (10), or a pharmaceutically acceptable salt or analog thereof.

In one aspect presented herein, methods are provided for screening a drug candidate for a specified condition. In one embodiment of the present invention, said processor-based method comprises (i) modeling, by computational molecular docking, binding or interaction, if any,

- 5 043367 xia, one or more three-dimensional structure (conformations) of a test molecule, which is supposed to be a drug for the specified condition, with a three-dimensional structure or model of at least one dopamine receptor; (ii) determining, using said computational method, the binding affinity or interaction structure of the test molecule with said structure or model of said at least one dopamine receptor; and (iii) comparing, by said computational method, said binding or interaction affinity of said test molecule to a predetermined threshold value, wherein modulation of said dopamine receptor by the test molecule comparable to or greater than the threshold value is indicative of a drug for said condition . In one embodiment of the present invention, said condition is cancer. In one embodiment of the present invention, said dopamine receptor is a receptor from the D2-like dopamine receptor family.

In another aspect provided herein, methods are provided for treating a subject having a condition. In one embodiment of the present invention, the method comprises administering an effective amount of a therapeutic agent that targets at least one dopamine receptor or G protein-coupled receptor (GPCR). In one embodiment, said therapeutic agent is a neutralizing agent. In one embodiment, said therapeutic agent is a receptor antagonist. In one embodiment, said therapeutic agent is a receptor agonist. In one embodiment, the therapeutic agent is a competitive inhibitor of the receptor for dopamine. In one embodiment, the therapeutic agent is a non-competitive receptor inhibitor for dopamine. In one embodiment, the therapeutic agent is selective for the D2-like dopamine receptor family over the D1-like dopamine receptor family. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said dopamine receptor is a receptor from the D2-like dopamine receptor family. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132. In one embodiment of the present invention, said GPCR is GPR91. In one embodiment of the present invention, said GPCR is MTNR1A. In one embodiment of the present invention, said GPCR is CXCR7. In one embodiment of the present invention, said GPCR is GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, or combinations thereof. In one embodiment of the present invention, said therapeutic agent is a monoclonal antibody (eg, a chimerized or humanized monoclonal antibody), a polyclonal antibody (eg, a chimerized or humanized polyclonal antibody), or a bispecific antibody. In one embodiment of the present invention, said therapeutic agent is a drug or active agent, such as an anticancer agent, conjugated to an antibody. In one embodiment of the present invention, said therapeutic agent is a radioactively conjugated antibody or an antibody conjugated to a small molecule. In one embodiment of the present invention, said therapeutic agent is a vector expressing a recombinant anti-dopamine receptor or GPCR antibody. In one embodiment of the present invention, the therapeutic agent is a fusion protein or peptide that targets a dopamine receptor or GPCR. In one embodiment of the present invention, the therapeutic agent is an siRNA, shRNA, or antisense oligonucleotide that targets a dopamine receptor or GPCR. In one embodiment of the present invention, said dopamine receptor or GPCR is targeted by CRISPR interference.

In another aspect, provided herein, methods are provided for treating and assessing the effectiveness of treatment in a subject having a condition. In one embodiment of the present invention, the method comprises (i) treating a subject in accordance with a treatment method described herein, (ii) assessing the effectiveness of the treatment as described herein. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10) or a pharmaceutically acceptable salt or analog thereof. In one embodiment, the dosage of drug administered, the frequency of administration of a compound (e.g., a compound of formula (10)), or both, is selected or adjusted based on gene expression levels or from

- 6 043367 measured number of gene copies, or detected mutations.

Brief description of graphic materials

The above brief description of the invention, as well as the following detailed description of embodiments of the invention, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present invention is not limited to the exact combinations and instrumentation described herein. The figures show:

Fig. 1. Antagonism of dopamine receptors (DRD1, DRD2S, DRD2L, DRD3, DRD4 and DRD5) via ONC201.

In fig. Figure 2 shows soluble prolactin detected by ELISA in peripheral blood from patients with advanced solid tumor at baseline and after a single dose of ONC201 (PO 125,625 mg). Post-treatment samples were collected at 6 hours, 1, 2, 7 and 21 days after treatment.

Fig. 3. Sensitivity of tumors of different types in the collection of cell lines of the Genomics of Drug Sensitivity in Cancer (GDSC) program. Average sensitivity was determined by the average estimated IC ₅₀ values from cell viability assays performed 72 hours after treatment. The numbers above the bars indicate the number of cell lines per tumor type.

Fig. 4. ONC201 is a selective DRD2 antagonist. (A) Agonism of orphan or known GPCRs or antagonism of known GPCRs using an arrestin-recruiting reporter gene assay (10 μM ONC201). (B) Antagonism of ligand-stimulated dopamine receptors by ONC201 using an arrestin-recruitment reporter gene assay. Schild assay of DRD2L antagonism by ONC201 using (C) arrestin recruitment or (D) cAMP modulation reporters.

Fig. 5. ONC201 antagonism of DRD2 is highly specific among GPCRs and other anticancer drug targets. (A) GPCR antagonism using an arrestin-recruiting reporter gene assay (10 μM ONC201). Competition of ONC201-mediated DRD2L dopamine antagonism in (B) arrestin recruitment or (C) cAMP modulation reporters. (D) Antagonism or agonism of nuclear hormone receptors by ONC201 (2 or 20 μM) with nuclear translocation reporter gene assay. (E) Inhibition of enzymatic kinase activity in vitro by ONC201 (1 μM). (F) Antagonistic activity of ONC201 or the linear isomer of ONC201 against DRD2L without any biological activity using an arrestin-recruitment reporter gene assay.

Fig. 6. GBM cell lines with higher DRD2 expression are more sensitive to ONC201. (A) Inhibition of NCI60 GBM cell lines as a function of ONC201 concentration. (B) Log ONC201 GI ₅₀ (M) as a function of DRD2 expression for each GBM cell line. R ² =0.8707.

Fig. 7. ONC201 exhibits superior selectivity among GPCRs for DRD2 compared to other DRD2 antagonists such as risperidone.

Fig. 8. ONC201 has higher tumor cell selectivity than the antipsychotic DRD2 antagonist thioridazine.

Fig. 9. Optimization of DRD2 calcium flux inhibition using ONC201. HEK293T cells were transfected with expression constructs for wild-type DRD2 (A) or control GPCR (B). Inhibition of DRD2-specific calcium flux was examined with 0.1 and 1 nM dopamine for ONC201 concentrations ranging from 100 to 100 μM. 100 μM ONC201 completely inhibited DRD2 dopamine-induced calcium flux but had no effect on the control GPCR.

Fig. 10. Comparison of DRD2 inhibitors. Inhibition of DRD2-specific calcium flux was examined at 1 nM dopamine, using the inhibitors spiperone (squares), domperidone (triangles), and ONC201 (circles) over a range of concentrations. Data from individual assays were normalized using the no-inhibitor value (shown as 10 ^-11 M) as 100% activity.

Fig. 11. Identification of DRD2 residues critical for dopamine-induced calcium flux. (A) Dopamine-induced calcium flux was analyzed as before at 1 nM dopamine across the entire alanine scan library for DRD2. Data represent the average of three experiments. Mutant clones were considered calcium flux deficient if they exhibited flux values less than 2 standard deviations below the average calcium flux value (AV - 2SD) for the entire library. (B) The identified locations of the 28 mutated residues are shown (spheres) on the crystal structure of DRD3 (PDB id 3PBL; Chien, E.Y. et al. (2010) Science 330:1091-5). The D2R/D3R antagonist ethiclopride is also indicated.

Fig. 12. Identification of DRD2 residues critical for inhibition of dopamine-induced calcium flux. (A) Dopamine-induced calcium flux was analyzed as before with 1 nM dopamine but in the presence of 100 μM ONC201 across the entire alanine scan library for DRD2. Data are the average of three experiments normalized to the flux value with wild-type DRD2 (% WT). Mutant clones were considered critical for ONC201 inhibition if they exhibited flux values more than 2 standard deviations above the mean calcium flux value (AV+2SD) for the entire library. (B) The locations of the identified 8 mutated residues (red spheres) on the crystal structure of DRD3 are determined.

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Fig. 13. The reference compound, (+)butaclamol, and the test compound, ONC201 dihydrochloride, successfully competed for [ ^3H ]methylspiperone, with _IC50 values of

2.5 nM and 21 µM, respectively.

Fig. 14. Kinetic association curves for ONC201 dihydrochloride to the DRD2S receptor for the determination of K _on and Kof.

Fig. 15. Compound activity with selected GPCR and orphan GPCR biosensor assays. The compound was tested in antagonist and agonist modes using biosensor assays with desired GPCRs and orphan GPCRs. For agonist analysis, data were normalized to the maximum and minimum responses observed in the presence of control ligand and vehicle. For antagonist analysis, data were normalized to the maximum and minimum responses observed in the presence of EC ₈₀ ligand and vehicle. The following concentrations of EC ₈₀ were used: CCR4 arrestin: 0.0078 μM CCL22; CHRM2 arrestin: 26 µM acetylcholine; and MC4R arrestin: 0.0026 µM melanotan II.

Fig. 16. ONC206 and ONC212 demonstrated antitumor efficacy among various tumor types in the NCI60 panel of cancer cell lines. ONC203 is an inactive negative control.

Fig. 17. ONC206 is an imipridone with improved DRD2 antagonism. ONC2 0 6, an analogue of ONC201, demonstrates superior antagonism of the D2-like dopamine receptor family and retains highly selective D2-like dopamine receptor antagonism compared to other antipsychotics such as haloperidol.

Fig. 18. Bone cancer is more sensitive to ONC206 than to ONC201.

Fig. 19: Of the bone cancer subtypes, Ewing's sarcoma is the most sensitive to ONC206.

Fig. 20. The anticancer efficacy of ONC206 is in the nanomolar range in 14 of 16 Ewing's sarcoma cell lines. ONC206 demonstrated superior efficacy compared to ONC201 in all cell lines.

Fig. 21. Imipridone ONC212 targets orphan GPCR. It is a highly selective agonist of the orphan GPCR tumor inhibitor GPR132, and it does not affect DRD2.

Fig. 22. ONC212 at nanomolar concentrations induced cell death in cancer cells (HCT116) but not in normal cells (MRC5).

Fig. 23. ONC212 induces an integrated stress response and inhibits Akt/ERK phosphorylation at nanomolar concentrations and at earlier time points compared to ONC201.

Fig. 24. ONC212 demonstrates oral and intravenous antitumor efficacy in mouse xenograft models of colorectal and breast cancer.

Fig. 25. Leukemia is more sensitive to ONC212 than to ONC201.

Fig. 26. ONC212 demonstrates antitumor efficacy (and superior efficacy compared to ONC201) in the nanomolar range in 55 leukemia cell lines, regardless of subtype.

Fig. 27. GPCRs were agonized or antagonized (>50%) by the 9 imipridones tested. Imipridones selectively target class A rhodopsin-like GPCRs.

Fig. 28. Case Study of a Subject with Recurrent Glioblastoma (Example 16). (A) Tumor size of the subject's total tumor burden relative to baseline (%). One cycle is 3 weeks. (B) Contrast-enhanced MRI scan: baseline, 21, 27, and 36 weeks after ONC201 initiation of one of the 2 malignant lesions.

Fig. 29. ONC212 demonstrates anticancer effects in acute myeloid leukemia (AML) cell lines. (A) Comparison of viability of MV411 AML cells treated with ONC212 or cytarabine. (B) Comparison of viability of MOLM14 cells, MV411 AML cells, MRC5 lung fibroblasts, and ONC212-treated Hs27a bone marrow cells. (C) Viability of MOLM14 cells and MV411 AML cells treated with ONC212 (250 nm) for 4, 8, 24, 48, 72, and 96 h.

Fig. 30. Efficacy of ONC212 in an ONC201-resistant AML xenograft model (MV411 AML cells (5x106) subcutaneously implanted in the flank of athymic nude mice). ONC212 and ONC201 were administered orally (PO) as indicated. Tumor volume (A and B) and body weight (C) (n=10) were measured on the indicated days. * represents p < 0.05 relative to vehicle.

Fig. 31. Efficacy of ONC206 in a xenograft model of Ewing's sarcoma (MHH-ES-1 Ewing's sarcoma cells (5x106) subcutaneously implanted in the flank of athymic nude mice). ONC206 (PO) and methotrexate (IV) were administered on day 1 and day 13 as indicated. Tumor volume (A) and body weight (B) (n=4) were measured on the indicated days.

Fig. 32. Profile of GPCR ONC213 (10 µM) by reporter gene assay with β-arrestin recruitment.

Fig. 33. ONC213 demonstrated in vitro anticancer activity in HCT116/RPMI8226 cancer cells similar to ONC212, but in vitro toxicity to normal cells was reduced compared to ONC212.

Fig. 34. Profile of GPCR ONC237 (10 µM) by β-arrestin recruitment reporter gene assay.

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Fig. 35. Profile of GPCR ONC236 (10 µM) by β-arrestin recruitment reporter gene assay.

Fig. 36. GPCR profile of ONC234 (10 µM) by β-arrestin recruitment reporter gene assay.

Fig. 37. GPCR profile of linear isomer ONC201 (TIC-10) (10 µM) by β-arrestin recruitment reporter gene assay.

Fig. 38. Number of GPCRs hits for some imipridones.

Detailed Description of the Invention

The scientific and technical terms used herein are intended to have the usual meanings understood by those of ordinary skill in the art. Such terms are defined and used in the context of various standard references, including but not limited to the following: J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; ^3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W. H. Freeman & Company, 2004; Engelke, D.R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LlC, Eagleville, PA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R. Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, ^3rd edition, Cold Spring Harbor Laboratory Press; Dec. 15, 2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002;185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808, and US Patent No. 8673923. The contents of each of the above references are incorporated herein by reference in their entirety.

The term substituted means that any one or more hydrogen atoms on the designated atom are replaced by a substituent selected from the specified group, provided that the normal valence of the specified atom is not exceeded and that the specified substitution results in the formation of a stable compound. If the substituent is keto (i.e. =O), then 2 hydrogen atoms are replaced per atom. Keto substituents are not present on aromatic moieties. Ring double bonds are double bonds that form between two adjacent atoms in the ring (such as C=C, C=N or N=N).

When any variable (for example, R ⁴ ) appears more than once in the composition or formula of a compound, its definition in each occurrence is independent of its definition in each other occurrence. Thus, for example, if a group is indicated to be substituted with 0-3 R ⁴ moieties, then said group need not be substituted with up to three R ⁴ groups, and R ⁴ in each case is selected independently from the definition of R ⁴ . In addition, combinations of substituents and/or variables are acceptable, but only if such combinations result in stable compounds.

When an atom or chemical fragment is followed by an indexed numerical range (for example, C1.6), it will be understood that it is intended to cover every number within the range, as well as all ranges in between. For example, C ₁ . ₆ alkyl denotes alkyl groups containing 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4 , 2-3, 3-6, 3-5, 3-4, 4-6, 4-5 and 5-6 carbon atoms.

The term alkyl includes both branched and unbranched saturated aliphatic hydrocarbon groups containing the specified number of carbon atoms. For example, C ₁ . ₆ alkyl includes C1, _C2 , _C3 , _C4 , _C5 and _C6 alkyl groups. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, sec-pentyl, neopentyl, and n-hexyl. In some cases, straight-chain or branched-chain alkyl contains six or fewer carbon atoms in its backbone (e.g., C1-C6 for straight-chain, _C3 - _C6 for branched-chain), and in other cases, straight-chain or branched-chain alkyl contains four or fewer carbon atoms. Likewise, cycloalkyls contain three to eight carbon atoms in their ring structure, and in other embodiments, cycloalkyls contain five or six carbon atoms in their ring structure. Most preferred is C ₁ . ₆ alkyl, in particular ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term substituted alkyl means alkyl, as defined above, substituted with one, two or three substituents selected from halogen, -OH, alkoxy, -NH2, -N(CH ₃ ) ₂ , -C(=O)OH, trifluoromethyl, -C=N, -C(=O)O(C ₁ -C ₄ )alkyl, -C(=O)NH ₂ , -SO2NH2, C(=NH)NH2 and -NO2, preferably containing one or two substituent selected from halogen, OH, alkoxy, -NH2, trifluoromethyl, -N(CH ₃ ) ₂ and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2 difluoropropyl, 2 carboxycyclopentyl and 3-chloropropyl.

Unless a different number of carbon atoms is specified, lower alkyl includes an alkyl group as defined above but containing from one to six carbon atoms, preferably from one to four, in its basic structure. Lower alkenyl and lower alkynyl have a chain length of 2-6 carbon atoms and preferably 2-4 carbon atoms.

Alkenyl includes unsaturated aliphatic groups similar in length, and possible substitution with alkyls, described above, but which contain at least one double bond.

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For example, the term alkenyl includes straight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched-chain alkenyl groups, cycloalkenyl (e.g., alicyclic) groups (e.g., cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl- or alkenyl-substituted cycloalkenyl groups and cycloalkyl- or cycloalkenyl-substituted alkenyl groups. In some cases, a straight or branched chain alkenyl group contains six or fewer carbon atoms in its backbone (eg, C2-C6 for straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups can contain from three to eight carbon atoms in their ring structure, and in some embodiments, cycloalkenyl groups contain five or six carbon atoms in their ring structure. The term C2-C6 includes alkenyl groups containing from two to six carbon atoms. The term C3-C6 includes alkenyl groups containing three to six carbon atoms.

The term alkynyl includes unsaturated aliphatic groups similar in length and possible substitution to the alkyls described above, but which contain at least one double bond. For example, the term alkynyl includes straight chain alkynyl groups (eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, nonynyl, decynyl), branched chain alkynyl groups, and cycloalkyl or alkynyl groups substituted with cycloalkenyl. In some embodiments, a straight or branched chain alkynyl group contains six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing from two to six carbon atoms The term C3-C6 includes alkynyl groups containing from three to six carbon atoms.

The term cycloalkyl refers to a monocyclic or polycyclic non-aromatic radical where each of the atoms forming the ring (ie, the backbone atoms) is a carbon atom. In one case, said cycloalkyl group is saturated or partially unsaturated. In another case, said cycloalkyl group is fused to an aromatic ring. Cycloalkyl groups include groups containing from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following:

Monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes unsaturated non-aromatic carbocyclyl or non-aromatic unsaturated carbocyclyl groups, and both refer to a non-aromatic carbocycle as defined herein that contains at least one carbon-carbon double bond or one carbon-carbon triple bond.

The term cycloalkylalkyl refers to an alkyl group substituted with a cycloalkyl group. Examples of cycloalkylalkyl groups include cyclopropylalkyl, cyclohexylalkyl.

The term heterocycloalkyl refers to a non-aromatic heterocycle where one or more of the ring-forming atoms is a heteroatom, such as an O, N, or S atom. Heterocycloalkyl groups include mono- or polycyclic (eg, containing 2, 3, or 4 fused rings) ring systems, and also spirocycles. Examples of heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidine yl and imidazolidinyl. Also included in the definition of heterocycloalkyl are moieties containing one or more aromatic rings fused (i.e., sharing a common bond) with a non-aromatic heterocyclic ring, for example, quinolyl, isoquinolyl and benzo derivatives of heterocycles. A heterocycloalkyl group containing one or more fused aromatic rings is attached by either an aromatic or non-aromatic moiety. Also included under the definition of heterocycloalkyl are fragments in which one or more ring-forming atoms can be replaced by 1 or 2 oxo groups or sulfide groups. In some cases, said heterocycloalkyl group contains from 1 to about 20 carbon atoms and in other cases from about 3 to about 20 carbon atoms. In some cases, the heterocycloalkyl group contains from 3 to about 20, from 3 to

- 10 043367 about 14, from 3 to about 7 or from 5 to 6 ring-forming atoms. In some cases, the heterocycloalkyl group contains from 1 to about 4, from 1 to about 3, or from 1 to 2 heteroatoms. In some cases, a heterocycloalkyl group contains from 0 to 3 double bonds. In some cases, the heterocycloalkyl group contains from 0 to 2 triple bonds.

The term heterocycloalkylalkyl refers to an alkyl group substituted with heterocycloalkyl. Examples of heterocycloalkylalkyls include morpholinoalkyl and piperazinylalkyl.

The term aryl refers to monocyclic or polycyclic (eg, containing 2, 3 or 4 fused rings) aromatic hydrocarbons, such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl. In some cases, the aryl group contains from 6 to 20 carbon atoms.

The term arylalkyl refers to an alkyl group substituted with an aryl group. Examples of arylalkyl groups include benzyl and phenylethyl.

The term heteroaryl refers to an aromatic heterocycle containing at least one heteroatom ring element, such as an O, S or N atom. Heteroaryl groups include monocyclic and polycyclic (eg, containing 2, 3 or 4 fused rings) systems. The N atom forming a ring in a heteroaryl group can also be oxidized to form an N-oxo moiety. Examples of heteroaryl groups include pyridyl, N-oxopyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl and indolinyl. In some cases, the heteroaryl group contains from 1 to about 20 carbon atoms and in other cases from about 3 to 20 carbon atoms. In some cases, the heteroaryl group contains from 3 to about 14, from 3 to about 7, or from 5 to 6 ring-forming atoms. In some cases, the heteroaryl group contains from 1 to about 4, from 1 to about 3, or from 1 to 2 heteroatoms.

The term heteroarylalkyl group refers to an alkyl group substituted with a heteroaryl group. An example of a heteroarylalkyl group is pyridylmethyl.

The terms halo or halogen refer to a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom, preferably F, Cl or Br, more preferably F or Cl. The term perhalogenated refers to a moiety in which all of the hydrogen atoms have been replaced by halogens. The term haloalkyl refers to alkyl moieties in which a halogen replaces hydrogen on one or more carbons of the hydrocarbon backbone. C ₁ -C ₆ -haloalkyl includes straight or branched chain alkyl with six or fewer backbone carbon atoms, and halogen replacing hydrogen on one or more backbone carbons.

The term alkoxy or alkoxyl includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently attached to an oxygen atom. The term C ₁ -C ₆ -alkoxy refers to moieties containing six or fewer carbon atoms in the hydrocarbon backbone. Examples of alkoxy groups (or alkoxy radicals) include methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups. Preferred are (C ₁ -C ₃ )alkoxy, in particular ethoxy and methoxy. Examples of substituted alkoxy groups include halogenated alkoxy groups.

The term hydroxy or hydroxyl includes groups with -OH or -O-.

The term pharmaceutically acceptable salts refers to derivatives of the compounds of the present invention wherein the parent compound is modified into a salt form by an existing acid or base moiety. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of inorganic or organic acids of alkali radicals such as amines; alkaline or organic salts of acidic residues such as carboxylic acids.

Pharmaceutically acceptable salts include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

A pharmaceutically acceptable salt can be synthesized from a starting compound that contains a basic or acidic moiety by conventional chemical methods. In general, said salts can be prepared by reacting the free acidic or basic forms of said compounds with a stoichiometric amount of the corresponding base or acid in water or an organic solvent or a mixture of the two; Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, ^17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, Journal of Pharmaceutical Science, 66, 2 (1977), and in P. H. Stahl and C. G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, ^2nd Revised edition, Weinheim/Zurich:Wiley-VCH/VHCA ( 2011), each publication is incorporated herein by reference in its entirety.

Examples of suitable inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid or hydrobromic acid, while examples of suitable organic acids may include carboxylic acid, sulfonic acid or sulfonic acid such as acetic acid, tartaric acid, lactic acid, propionic acid, glycolic acid, malonic acid, maleic acid, fumaric acid, tannic acid, succinic acid, alginic acid, benzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid, maleic acid, salicylic acid, trifluoroacetic acid, 3-aminosalicylic acid, ascorbic acid, embonic acid, nicotinic acid, isonicotinic acid, oxalic acid, gluconic acid, amino acids, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid or naphthalene-2-sulfonic acid. Examples of suitable inorganic bases may include sodium hydroxide, potassium hydroxide and ammonia, while examples of suitable organic bases include amines, for example tertiary amines such as trimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline, isoquinoline, α- picoline, β-picoline, γ-picoline, quinaldine or pyrimidine.

The term antibody covers a structure that is the natural biological form of an antibody. In most mammals, including humans and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, with each pair containing one light and one heavy chain, each light chain containing immunoglobulin domains V _L and C _L , and each heavy chain the chain contains immunoglobulin domains VH, Ογ1, Cy2 and Cy3. In each pair, the light and heavy chain variable regions ( _VL and VH) are jointly responsible for antigen binding, and the constant regions ( _CL , Cy1, Γγ2 and Γγ3, especially Γγ2 and Γγ3) are responsible for the effector functions of the antibody. In some mammals, such as camels and llamas, full-length antibodies may consist of only two heavy chains, with each heavy chain containing the immunoglobulin domains VH, Cy2 and Γγ3. As used herein, immunoglobulin (Ig) refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have several structural forms, including full-length antibodies, antibody fragments, and individual immunoglobulin domains, including V _H , Cy1, Cy2, Γγ3, V _l and C _L.

Intact antibodies can be divided into different classes based on the amino acid sequence of the heavy chain constant region. There are five main classes (isotypes) of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains that correspond to different classes of antibodies are called alpha, delta, epsilon, gamma and mu, respectively. Those skilled in the art are well aware of the subunit structures and three-dimensional configurations of the various classes of immunoglobulins.

The terms antibody or antigen-binding fragment respectively refer to the intact molecules as well as functional fragments thereof, such as Fab, scFv-Fc divalent molecule, F(ab')2 and Fv, which are capable of specifically interacting with the desired target. In some cases, antigen binding fragments contain:

(1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule that can be obtained by digestion of a whole antibody with the enzyme papain to produce a whole light chain and a portion of one heavy chain;

(2) Fab', a fragment of an antibody molecule that can be obtained by treating the entire antibody with pepsin, followed by reduction to obtain the entire light chain and part of the heavy chain; two Fab' fragments are obtained per antigen molecule;

(3) (Fab')2, an antibody fragment that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv is a genetically modified fragment containing a light chain variable region and a heavy chain variable region, expressed as two chains;

(5) a single chain antibody (SCA), a genetically modified molecule comprising a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker in the form of a genetically fused single chain molecule; and (6) scFv-Fc, obtained by fusion of a single chain Fv (scFv) with a hinge region from an immunoglobulin (Ig), such as IgG, and an Fc region.

In one embodiment of the present invention, the antibody provided is a monoclonal antibody. In one embodiment, the proposed antigen binding fragment is a single chain Fv (scFv), diabody, tandem scFv, divalent scFvFc molecule, Fab, Fab', Fv, F(ab')2, or an antigen binding scaffold (e.g., affibody, monobody, anticalin, DARPin , Knottin).

The terms binds, binding, or their grammatical equivalents refer to compositions that directly or indirectly have an affinity for each other. Specific binding means that the binding is selective between two molecules. A specific example of specific binding occurs between an antibody and an antigen. Generally, specific binding can be distinguished from nonspecific binding when the dissociation constant (K _D ) is less than about 1 x 10'5 M or

- 12 043367 less than about 1x10 ^-6 M or 1x10 ^-7 M. Specific binding can be determined, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical cross-linking, and two-hybrid assays. Suitable controls can be used to distinguish between specific and nonspecific binding. Affinity is the strength of the binding interaction between two molecules, such as an antigen and its antibody, and is defined for antibodies and other molecules with more than one binding site as the strength of the binding of a ligand at one specific binding site. Although noncovalent attachment of a ligand to an antibody or other molecule is usually not as strong as covalent attachment, high affinity refers to a ligand that binds to an antibody or other molecule having an affinity constant (Ka) greater than 10 ⁴ M ^-1 , typically 10 ⁵ 10 ¹¹ M ^-1 as determined by inhibition ELISA or equivalent affinity determined by comparable methods such as Scatchard plots or using the dissociation constant Kd, which is the reciprocal of Ka.

The term selective for binding, inhibition, stimulation, or modulation means preferentially binding, inhibiting, stimulating, or modulating, respectively, a first activity relative to a second activity (e.g., preferential binding of one receptor to another receptor; preferential inhibition over other receptors or preferential inhibition mutant type versus wild type or vice versa). In some cases, the binding is more than two times more selective, more than five times more selective, more than ten times more selective, more than fifty times more selective, more than 100 times more selective, or more than 1000 times more selective for a desired molecular target or pathway versus an undesired molecular target or pathway. In some cases, the compound binds the first molecular target or affects the pathway by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100 times relative to the second target or path under the same conditions. It is understood that in preferred embodiments, binding to the D2-like dopamine receptor family or individual receptors thereof will be selective for the D1-like dopamine receptor family or receptor thereof in any of the above ratios. The activity of a molecular target or pathway in vitro or in vivo can be measured using any suitable reproducible method.

The term modulation refers to the stimulation or inhibition of the activity of a molecular target or pathway. For example, a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of that target or pathway by at least 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least at least about 90%, at least about 95%, at least about 98%, or about 99% or more relative to the activity of that molecular target or pathway under the same conditions, but without the presence of the specified composition. In another example, a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of that target or pathway by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the activity of this target or pathway under the same conditions, differing only in the absence of the specified composition. The activity of a molecular target or pathway can be measured using any suitable reproducible means. For example, the activity of a molecular target or pathway can be measured in vitro or in vivo using a suitable assay known in the art for measuring activity. Control samples (untreated with the specified composition) can be assigned a relative activity value of 100%.

In one embodiment, the antibody, antigen binding fragment, or affinity tag binds its target with a KD of 0.1 nM to 10 mM, 0.1 nM to 1 mM, or within the range of 0.1 nM. In one embodiment, the antibody, antigen binding fragment, or affinity tag binds its target with a KD of 0.1-2 nM, 0.1-1 nM, 0.05-1 nM, 0.1-0.5 nM, or 0.1- 0.2 nM. In one embodiment, the antibody, antigen binding fragment, or affinity tag directly binds its target. In one embodiment of the present invention, the antibody, antigen binding fragment, or affinity tag binds its target indirectly, for example, by binding as a secondary antibody that binds to an antibody bound to the target.

The word tag refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent, such as a nucleic acid probe or antibody, and facilitates detection of the reagent to which it is conjugated or fused. The label itself may be detectable (eg, a radioisotope or fluorescent label), or, in the case of an enzymatic label, may catalyze a chemical change in the substrate compound or composition that is detectable.

The term probe refers to synthetic or biologically produced nucleic acid

- 13 043367 there, which contain specific nucleotide sequences that hybridize under stringent conditions to target nucleic acid sequences. The terms labeled probe, nucleic acid probe operably linked to a detectable label, or nucleic acid strand operably linked to a detectable label refer to a probe containing a marker fragment or detectable label for detection. The marker portion is attached at the 5' end, 3' end, internally, or a combination thereof. Thus, one probe can be attached to several marker fragments. The preferred moiety is an identifying tag such as a fluorophore. The labeled probe may also contain a variety of different nucleic acid sequences, each labeled with one or more marker fragments. Marker fragments can be the same or different. It may be advantageous to label each of the different probes (eg, nucleic acid sequences) with a different marker group. This can be achieved by having one distinguishable fragment on each probe. For example, probe A is attached to fragment X and probe B is attached to fragment Y. Alternatively, probe A is attached to fragments X and Y, while probe B is attached to fragments Z and W. Alternatively, probe A is attached to fragments X and Y. while probe B is attached to fragments Y and Z. All probes A and B above will be distinguishable and uniquely labeled.

By tissue sample is meant a collection of similar cells obtained from tissue of a subject or patient, preferably containing cells with a nucleus containing chromosomal material. There are four main human tissues: (1) epithelium; (2) connective tissues, including blood vessels, bones, and cartilage; (3) muscle tissue; and (4) nervous tissue. The source of the tissue sample may be solid tissue from a fresh, frozen and/or preserved organ or tissue specimen, or from a biopsy or aspirate; blood or blood components; physiological fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from the time the subject was in utero or during development. The tissue sample may be primary or cultured cells or cell lines. The tissue sample may contain compounds that do not naturally mix with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, or antibiotics. A tissue sample section refers to one part or piece of a tissue sample, such as a thin piece of tissue or cells cut from a tissue sample. Multiple sections of tissue samples can be collected and analyzed. A cell line refers to a permanently established cell culture that will multiply given the appropriate fresh environment and space.

Determination methods

In various aspects, provided herein are methods for detecting or measuring a target receptor (eg, dopamine receptor or GPCR) in a biological sample. Targets are detected by bringing the sample into contact with a target detection reagent, such as an antibody or fragment thereof, and a labeling reagent. The presence or absence of targets is detected by the presence or absence of a labeling reagent. In some cases, the sample is contacted with a target detection reagent and a labeling reagent simultaneously, for example, the detection reagent is a primary antibody and the labeling reagent is a fluorescent dye conjugated thereto. Alternatively, said biological sample is contacted with a target detection reagent and a labeling reagent sequentially, for example, said detection reagent is a primary antibody and the labeling reagent comprises a secondary antibody. For example, the sample is incubated with a detection reagent, in some cases together with a labeling reagent, under conditions that allow the formation of a complex between the detection reagent (and labeling reagent) and the target. After complex formation, the sample is optionally washed one or more times to remove unbound detection reagent (and labeling reagent). When said sample is further contacted with a labeling reagent that specifically binds the detection reagent associated with the target, said sample can optionally be washed one or more times to remove unbound labeling reagent. The presence or absence of the target in the sample is then determined by detecting a labeling reagent.

The methods described herein provide detection of multiple targets in a sample. Multiple targets are identified by contacting the biological sample with additional detection reagents and then with an additional labeling reagent specific to the additional detection reagents using the methods described.

The detectable fragment, i.e. a detectable label is a substance used to facilitate the identification and/or quantification of a target.

The detected fragments are observed or measured directly or observed or measured indirectly. Detectable fragments include, but are not limited to, radioactive tracers that can be detected using radiation counting devices; pigments, dyes or other chromogens that can be observed visually or measured using a spectrophotometer; spin labels, which can be measured using a spin label analyzer;

- 14 043367 and fluorescent moieties, where the output signal is generated upon excitation of a suitable molecular adduct and which can be visualized upon excitation by light that is absorbed by the dye, or can be measured by standard fluorimeters or imaging systems. The detectable fragment may be a luminescent substance, such as a phosphorescent substance or a fluorogen; bioluminescent substance; a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance or enzyme in which enzyme-dependent secondary signal generation occurs, such as the formation of a colored product from a colorless substrate. The detectable fragment may also take the form of a chemical or biochemical or inert particle, including colloidal gold, microspheres, quantum dots, or inorganic crystals such as nanocrystals or phosphorescent substances. The term detectable fragment or detectable label can also refer to a tag or hapten that can selectively bind to a labeled molecule such that the labeled molecules, when subsequently added, are used to generate a detectable signal. For example, biotin, iminobiotin, or desthiobiotin can be used as a tag, and then an avidin or streptavidin horseradish peroxidase (HRP) conjugate can be used to bind to the tag, and then a chromogenic substrate (such as tetramethylbenzidine) or a fluorogenic substrate such as Amplex Red can be used. or Amplex Gold (Molecular Probes, Inc.) to detect the presence of HRP. Likewise, the tag may be a hapten or an antigen (eg, digoxigenin), and an enzymatically, fluorescently, or radiolabeled antibody may be used to bind to the tag. Numerous labels are known to those skilled in the art and include, but are not limited to, particles, fluorescent dyes, haptens, enzymes and their chromogenic, fluorogenic and chemiluminescent substrates.

A fluorophore is a chemical moiety that exhibits an absorption maximum above 280 nm and retains its spectral properties when covalently attached to a labeling reagent. Fluorophores include pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzidole, oxazole or benzoxazole, thiazole or benzothiazole, porphyrin, cyanine, perylene, 4-amino-7-nitrobenz2-oxa-1,3-diazole (NBD), carbocyanine , carbostyryl, salicylate, anthranilate, azulene, pyridine, quinoline, borapolyazaindacene, xanthene, oxazine or benzoxazine, carbazine, phenalenone, coumarin, benzofuran and benzphenalenone and their derivatives. Oxazines include resorufins, aminooxazinones, diaminooxazines and their benzo-substituted analogues.

When the fluorophore is xanthene, the fluorophore may be fluorescein, rhodol or rhodamine. Fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins or naphthofluoresceins. Similarly, rhodol includes seminafluorodafluors. Alternatively, the fluorophore is xanthene, which is linked through a single covalent bond at the 9-position of xanthene. Preferred xanthenes include 3H-xanthene-6ol-3-one derivatives, 6-amino-3H-xanthene-3-one derivatives or 6-amino-3H-xanthene-3-imine derivatives. Fluorophores include xanthene (rhodol, rhodamine, fluorescein and their derivatives), coumarin, cyanine, pyrene, oxazine and borapolyazaindacene. In addition, said fluorophore may be sulfonated xanthenes, fluorinated xanphenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines. The absorption and fluorescence properties of a labeling reagent will be determined by the choice of fluorophore for said labeling reagent. The physical properties of a fluorophore label include spectral characteristics (absorbance, emission and Stokes shift), fluorescence intensity, lifetime, polarization and photobleaching rate, which can be used to distinguish one fluorophore from another.

Typically, a fluorophore contains one or more aromatic or heteroaromatic rings, which are optionally substituted with one or more of a variety of substituents including halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo or other substituents commonly found on fluorophores known in the art.

Preferably, the detectable moiety is a fluorescent dye. Fluorescent dyes include, for example, fluorescein, rhodamine, Texas red, Cy2, Cy3, Cy5, Cy0, Cy0.5, Cy1, Cy1.5, Cy3.5, Cy7, VECTOR red, ELF™ (enzyme-labeled fluorescence), FluorX , calcein, calcein-AM, CRYPTOFLUOR™'S, orange (42 kDa), tangerine (35 kDa), gold (31 kDa), red (42 kDa), crimson (40 kDa), BHMP, BHDMAP, Br-Oregon, Lucifer yellow, Alexa family of dyes, N-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl) (NBD), BODIPY™, boron dipyrromethene difluoride, Oregon green, MITOTRACKER ™ red, DiOC7 (3), DiIC18, phycoerythrin, phycobiliproteins BP (240 kDa) RPE (240 kDa) CPC (264 kDa) APC (104 kDa), Spectrum blue (Spectrum Blue), Spectrum blue (Spectrum Aqua), Spectrum green , Spectrum gold, Spectrum orange, Spectrum red, NADH, NADPH, FAD, infrared (IR) dyes, cyclic GDP-ribose (cGDPR), calcofluor white, tyrosine and tryptophan. Many fluorophores can also function as chromophores and are therefore also preferred chromophores.

- 15 043367

In addition to fluorophores, enzymes are also used as detectable moieties. Enzymes are desirable detectable moieties because amplification of the detectable signal can be achieved, thereby increasing the sensitivity of the assay. The enzyme itself does not produce a detectable response, but when brought into contact with an appropriate substrate, it destroys the substrate so that the converted substrate produces a fluorescent, colorimetric, or luminescent signal. Enzymes enhance the detectable signal because a single enzyme in a labeling reagent can cause multiple substrates to be converted into a detectable signal. Thus, there is an advantage when a small amount of the target is present in the sample, or there is no fluorophore that gives a comparable or stronger signal than the enzyme. However, fluorophores are preferred because they do not require additional analysis steps and thus reduce the overall time required to complete the analysis. The substrate for the enzyme is selected to produce a preferred measurable product, for example, colorimetric, fluorescent or chemiluminescent. Such substrates are widely used in the art.

A preferred combination of colorimetric or fluorogenic substrate and enzyme uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which produce a distinguishable color (brown and red, respectively). Other colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3', 5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplexe red reagent and variants thereof, and reduced dihydroxanthenes, including dihydrofluoresceins and dihydrorhodamines, including dihydrorhodamine 123. Peroxidase Tyramide substrates are a unique class of peroxidase substrates in that they can essentially be detected prior to enzyme action, but are locked in place by the action of peroxidase in a process described as tyramide signal amplification (TSA). These substrates are widely used to label targets in samples that are cells, tissues, or arrays for subsequent detection by microscopy, flow cytometry, optical scanning, and fluorometry.

Additional colorimetric (and in some cases fluorogenic) substrate-enzyme combinations use a phosphatase enzyme such as acid phosphatase, alkaline phosphatase, or a recombinant version of such a phosphatase in combination with a colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate ( BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4 -methylcoumarinyl phosphate (DiFMUP), fluorescein diphosphate, 3-0-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9dimethylacridin-2-on-7-yl) phosphate (DDAO-phosphate) or ELF 97, ELF 39 or related phosphates.

Additional suitable enzymes are glycosidases, in particular β-galactosidase, β-glucuronidase and β-glucosidase. Suitable colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl e-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides and glucuronides, o-nitrophenyl e-O-galactopyranoside (ONPG) and p -nitrophenyl e-Dgalactopyranoside. Preferred fluorogenic substrates include resorufin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and structural variants thereof, 4-methylumbelliferyl β-O-galactopyranoside, carboxyumbelliferyl β-O-galactopyranoside, and fluorinated coumarin-β-O-galactopyranosides. Additional enzymes include hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

For some assays, enzymes and their suitable substrates that produce chemiluminescence are preferred. These include, but are not limited to, natural and recombinant forms of luciferase and aequorins. Additionally suitable are substrates that produce chemiluminescence for phosphatases, glycosidases and oxidases, such as those containing stable dioxates, luminol, isolinol and acridinium esters. For example, the enzyme is luciferase or aquorin. The substrates are luciferin, ATP, Ca++ and coelenterazine.

In addition to enzymes, haptens such as biotin are useful detectable moieties. Biotin is useful because it is found in an enzyme system that can further enhance the detectable signal and can serve as a tag in affinity chromatography for isolation. Detection uses an enzyme conjugate that has an affinity for biotin, such as avidin-HRP. A peroxidase substrate is then added to produce a detectable signal. Haptens also include hormones, natural and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymph

- 16 043367 kines, amino acids, peptides, chemical intermediates or nucleotides.

In some cases, the detected fragment is a fluorescent protein. Exemplary fluorescent proteins include green fluorescent protein (GFP), phycobiliproteins and derivatives thereof, luciferase, or aequorin. Fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dyes, labeled labeling reagents. These tandem dyes contain a fluorescent protein and a fluorophore to produce a larger Stokes shift, in which the emission spectra are further shifted from the absorption spectra of the fluorescent protein. This provides advantages for detecting a small amount of target in a sample where the emitted fluorescent light is maximally optimized, in other words, the fluorescent protein absorbs virtually none of the emitted light. The fluorescent protein and fluorophore function as an energy transfer pair, where the fluorescent protein emits at a wavelength that the fluorophore absorbs, and the fluorophore then emits at a wavelength further away from the fluorescent protein than can be obtained with the fluorescent protein alone. A particularly useful combination is phycobiliproteins and sulforhodamine fluorophores or sulfonated cyanine fluorophores; or sulfonated xanthene derivatives. Alternatively, the fluorophore is an energy donor and the fluorescent protein is an energy acceptor.

Methods for visualizing the detected fragment depend on the label.

In some cases, the sample is illuminated with light at a wavelength selected to produce a detectable optical response and observed by means of detecting that response. Equipment suitable for illuminating fluorescent compounds includes hand-held ultraviolet lamps, mercury arc lamps, xenon lamps, lasers, and laser diodes. These illumination sources are optically integrated into laser scanners, microplate readers for fluorescence detection, or standards or microfluorometers. The degree or location of the signal compared to the standard or expected response indicates whether and to what extent the specified pattern has a certain characteristic or desired target.

The optical response is detected by visual inspection or using one of the following devices: CCD camera, video camera, photographic film, laser scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or enhancement tools signal, such as photomultiplier tubes. When a sample is examined using a flow cytometer, examining it does not necessarily involve sorting its parts according to their fluorescence response.

When an indirectly detectable label is used, illumination typically involves the addition of a reagent to produce a detectable signal, such as a colorimetric enzyme substrate. Radioisotopes are also considered indirectly detectable when no additional reagent is needed, rather the radioisotope is exposed to the x-ray film or the effect is detected by another mechanism to record and measure the signal. This is true for some chemiluminescent signals that can be observed after exposure of the film.

I. ONC201 (compound (1)), its salts and methods of synthesis.

This document describes the ONC201 (connection (1))

and its analogs and their pharmaceutically acceptable salts, as well as methods for their preparation. In in vitro models, animal models, and human clinical trials, ONC201 has broad-spectrum anticancer activity, low toxicity including few, if any, side effects, low genotoxicity, and high bioavailability including oral bioavailability. Thanks to these features, ONC 201 and its various analogues are particularly well suited for a variety of applications. ONC201 can be prepared using the synthesis method shown in Scheme 1.

- 17 043367

Scheme 1

ST WW ^to Stage 3 ..... g

Connection (1) ।

The synthesis of the ONC201 dihydrochloride salt begins with a commercially available intermediate, N-benzyl-3-carbomethoxy-4-piperidone hydrochloride, compound (3). In one embodiment, the synthesis involves neutralizing compound (3) with a base (step 1) to obtain compound (4), the free base. In one embodiment, compound (3) is neutralized with an inorganic base to obtain compound (4). In one embodiment, compound (3) is neutralized with an organic base to obtain compound (4). In one embodiment, intermediate (3) is neutralized in the presence of an alcohol, such as nbutanol. In one embodiment of the present invention, intermediate (3) is neutralized in the presence of at least one organic solvent. For example, intermediate (3) is neutralized in the presence of n-butanol and/or ethyl acetate. In one embodiment of the present invention, intermediate (3) is neutralized in the presence of a base and at least one organic solvent, for example NaHCO ₃ and n-butanol. In one embodiment of the present invention, said intermediate (3) is neutralized in the presence of n-butanol and triethylamine (Et ₃ N).

In one embodiment of the present invention, the synthesis involves reacting said compound (4) with compound (5) (Step 2) to produce intermediate (1). In one embodiment, the interaction in step 2 involves heating said compound (4) with compound (5). In one embodiment of the present invention, said reaction in step 2 involves refluxing said compound (4) and compound (5) in the presence of a solvent. In one embodiment, the step 2 reaction involves using a Dean-Stark trap to remove water and/or methanol (MeOH) produced in the reaction.

In one embodiment of the present invention, the dihydrochloride salt of ONC201 is synthesized (step 3). In one embodiment of the present invention, said reaction in step 3 involves treating ONC201 with HCl in dioxane. In one embodiment of the present invention, said interaction in step 3 involves treating ONC201 with 4H. HCl in dioxane. In one embodiment of the present invention, said synthesis process optionally involves recrystallization of the ONC201 di-salt. In a preferred embodiment of the present invention, the dihydrochloride salt of ONC201 is synthesized as illustrated in Scheme 2.

Scheme 2

¹ _% \n-outanol 'MC.ll PvNSS'-' ₊ G „S. ,Λ"ΛΛΛΛΛΛ"ΛΛΛΛΛΛ"ΛΛΛΛΛ n-b\tanol or - and arr. cold.

. ethyl acetate ·

P) I) (5)

Connection (1)

II. TNF-related apoptosis-inducing ligand (TRAIL).

TRAIL protein can be analyzed in a test sample obtained from a subject to detect TRAIL expression induced by the compounds and their salts described herein. Immunological methods can be used to analyze TRAIL in a sample, including enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay

- 18 043367 (ELIFA), flow cytometry, immunoblot, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescence immunoassay (LIA), fluorescence immunoassay (FIA) and radioimmunoassay. Assays can be used to obtain qualitative and/or quantitative results. Specific details of suitable methods for both qualitative and quantitative sample analysis are described in standard references, including E. Harlow & D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling & S. Diibel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Basics: From Background to Bench, BIOS Scientific Publishers, 2000; BKC Lo, Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; F. M. Ausubel et al., Eds., Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook: Functional Oligonucleotides and Their Applications, Wiley, 2006; Ormerod, M.G., Flow Cytometry: a practical approach, Oxford University Press, 2000; Givan, AL, Flow Cytometry: first principles, Wiley, New York, 2001; Gorczyca, W., Flow Cytometry in Neoplastic Hematology: morphologicimmunophenotypic correlation, Taylor & Francis, 2006; Crowther, JR, The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, ^3rd Edition, Elsevier Science, 2005, and J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ^3rd ed., 2001.

Protocols for detecting and analyzing a sample on TRAIL to determine the effect of a pharmaceutical composition are described in US Pat. No. 8,673,923 to Wafik S. El-deiry et al., which is incorporated herein by reference in its entirety.

In one embodiment, TRAIL assays are used to monitor a subject. For example, a sample is collected from a subject prior to initiation of treatment with the pharmaceutical composition and one or more times during and/or after administration to assess the effectiveness of the treatment. In another example, a sample is collected from a subject at various points in time to assess the progress or development of a disease or treatment. In one embodiment, death receptors of circulating tumor cells are analyzed to see whether a treatment described herein increases the number or type of death receptors.

The types of cancer that are treated using the methods and compositions described herein are characterized by abnormal cellular proliferation, including preneoplastic hyperproliferation, in situ cancer, neoplasms and metastases. The methods and compositions described herein can be used to prevent, as well as alleviate, signs or symptoms of cancer. Treating cancer in a subject includes: preventing, inhibiting or ameliorating said cancer in the subject, for example, slowing the progression of cancer or reducing or ameliorating the signs and/or symptoms of cancer. Examples of cancers that are treated using the methods and compositions described herein include breast cancer, CNS cancers, colon cancer, ovarian cancer, prostate cancer, leukemia, lung cancer and lymphoma.

III. Compound of formula (10) and its salt.

In one aspect, compounds and salts of formula (10) and methods for their preparation are provided. Those skilled in the art will appreciate that the general principles and concepts described herein with respect to ONC201 (compound (1)) and its salts, including the principles and concepts relating to methods and pharmaceutical compositions, apply equally to compounds of formula (10) and their salts.

In one embodiment of the present invention, compounds of formula (10) are provided:

(10) wherein R1 and R2 are independently selected from H, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio and acyl radicals. In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(2-CH ₃ -Pb) (ie ONC201). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH2-(2,4-di F-Ph) (ie ONC206). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(4-CF ₃ -Ph) (ie ONC212). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(3,4-diF-Ph) (ie ONC213). In one embodiment of the present invention, R1 is CH ₂ (3,4-di-C1Ph and R2 is CH ₂ -(4-CF ₃ -Ph) (i.e., ONC234). In one embodiment of the present invention, R1 is is CH ₂ -3-thienyl and R2 is CH ₂ -(4-CF ₃ -Ph) (ie ONC236).

In one embodiment of the present invention, R1 and R2 are independently selected from the group consisting of H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzylpiperazine, _C1-4 alkyl

- 19 043367 thienyl, C _1-4 alkylpyridinyl, C _1-4 alkylisoxazolidinyl, C _1-4 alkylmorpholinyl, C _1-4 alkylthiazolyl and C _1-4 alkylpyrazinyl, where C _1-4 alkyl, C _1-4 alkylphenyl, C _{1 -4} alkylphenyl ketone, C _1-4 benzylpiperazine, C _1-4 alkylthienyl, C _1-4 alkylpyridinyl, C _1-4 alkylisoxazolidinyl, C _1-4 alkylmorpholinyl, C _1-4 alkylthiazolyl and C _1-4 alkylpyrazinyl are optionally substituted with C _1-4 alkyl, C _1-4 alkoxyl, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment, R1 and/or R2 is substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment of the present invention, said heteroarylalkyl is selected from _C1-4 alkylpyrrolyl, _C1-4 alkylfuryl, _C1-4 alkylpyridyl, _C1-4 alkyl-1,2,4-thiadiazolyl, _C1-4 alkylpyrimidyl, _{C1 -4} alkylthienyl, C _1-4 alkyl isothiazolyl, C _1-4 alkyl imidazolyl, C _1-4 alkyl tetrazolyl, C _1-4 alkyl pyrazinyl, C _1-4 αlkylpyrimidyl, C _1-4 alkyl quinolyl, C _1-4 αlkyl isoquinolyl, C _1-4 alkylthiophenyl, C _1-4 alkylbenzothienyl, C _1-4 alkylisobenzofuryl, C _1-4 alkylpyrazolyl, C _1-4 alkylindolyl, C _1-4 αlkylpurinyl, C _1-4 alkylcarbazolylα, C _1-4 alkylbenzimidazolyl and C _1-4 αlkylisoxazolyl.

In one embodiment, R1 and/or R2 is benzyl, optionally substituted with one or more of the following substituents on the benzyl ring: X, -CH3, _-NO2 , -OCH3, -CN, -CHH2, -CH2H, C2-C4- alkyl, -СХ3, -СШСХз), -СН(СХз)2, -С(СХз)з, -СрХ2р+1, -ОСХз, -ОСрН2р+1, -ОСрХ2р+1, OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO2R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected from H or C1-C4 alkyl; and where p is an integer from 2 to 20 and X is a halogen, including F, Cl, Br or I; preferably F, Cl or Br; more preferably F or Cl.

In one embodiment of the present invention, R1 is selected from H, CH3, CH ₂ Ph, CH ₂ -(4-CF ₃ -Ph), CH ₂ -(4-F-Ph), CH ₂ -(4-Cl-Ph) , CH ₂ -(OSH ₃ -Pd), CH ₂ -((2-Cl)-Ph), CH2-(2-thienyl), CH ₂ -(3-thienyl), CH2-2pyridinyl, CH2-4-methyl -2-thiazolyl, CH2-2-pyrazinyl, CH ₂ CH ₂ Ph, CH ₂ CH ₂ (4-N-benzyl-niperazine), CH2-(2,4-di F-Ph), CH ₂ -(3, 4-di Cl-Ph), CH ₂ -(3,4-di F-Ph), CH ₂ -(3,5-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH2CH(OH)Ph, (4-F-Ph)-4-oxobutyl, CH2CH2NHCOOC(CH3% CH2CH2CH2NH2 and CD2C6D5. In one embodiment of the present invention, R2 is selected from H, _CH3 , _CH2Ph , _CH2 -(4- CF ₃ -Ph), CH ₂ -((2-Cl)-Ph), CH2-((2-F)-Ph), CH2-(2-thienyl), CH ₂ CH ₂ Ph, adC^^-benzyl -piperazine), CH2-(2,4-di F-Ph), CH ₂ -(2,4-di Cl-Ph), CH ₂ -(3,4-di Cl-Ph), CH ₂ -(3,4 -di F-Ph), CH ₂ -(3,5-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH2(2-CH ₃ , 4-F-Ph), CH ₂ -((4-OSH ₃ )-Ph), CH ₂ -(3-pyridinyl), CH ₂ -(3-isoxazolidinyl), CH ₂ CH ₂ -(4-morpholinyl), CH2-(2-F, 4CF3 -Ph), CH2CH(OH)Ph, (CH2) _3CO -4F-Ph, (4-F-Ph)-4-oxobutyl, CH2CH2NHCOOC( _CH3 ) ₃ , CH2CH2CH2NH2 and CD2C6D5.

In one embodiment of the present invention, R1 is hydrogen. In one embodiment of the present invention, R ₁ represents a substituted or unsubstituted arylalkyl, such as a benzyl (CH ₂ Ph) or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted arylalkyl, for example, benzyl or phenylethyl. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from halogen, CH3, CF3 or _OCH3 . In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted heterocycloalkylalkyl, for example, piperazinilalkyl or morpholinoalkyl. In one embodiment of the present invention, R2 is a substituted or unsubstituted heteroarylalkyl, for example, pyridylmethyl or isoxazolidinylmethyl. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from halogen, CH ₃ , CF ₃ or OCH ₃ .

In one embodiment of the present invention, compound (10) has the structure of formula (80):

where each of R _a1 , R _a2 , R _a3 , R _a4 , R _a5 , R _b1 , R _b2 , R _b3 , R _b4 and R _b5 is independently selected from the group consisting of H, X, -CH3, -NO ₂ , -OSH3, -CN, -СХН2, -СХ2Н, С2-С4-alkyl, -СХ3, -СН ₂ (СХз), -СН(СХз)2, -С(СХз)з, -СрХ2р+1, -OCХ3, -OSрН2р+1, -ОСрХ2р+1, OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO2R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected from H or C1-C ₄ -alkyl; and where p is an integer from 2 to 20 and X is a halogen.

- 20 043367

In one embodiment of the present invention, compound (10) has the structure of formula (90)

wherein R2 is as defined above, and wherein each of _Rb1 , _Rb2 , _Rb3 , _Rb4 and _Rb5 is independently selected from the group consisting of H, X, _-CH3 , -NO2, _-OCH3 , -CN , -СХН ₂ , -СХ ₂ Н, С ₂ -С ₄ -alkyl, -СХ ₃ , -СН ₂ ( _СХ3 ), -СН(СХз)2, -С(СХз)з, -СрХ2р+1, - OSKh3, -OSrH _2p + _b -OsrKh _2p + _b OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO2R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected from H or C1-C ₄ -alkyl; and where p is an integer from 2 to 20 and X is a halogen.

In one embodiment of the present invention, compound (10) has the structure of formula (40):

where R1 is as defined above, and where each of R _a1 , R _a 2, R _a3 , R _a4 and R _a 5 is independently selected from the group consisting of H, X, -CH3, -NO2,

-OCH3, -CN, -CHH2, -CH2H, C _2-4 alkyl, -CH3, -CH ₂ (CH ₃ ),

-СН(СХз)2, -С(СХз)з, -CpX2p+1, -OCX3, -OCpH2p+1, -OCX^, OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO2R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected from H or C1-C ₄ -alkyl; and where p is an integer from 2 to 20 and X is a halogen. In one embodiment of the present invention, R1 is hydrogen. In one embodiment of the present invention, R1 is a substituted or unsubstituted arylalkyl, such as benzyl or phenylethyl. In one embodiment of the present invention, said arylalkyl is substituted with C1. ₄ alkyl, C1. ₄ alkoxyl, hydroxyl, perhalogenated C1. ₄ alkyl or halogen. In one embodiment of the present invention, said benzyl is substituted with one or more halogens. In one embodiment of the present invention, said benzyl is substituted with one or more substituents selected from halogen, _CH3 , _CF3 , OCH3. In one embodiment of the present invention, said benzyl is substituted with one halogen, for example, fluorine in the ortho or para position. In one embodiment of the present invention, said benzyl is substituted with two halogens, for example, F in both meta positions.

In one embodiment of the present invention, compound (40) has the structure of compound (45):

where Ra1, Ra2, Ra3, Ra4 and Ra5 are as defined above. In one embodiment of the present invention, said benzyl is substituted with one or more halogens. In one embodiment of the present invention, said benzyl is substituted with one or more substituents selected from halogen, CH3, CF3, OCH3. In one embodiment, R _a1 or R _a 5 is a halogen, such as fluorine. In one embodiment, both R _a 2 and R _a3 are halogen, such as F.

In one embodiment of the present invention, compound (10) has the structure of compound (50)

where R1 is as defined above, and where R _b is selected from the group consisting of H, X, -CH3, -NO2, -OCH3, -CN, -CHH2, -CH2H, C2-4alkyl, -CH3, _-CH2 (СХз), -СН(СХз)2, -С(СХз)з, -СрХ _2р + _ь -ОСХ3, -ОСрНр+ь -ОС _р Х _2р+1 , OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO ₂ R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected

- 21 043367 ny from H or C ₁ -C ₄ -alkyl; and where p is an integer from 2 to 20 and X is a halogen, and where each of R _a1 , R _a2 , R _a4 and R _a 5 are independently selected from the group consisting of H, X, -CH ₃ , -NO ₂ , -OCH ₃ , -CN, -СХН ₂ , -СХ ₂ Н, С _2-4 alkyl, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -C _p X _2p+1 , -OCX3, -OS _p H _2p+1 , -OCpX2p+1, OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO2R ^m , C(O)R ^m and C(O)OR ^m ; R ^m and R ⁿ are independently selected from H or C ₁ -C ₄ -alkyl; and where p is an integer from 2 to 20 and X is a halogen. In one embodiment of the present invention, R ₁ is hydrogen. In one embodiment of the present invention, R ₁ represents a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C ₁ . ₄ alkyl, C ₁ . ₄ alkoxyl, hydroxyl, perhalogenated _C1 . ₄ alkyl or halogen. In one embodiment of the present invention, R _b is selected from halogen, CH ₃ , CF 3 and OCH ₃ . In one embodiment of the present invention, one or more of R _a1 , R _a2 , R _a4 and R _a5 are selected from halogen, CH ₃ , CF ₃ and OCH ₃ . In one embodiment of the present invention, R _a1 , R _a2 , R _a4 and R _a 5 are hydrogen and R _b is selected from halogen, CH ₃ , CF3 and OCH ₃ . In one embodiment of the present invention, Rb is a halogen, such as F, and Ra1 is CH3. In one embodiment of the present invention, Rb is F or Cl, and R _a2 is F or Cl. In one embodiment of the present invention, R _b is CF3. In one embodiment of the present invention, Rb is OCH3. In one embodiment of the present invention, Rb and Ra1 are Cl.

In one embodiment of the present invention, compound (50) has the structure of compound (55):

where R _a1 , R _a 2, R _a4 , R _a 5 and R _b are as defined above. In one embodiment of the present invention, R _b is selected from halogen, CH3, CF3 and OCH3. In one embodiment of the present invention, one or more of R _a1 , R _a 2 , R _a4 and R _a 5 is selected from halogen, CH3, CF3 and OCH3. In one embodiment of the present invention, R _a1 , R _a 2 , R _a4 and R _a 5 are hydrogen and R _b is selected from halogen, CH3, CF3 and OCH3. In one embodiment of the present invention, R _b is a halogen, such as F, and Ra1 is CH3. In one embodiment of the present invention, Rb is F or Cl, and R _a 2 is F or Cl. In one embodiment of the present invention, R _b is CF ₃ . In one embodiment of the present invention, R _b is OCH3. In one embodiment of the present invention, Rb and Ra1 are Cl.

In one embodiment of the present invention, compound (10) has the structure of compound (60)

In one embodiment of the present invention, R ₁ is H. In one embodiment of the present invention, R ₁ is a substituted or unsubstituted arylalkyl, such as benzyl or phenylethyl. In one embodiment of the present invention, R1 is substituted or unsubstituted heterocycloalkylalkyl or substituted or unsubstituted heteroarylalkyl, such as CH2-(2-thienyl), _CH2- (3-thienyl), CH2-4-methyl-2-thiazolyl, CH2- 2-pyrazinyl _, CH2CH2 (4-N-benzylpiperazine), _CH2- (3-isoxalidinyl), CH2-2-pyridinyl, _CH2-3 -pyridinyl and _CH2CH2- (4-morpholinyl). In one embodiment of the present invention, said arylalkyl is substituted with C ₁ . ₄ alkyl, C ₁ . ₄ alkoxyl, hydroxyl, perhalogenated _C1 . ₄ alkyl or halogen. In one embodiment of the present invention, said benzyl is substituted with one or more halogens. In one embodiment of the present invention, said benzyl is substituted with one or more substituents selected from halogen (eg, F), CH ₃ , CF 3 and OCH ₃ . In one embodiment, said benzyl is substituted in the para position with halogen, CH ₃ , CF 3 or OCH ₃ substituents. In one embodiment of the present invention, R1 is fluorophenyloxobutyl or hydroxyphenylethyl.

- 22 043367

Scheme 3 shows the synthesis of compounds of formula (10).

Scheme 3

Methods: a. NaH, dimethyl carbonate, toluene, 80C, 4 hours; b. IN NaOH/CEECh convert to free base, then heat in dioxane at 70C; With. 1-butanol/heating with ref.cold. 3-6 hours (Dean-Stark trap) PPTS; d. dioxane 70C; e. HC1 in dioxane - 25C - _ar ^ room temp. to obtain HCl salt; f. Na ₂ CO3, D1EA 80С; g. Convert NaOH/CH ₂ Cl ₂ into free base, then heat with MeOH. cold, 3.5 hours

Compounds of formula (10) (ie, imipridones) were synthesized starting with a substituted piperidone, which was modified by reacting with a substituted aminoimidazoline to give the parent compound (10). There are two routes: one in which the R1 substituent is present in piperidone (ie 68). In this method, compound (68) is acylated with dimethyl carbonate using sodium hydride in toluene at 80°C to obtain piperidone ester (69). Commercially available methylthioimidazoline HI salt (63) was reacted with an amine in dioxane at 70°C to give R ₂ -substituted aminoimidazoline (64) as its HI salt. Direct reaction of compound (64) with piperidone ester (69) in 1-butanol under reflux and removal of water through a Dean-Stark trap for 3-6 hours afforded tricyclic compound (10). In a variation of this scheme, the N-BOC-protected piperidone (61) was converted by the same methods to the BOC-protected compound (65), which was treated with HCl in dioxane to remove the BOC group and then converted to the free base (66) using 1N NaOH with extraction with methylene chloride. Subsequent treatment (66) with halide (67) or epoxide (70) provided the desired compound (10).

The crude products can be purified by column chromatography, eluting with methylene chloride:methanol, or by HPLC using acetonitrile:TFA:H ₂ O, to obtain the final products as free bases or TFA salts. Treatment of these free bases with HCl in dioxane or lyophilization of TFA salts gives products (10) as HCl or TFA salts. Alternatively, said free base may be treated with another inorganic or organic acid to obtain other salts, usually selected from those known as pharmaceutically acceptable salts. The salts of compound (10) are generally solids, and examples are crystallized from ethanol or other solvents to obtain high quality crystals. The tricyclic structure was conclusively confirmed in the case of compound (1) by X-ray crystal structure and NMR.

The compounds described herein can be used, with or without an aminoalkyl linker (eg, compounds (33)), to identify molecules (eg, proteins) that interact with them in the context of a cell. The expression of these binding targets can be used to predict response to imipridones or their analogs (ie, as biomarkers). These compounds can be used to screen structurally unrelated molecules using competition assays known in the art to identify drugs that can displace interaction with a higher affinity target. In addition, these molecules may have improved drug properties or allow additional use by changing the properties of the drug, including safety, activity, pharmacokinetics, biodistribution or metabolism.

- 23 043367

Table 1

Examples of compounds of formula (10)

No. ONC number Ri r ₂ 1 ONC201 _CH2Ph CH ₂ - ((2-CH ₃ ) -Ph) 13 _CH2Ph CH ₃ 14 ONC202 _CH2Ph CH ₂ -((2-C1)-Ph) 15 ONC203 _CH2Ph CH ₂ -(2-thienyl) 16 ONC204 _CH2Ph CH ₂ CH ₂ Ph 17 ONC205 _CH2Ph CH ₂ CH ₂ (4-N-benzylpiperazine) 18 ONC206 _CH2Ph CH ₂ -(2,4-di F-Ph) 19 ONC207 H CH ₂ - ((2-CH ₃ ) -Ph) 20 ONC208 CH ₃ CH ₂ - ((2-CH ₃ ) -Ph) 21 ONC209 CH ₂ CH ₂ Ph CH ₂ - ((2-CH ₃ ) -Ph) 22 CH ₂ CH ₂ - (4-N-benzylpiperizine) CH ₂ - ((2-CH ₃ ) -Ph) 23 CH ₂ CHOHPh CH ₂ - ((2-CH ₃ ) -Ph) 24 (CH ₂ ) ₃ CO-4F-Ph CH ₂ - ((2-CH ₃ ) -Ph) 32 ONC215 CH ₂ CH ₂ NHCOOC (CH ₃ ) 3 CH ₂ - ((2-CH ₃ ) -Ph) 33 ONC216 CH ₂ CH ₂ CH ₂ NH ₂ CH ₂ - ((2-CH ₃ ) -Ph) 41 ONC210 _CH2Ph CH ₂ -(3,5-di F-Ph) 51 ONC211 _CH2Ph CH ₂ -(3,4-di Cl-Ph) 52 ONC212 _CH2Ph CH ₂ - (4-CF ₃ -Ph) 53 ONC213 _CH2Ph CH ₂ -(3,4-di F-Ph) 54 ONC214 cd ₂ c ₆ d ₅ CH ₂ - ((2-CH ₃ ) -Ph) 43 ONC217 _CH2Ph CH ₂ - (2-F-Ph) 55 ONC218 _CH2Ph CH ₂ (2-CH ₃ , 4-F-Ph) 56 ONC219 _CH2Ph CH ₂ -(2,4-di Cl-Ph) 57 ONC220 _CH2Ph CH ₂ - ((4-OSH ₃ ) -Ph) 34 ONC226 _CH2Ph CH ₂ -(3-pyridinyl) 35 ONC222 _CH2Ph CH ₂ -(3isoxalidinyl) 36 ONC224 _CH2Ph CH ₂ CH ₂ - (4morpholinyl) 37 ONC223 _CH2Ph CH ₂ - (4-CH ₃ -Ph) 38 ONC221 H CH ₂ - (4-CF ₃ -Ph) 73 ONC227 CH ₂ - (4-CF ₃ -Ph) CH ₂ - (4-CF ₃ -Ph) 72 ONC225 _CH2Ph CH ₂ -(2-F, 4-CF ₃ -Ph) 74 ONC228 CH ₂ - (4-F-Ph) CH ₂ - (4-CF ₃ -Ph) 75 ONC229 CH ₂ - (OCH ₃ -Ph) CH ₂ - (4-CF ₃ -Ph) 76 ONC230 (4-F-Ph)-4oxobutyl CH ₂ - (4-CF ₃ -Ph) 77 ONC231 CH ₂ -3-pyridyl CH ₂ - (4-CF ₃ -Ph) 78 ONC232 CH ₂ -4-methyl-2thiazolyl CH ₂ - (4-CF ₃ -Ph) 79 ONC233 CH ₂ -2-pyrazinyl CH ₂ - (4-CF ₃ -Ph) 81 ONC234 CH ₂ -(3,4-di Cl-Ph) CH ₂ - (4-CF ₃ -Ph) 82 ONC235 CH ₂ -(4-Cl-Ph) CH ₂ - (4-CF ₃ -Ph) 83 ONC236 CH ₂ -3-thienyl CH ₂ - (4-CF ₃ -Ph) 84 ONC237 CH ₂ CH(OH)Ph CH ₂ - (4-CF ₃ -Ph)

IV. Assessing the sensitivity and effectiveness of treatment regimens.

Measurement of dopamine receptor or other G protein-coupled receptor (GPCR) gene expression, gene mutations, or copy number may be used to predict response or sensitivity to a treatment described herein and to identify whether subjects will respond to a method of treatment described herein, such as treatment with a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one aspect provided herein, methods are described for determining whether a subject having a condition may respond to a treatment regimen described herein. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring expression levels of at least one dopamine receptor or G protein-coupled receptor (GPCR) in the sample; (iii) comparing the specified measured levels in the sample with the levels for a predetermined standard; and (iv) determining the likelihood of a subject's response to the treatment regimen based on the measured levels in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the step

- 24 043367 measuring the level of expression of a dopamine receptor or GPCR in a sample includes the steps of (i) contacting the sample with an antibody or antigen binding fragment that specifically binds to the receptor to form a complex of the antibody or antigen binding fragment with the receptor; and (ii) measuring the amount of complex. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment, said cancer is a neuro-oncological disease. In one embodiment, said cancer is a neuroendocrine tumor. In one embodiment, said cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, or diffuse brainstem glioma. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment, said mental disorder is selected from the group consisting of psychosis, schizophrenia, bipolar disorder or major depressive disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said gram-positive bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection). In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family. In one embodiment of the present invention, said dopamine receptor is DRD2. In one embodiment of the present invention, said dopamine receptor is DRD3. In one embodiment of the present invention, said dopamine receptor is DRD4. In one embodiment of the present invention, said dopamine receptor is DRD2, DRD3, or both. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132. In one embodiment of the present invention, said GPCR is selected from the group consisting of GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, and combinations thereof. In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprising administering an effective amount of a drug, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein the level of DRD5 expression measured in the sample is increased when compared to a predetermined standard indicates whether a subject is likely to respond to a treatment regimen.

In another aspect provided herein, methods are described for assessing the effectiveness of a treatment regimen described herein, monitoring, or determining a prognosis for a subject with a condition. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring expression levels of at least one dopamine receptor or G protein-coupled receptor (GPCR) in the sample; (iii) comparing the specified measured levels in the sample with the levels for a predetermined standard; and (iv) determining a prognosis or determining the likelihood of a subject's response to a treatment regimen based on the measured levels in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the step of measuring the expression level of a dopamine receptor or GPCR in a sample includes the steps of (i) contacting the sample with an antibody or antigen binding fragment that specifically binds to the receptor to form a complex of the antibody or antigen binding fragment with the receptor; and (ii) measuring the amount of complex. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring the number of gene copies or mutations found in at least one dopamine receptor in the sample; (iii) comparing the specified measured number of copies or mutations in the sample with levels for a predetermined standard; and (iv) determining the likelihood of a subject's response to a specified treatment regimen based on the measured number of copies or mutations found in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment, said cancer is a neuro-oncological disease. In one embodiment, said cancer

- 25 043367 is a neuroendocrine tumor. In one embodiment, said cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, or diffuse brainstem glioma. In one embodiment of the present invention, the subject has a mental disorder or is at risk of developing a mental disorder. In one embodiment, said mental disorder is selected from the group consisting of psychosis, schizophrenia, bipolar disorder or major depressive disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said gram-positive bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection). In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor is selected from DRD2, DRD2S, DRD2L and DRD3. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family. In one embodiment of the present invention, said dopamine receptor is a D1-like dopamine receptor. In one embodiment of the present invention, said dopamine receptor is DRD1. In one embodiment of the present invention, said dopamine receptor is DRD2. In one embodiment of the present invention, said dopamine receptor is DRD3. In one embodiment of the present invention, said dopamine receptor is DRD4. In one embodiment of the present invention, said dopamine receptor is DRD5. In one embodiment of the present invention, said dopamine receptor is DRD2, DRD3, or both. In one embodiment, said GPCR is a class A GPCR. In one embodiment of the present invention, said GPCR is GPR132. In one embodiment of the present invention, said GPCR is selected from the group consisting of GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, and combinations thereof.

In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprises administering an effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein an increased level of DRD5 expression measured in the sample when compared to a predetermined standard indicates whether the treatment regimen is effective. In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprises administering an effective amount of a drug, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein mutations in the DRD5 gene measured in the sample indicate that whether the treatment regimen is effective. In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprising administering an effective amount of a drug, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein the Q366R missense mutation in the DRD5 gene measured in the sample is indicate whether the treatment regimen is effective.

In another aspect provided herein, methods are described for determining the likelihood of a subject having a condition responding to a treatment regimen described herein. In one embodiment of the present invention, said methods comprise (i) collecting a biological sample from said subject; (ii) measuring the number of gene copies or mutations found in at least one dopamine receptor in the sample; (iii) comparing the specified measured number of copies or mutations in the sample with levels for a predetermined standard; and (iv) determining the likelihood of a subject's response to a specified treatment regimen based on the measured number of copies or mutations found in the sample compared to those measured for a predetermined standard. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment, said cancer is a neuro-oncological disease. In one embodiment, said cancer is a neuroendocrine tumor. In one embodiment, said cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, or diffuse brainstem glioma. In one embodiment of the present invention, said subject has a mental

- 26 043367 construction or at risk of developing a mental disorder. In one embodiment, said mental disorder is selected from the group consisting of psychosis, schizophrenia, bipolar disorder or major depressive disorder. In one embodiment of the present invention, the subject has an infection or is at risk of developing an infection. In one embodiment of the present invention, said infection is a bacterial infection. In one embodiment of the present invention, said infection is a gram-negative bacterial infection. In one embodiment of the present invention, said infection is a gram-positive bacterial infection. In one embodiment of the present invention, said bacterial infection is an infection with a bacterium selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter. In one embodiment of the present invention, said gram-positive bacterial infection is a Staphylococcus infection. In one embodiment, said Staphylococcus infection is an S. aureus infection (eg, methicillin-resistant S. aureus (MRSA) infection). In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In one embodiment of the present invention, said dopamine receptor belongs to the D2-like dopamine receptor family. In one embodiment of the present invention, said dopamine receptor is DRD1. In one embodiment of the present invention, said dopamine receptor is DRD2. In one embodiment of the present invention, said dopamine receptor is DRD3. In one embodiment of the present invention, said dopamine receptor is DRD4. In one embodiment of the present invention, said dopamine receptor is DRD5. In one embodiment of the present invention, said dopamine receptor is DRD2, DRD3, or both. In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprises administering an effective amount of a drug, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein mutations in the DRD5 gene measured in the sample indicate that whether the subject is likely to respond to the treatment regimen. In one embodiment of the present invention, said dopamine receptor is DRD5, said treatment regimen comprising administering an effective amount of a drug, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, wherein the Q366R missense mutation in the DRD5 gene measured in the sample is indicate whether the subject is likely to respond to the treatment regimen.

Additionally, measurement of expression, post-translational modifications, or activity levels of eIF2alpha, ATF4, CHOP, DR5, or mutations in eIF2alpha, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used to predict response or sensitivity to the treatment described in herein, and to identify whether subjects will respond to a treatment described herein, such as treatment with a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analogue thereof. In addition, measurement of expression, post-translational modifications or activity levels of eIF2-alpha, ATF4, CHOP, DR5 or mutations in eIF2-a, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used to assess the effectiveness or monitor the treatment described in this document. In addition, measurement of expression, post-translational modifications, or activity levels of eIF2-alpha, ATF4, CHOP, DR5, or mutations in eIF2-a, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used for in vivo, in vitro, or in vitro screening. silico for structurally unrelated anticancer molecules. For example, competition and other assays known in the art can be used to identify drugs capable of displacing the interaction with a higher affinity target, comparing changes in these levels with the corresponding changes obtained by a compound of formula (10) or its analogs. Assays can also be performed on living mammalian cells, which more closely approximate the effect of a given serum drug level in the body, or on microsomal extracts obtained from cultured cell lines.

In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the treatment regimen includes administering an effective amount of imipridone, such as ONC201, or an analogue thereof. In one embodiment of the present invention, the treatment regimen includes administering an effective amount of ONC201. In one embodiment of the present invention, the treatment regimen comprises administering an effective amount of a compound of formula (10). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (40), for example, a compound of formula (45). In one embodiment of the present invention, said compound is in the form

- 27 043367 ly (10) is a compound of formula (50), for example, formula (55). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (80). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (90). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (60). In one embodiment of the present invention, analogues of compound (1) have a structure selected from formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31 ).

Levels for a predetermined standard may be, for example, mean or median levels measured in samples from subjects. Levels for a predetermined standard can be measured under the same or substantially similar experimental conditions as when measuring a sample from a specified subject. Levels for a predetermined standard can be obtained from subjects who respond to treatment with imipridone, such as ONC201, or an analogue thereof. In one embodiment of the present invention, a predetermined standard is obtained from subjects who respond to treatment with the specified compound, and if the levels in the sample from the subject are similar to the levels in the standard, then the subject can be classified as likely to respond to treatment. Levels for a predetermined standard can be obtained from subjects who do not respond to treatment with the specified compound. In one embodiment of the present invention, a predetermined standard is obtained from subjects who do not respond to treatment with the specified compound, and if the levels in the sample from the subject differ (for example, up or down) from the predetermined standard, then the subject can be classified as one of those who are likely to respond to treatment. Levels for a predetermined standard can be obtained from normal healthy subjects.

Immunoassays can be used to analyze protein levels or methylation in a sample, including enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), flow cytometry, immunoblotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescence immunoassay (LIA), fluorescence immunoassay (FIA) and radioimmunoassay. Methylation levels of m6A mRNA can be obtained using methylated RNA immunoprecipitation (Me-RIP) or other quantitative biochemical assays known in the art.

Nucleic acid mutations can be determined by any of a number of known methods. For example, a biological sample may first be obtained from an individual. Such biological samples include, but are not limited to, a body fluid (eg, urine, saliva, plasma or serum) or a tissue sample (eg, a buccal tissue sample or a buccal epithelial cell). The biological sample can then be sequenced or scanned using known methods. For example, DNA arrays can be used to analyze at least a portion of a subject's genomic sequence. In addition, complete or partial genome sequence data can be used. These sequences can be determined using standard sequencing methods, including chain termination (Sanger dideoxynucleotide), dye terminator sequencing, and SOLID™ sequencing (Applied Biosystems). Whole genomic sequences can be cut by restriction enzymes or cut (mechanically) into shorter fragments for sequencing. DNA sequences can also be amplified using known methods such as PCR and vector-based cloning methods (eg Escherichia coli). In one embodiment of the present invention, at least a portion of the subject's genetic material (e.g., DNA, RNA, mRNA, cDNA, other nucleotide bases, or derivatives thereof) is scanned or sequenced, using, for example, conventional DNA sequencers or microprocessor-based technologies, to determine the presence or absence of mutations or copy number variations.

In one aspect provided herein, methods are described for identifying and treating a subject having a condition that is likely to respond to a treatment regimen described herein. In one embodiment of the present invention, the method comprises (i) determining the likelihood of a subject having a condition responding to a treatment regimen described herein; and (ii) treating with said treatment regimen a subject who has been determined to be likely to respond to said treatment regimen. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the treatment regimen includes administering an effective amount of imipridone, for example, ONC201 or an analogue thereof. In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of compound (1). In one embodiment of the present invention, the treatment regimen comprises administering an effective amount of a compound of formula (10). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (40), for example, a compound of formula (45). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (50), for example, formula (55). In one embodiment

- 28 043367 tions of the present invention, said compound of formula (10) is a compound of formula (80). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (90). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (60). In one embodiment of the present invention, analogues of compound (1) have a structure selected from formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31 ).

Levels for a predetermined standard may be, for example, mean or median levels measured in samples from subjects. Levels for a predetermined standard can be measured under the same or substantially similar experimental conditions as when measuring a sample from a specified subject. Levels for a predetermined standard can be obtained from subjects who respond to treatment with imipridone, such as ONC201 or an analogue thereof. In one embodiment of the present invention, a predetermined standard is obtained from subjects who respond to treatment with the specified compound, and if the levels in the sample from the subject are similar to the levels in the standard, then the subject can be classified as likely to respond to treatment. Levels for a predetermined standard can be obtained from subjects who do not respond to treatment with the specified compound. In one embodiment of the present invention, a predetermined standard is obtained from subjects who do not respond to treatment with the specified compound, and if the levels in the sample from the subject differ (for example, up or down) from the predetermined standard, then the subject can be classified as one of those who are likely to respond to treatment. Levels for a predetermined standard can be obtained from normal healthy subjects. Immunoassays can be used to analyze protein levels in a sample.

In another aspect, provided herein, methods are provided for treating and assessing the effectiveness of treatment in a subject having a condition. In one embodiment of the present invention, the method comprises (i) treating a subject in accordance with a treatment method described herein, (ii) assessing the effectiveness of the treatment as described herein. In one embodiment of the present invention, the subject has cancer or is at risk of cancer. In one embodiment of the present invention, the treatment regimen includes administering an effective amount of imipridone, such as ONC201 or an analogue thereof. In one embodiment of the present invention, said treatment regimen comprises administering an effective amount of compound (1). In one embodiment of the present invention, the treatment regimen comprises administering an effective amount of a compound of formula (10). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (40), for example, a compound of formula (45). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (50), for example, formula (55). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (80). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (90). In one embodiment of the present invention, said compound of formula (10) is a compound of formula (60). In one embodiment of the present invention, analogues of compound (1) have a structure selected from formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31 ).

Other conditions that may be suitable for the methods described herein include 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, cardiac arrhythmias, inflammatory heart disease, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital defects heart disease, rheumatic heart disease; diabetes and light chain amyloidosis.

V. Compositions.

In one aspect of the present invention there are provided pharmaceutical compositions containing compounds of formula (10):

(10)

- 29 043367 or formulas (1):

and pharmaceutically acceptable salts thereof. In one embodiment, said salt is a pharmaceutically acceptable monosalt of said compound. In one embodiment, said salt is a pharmaceutically acceptable di-salt of said compound. In one embodiment, said salt is a pharmaceutically acceptable monosalt or multi-salt (e.g., di-salt or tri-salt) of said compound selected from the group consisting of hydrochloride, hydrobromide, bisulfate, sulfates, phosphates, fumarates, succinates, oxalates, and lactates , bisulfates, hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate, maleate, fumarate sulfonate, methyl sulfonate, formate, acetate and carboxylate. In one embodiment of the present invention, said salt is a salt selected from the group consisting of p-toluenesulfonate, benzenesulfonate, citrate, methanesulfonate, oxalate, succinate, tartrate, fumarate and maleate. In one embodiment of the present invention, said salt is a salt selected from the group consisting of ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/or with counterions such as methylamino, dimethylamino, diethylamino and triethylamino counterions. In one embodiment, said salt is a dihydrochloride or dihydrobromide salt of said compound.

Compound (1) (ONC201) has the same chemical structure as can be detected by structural analysis (eg, NMR, X-ray diffraction) of compound NSC 350625, available in the National Cancer Institute Drug Development Program Data Archive.

In one embodiment, said pharmaceutical composition comprises a di-salt (ie, dihydrochloride salt) of ONC201 or an analogue thereof (ie, imipridone). Salts (ie, di-salts or tri-salts) of the ONC201 analogue can be prepared from an analogue of the ONC201 compound, which can be synthesized as described herein or using standard chemical synthesis methods known to one skilled in the art.

In one embodiment of the present invention, said pharmaceutical composition contains at least one pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, those listed in the Handbook of Pharmaceutical Excipients, ^7th ed., edited by Raymond C. Rowe et al., American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and earlier editions. Exemplary pharmaceutically acceptable carriers, methods of preparing pharmaceutical compositions and various dosage forms, and methods of administration are well known in the art, for example, as described in Pharmaceutical Dosage Forms: Tablets, edited by Larry L. Augsburger & Stephen W. Hoag., London: Informa Healthcare, 2008; and in L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, ^8th ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; AR Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, ^21st ed., 2005, especially Chapter 89; and in J. G. Hardman et al., Goodman &Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, ^10th ed., 2001.

In one embodiment of the present invention, the pharmaceutical compositions are formulated for ocular administration. In one embodiment of the present invention, said pharmaceutical composition is formulated for topical administration. In one embodiment of the present invention, said pharmaceutical compositions are formulated in the form of drops, ointments or liquids. In one embodiment of the present invention, said pharmaceutical composition contains conventional pharmaceutical carriers, for example, an aqueous, powder or oil base, thickeners.

In one embodiment of the present invention, the pharmaceutical composition is a formulation for intravenous administration. In one embodiment of the present invention, said composition for intravenous administration contains a compound of formula (10) or a pharmaceutically acceptable salt thereof dissolved in a solvent. In one embodiment, said solvent comprises water. In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof at a concentration of about 0.05, about 0.25, about 0.5, about 2.5, about 5, about 25, or about 50 mg/ml . In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof at a concentration of from about 0.05, 0.5, or 5 mg/ml to about 1, 10, or 100 mg/ml. In one embodiment of the present invention, said intravenous formulation contains from about 0.005, 0.05, or 0.5% to about 0.1, 1, or 10% of said compound or a salt thereof. In one embodiment of the present invention, said intravenous formulation contains about 0.05, 0.5, or 5% of said compound or a salt thereof. In some embodiments of the present invention, said intravenous formulation contains said compound or a salt thereof at a higher or lower concentration.

In one embodiment of the present invention, said intravenous formulation has a pH of about 3. In one embodiment of the present invention, said intravenous formulation is adjusted to pH 3 using phosphate buffer. In one embodiment of the present invention, the intravenous composition contains dextrose or sodium chloride. In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof at a concentration of about 5 mg/ml and a pH of 3 and forms a stable solution. In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof at a concentration of about 5 mg/ml and a pH < 5 and forms a stable solution. In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof and one or more antioxidants. In one embodiment of the present invention, said intravenous formulation contains a mixture of mono- and dihydrochloride salts of said compound. In one embodiment of the present invention, said intravenous formulation contains said compound or a salt thereof as a 1% solution at a concentration of about 10 mg/ml. For example, said intravenous formulation is a solution having a pH of about 3.3. In one embodiment of the present invention, the pH is less than 4.0.

In one embodiment of the present invention, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In one embodiment, said pharmaceutically acceptable carrier comprises a water-based carrier. In one embodiment of the present invention, said water-based carrier contains sterile water. In one embodiment of the present invention, the composition contains dextrose and/or sodium. In one embodiment, said pharmaceutically acceptable carrier comprises an oil.

In one embodiment of the present invention, the intravenous formulation contains ONC201 or an analogue thereof or a dihydrochloride salt thereof dissolved in water at a concentration of 25 mg/ml. In one embodiment of the present invention, the composition is adjusted to pH 3 using a phosphate buffer. In one embodiment of the present invention, the composition contains dextrose, sodium chloride, or both. In one embodiment of the present invention, the composition contains the dihydrochloride salt of ONC201 or an analogue thereof at a higher or lower concentration. In one embodiment of the present invention, the composition contains ONC201 or an analogue thereof or a dihydrochloride salt thereof at a concentration of about 5 mg/ml. In one embodiment of the present invention, the composition at a concentration of approximately 5 mg/ml forms a stable solution and pH=3. In one embodiment of the present invention, the composition at a concentration of about 5 mg/ml has a pH < 5 and forms a stable solution. In one embodiment of the present invention, the intravenous formulation contains ONC201 or an analog thereof or a dihydrochloride salt thereof and one or more antioxidants. In one embodiment of the present invention, the intravenous formulation contains a mixture of mono- and dihydrochloride salts of ONC201 or an analogue thereof. In one embodiment of the present invention, the intravenous formulation contains ONC201 or an analogue thereof or a dihydrochloride salt thereof as a 1% solution at a concentration of about 10 mg/ml. For example, said intravenous formulation is a solution having a pH of about 3.3. In one embodiment of the present invention, the pH is less than 4.0.

In one embodiment of the present invention, the intravenous formulation contains from about 0.5% to about 10% (or from about 5 mg/ml to about 100 mg/ml) ONC201 or an analogue or di-salt thereof. In one embodiment of the present invention, the intravenous formulation contains about 5% (or about 50 mg/ml) ONC201 or an analogue or di-salt thereof. In one embodiment of the present invention, the rate of intravenous administration can be slowed down to reduce the side effects of ONC201 or an analog or disol thereof.

In one embodiment of the present invention, said pharmaceutical composition contains about 0.1-99% ONC201 salt or an analogue thereof; and a pharmaceutically acceptable carrier, for example, oil or sterile water or other aqueous carrier. In one embodiment of the present invention, the composition contains a mono- or di-salt of ONC201 or an analog thereof in the range of from about 5 to about 50% for oral dosage forms.

In one embodiment of the present invention, the pharmaceutical composition contains an antioxidant. Suitable antioxidants include: derivatives of ascorbic acid, such as ascorbic acid, erythorbic acid, sodium ascorbate, thiol derivatives, such as thioglycerin, cysteine, acetylcysteine, cystoeritritol, ditiotheol, glutation, tocopherols, butyl- 31,04367. Sianisol (VNA), butylated hydroxytoluene (BHT), sulfuric acid salts such as sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate and sodium thiosulfate, nordihydroguaiaretic acid. It should be noted that antioxidants used for aqueous formulations typically include: sodium sulfite, sodium metabisulfite, sodium formaldehyde sulfoxylate and ascorbic acid and combinations thereof, while antioxidants used in oil-based solutions and organic solvents include butylated hydroxytoluene (BHT ), butylated hydroxyanisole (BHA) and propyl gallate and combinations thereof. In other embodiments of the present invention, the antioxidant may be one or more of a flavonoid, isoflavone, monothioglycerol, L-cysteine, thioglycolic acid, α-tocopherol, ascorbic acid 6-palmitate, dihydrolipoic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole ( BHA), vitamin E, propyl gallate, β-carotene, ascorbic acid. Antioxidants are typically used in an amount of about 0.1 to 1.0% by weight, most commonly about 0.2%.

In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201 or an analogue thereof, or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent. For example, said other therapeutic agent is selected from hormone analogs and antihormones, aromatase inhibitors, LH-RH agonists and antagonists, growth factor inhibitors, anti-growth factor antibodies, anti-growth factor receptor antibodies, tyrosine kinase inhibitors; antimetabolites; antitumor antibiotics; platinum derivatives; alkylating agents; antimitotic agents; tubulin inhibitors; PARP inhibitors, topoisomerase inhibitors, serine/threonine kinase inhibitors, tyrosine kinase inhibitors, protein-protein interaction inhibitors, RAF inhibitors, MEK inhibitors, ERK inhibitors, IGF1R inhibitors, ErbB receptor inhibitors, rapamycin analogs, BTK inhibitors, CRM1 inhibitors (eg, KPT185), P53 modulators (eg, nutlins), antiangiogenic agents (eg, axitinib, aflibercept, sorafenib and regorafenib), amifostine, anagrelide, clodronate, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, 2 -chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-azaepothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomin, aranose, arglabin, arzoxifene, atamestane, atrasentan, auristatin RE, AVT-199 (venetoclax), AVT-263 ( navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib), ARRY-704/AZD-8330, AR-12 , AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD6244, ARQ-736, ARQ 680, AS-703026 (primasertib), Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (afatiniba, tomtovoka), BIBF 1120 (vargatefa), BI 836845, BI 2536, BI 6727, BI 83 6845, BI 847325, BI 853520, BUB-022, bleomycin acid, bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin, busulfan, BYL-719, CA-4 prodrug, CA-4, CapCell, calcitriol, canertinib, canfosfamide, capecitabine, carboxyphthalatoplatinum, CCl-779, CC-115, CC-223, CEP-701, CEP-751, SVT-1 cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleuquin, cemadotin, CH4987655/RO-4987655, chlorine trianisene, cilengitide , cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, combretastatin A4, Cot inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, STP-37 , monoclonal antibodies CTLA-4, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diphlomothecan, didoxa, DMDC, dolastatin 10, doranidazole, DS-7423, E7010, E-6201, edatrexate, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib, ET- 18-OCNZ, ethinylcytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, gallium maltolate, gefitinib, heme tuzumab, gimatecan, glufosfamide, GCS -100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, immunogen G17DT, GMK, GPX-100, gp100-peptide vaccines, GSK- 5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homogarringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibrutinib, ibritutomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), immunol, indisulam, interferon alpha-2a, interferon alpha- 2b, pegylated interferon alpha-2b, interleukin-2, INK1117, INK-128, INSM-18, ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin

- 32 043367

B, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, cahalide F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuprorelin, lexidronam, LGD- 1550, linezolid, lutetium texafirin, lometrexol, losoxantrone, LU 223651, lurtothecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechlorethamine, MEK inhibitors, MEK-162, methyltestosterone, methylprednisolone, MEDI-573 , MEN-10755, MDX -H21O, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin, mivobulin, MK2206, MK-0646 (dalotuzumab), MLN518, motexafine gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906 (linsitinib), antibodies 4-1BB, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF0519 7281 , PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors, PG-TXL, PG2, PLX-4032/RO-5185426 (VEMurafeniba), PLX-3603/RO-5212054, RT-100, PWT-33597, PX-866, Picoplatin, Pivaloiloximethyrate, Pixanthron, Fenxiola O, PKI166, PKI-OD on, polyprenic acid , porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA436, rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors, regorafenib, revimid, RG-7167, RG- 7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecana, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim, satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101, semustine, seocalcitol, SM-11355, SN-38 , SN4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatinum, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tatsedinalin, thalaporfin, tarcev a, tariquitara, tasisulam, taxotera, taxoprexin, tazarotene, tegafur, temozolomide, tesmilifen, testosterone, testosterone propionate, tesmilifen, tetraplatin, tetrodotoxin, tezacitabine, thalidomide, teralux, terarubicin, thymalfasin, timektacin, tiazofurin, tipifarnib, tirapazamine, tocladesine, tomex, toremofin , trabectedin, TransMID-107, transretinoic acid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidine, valrubicin, vatalanib, vincristine, vinflunine, viruliz ina, WX- UK1, WX-554, Vectibixa, Xeloda, XELOX, XL-147, XL-228, XL-281, XL518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD9331, ZD1839, ZSTK-474, zoledronate, zosuquidar and their combinations.

In one embodiment of the present invention, said other therapeutic agent comprises a hormone analog and/or an antihormone, or both, selected from tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone, octreotide and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more LH-RH agonists and/or antagonists selected from goserelin acetate, leuprolide acetate, triptorelin pamoate and combinations thereof, and wherein the LH-RH antagonists are selected from Degarelix, Cetrorelix, Abarelix , Ozarelix, their combinations with Degarelix. In one embodiment of the present invention, said other therapeutic agent comprises one or more growth factor inhibitors selected from: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) , insulin-like growth factors (IGF), human epidermal growth factor (HER) and hepatocyte growth factor (HGF). In one embodiment of the present invention, said other therapeutic agent comprises one or more human epidermal growth factor inhibitors selected from HER2, HER3, and HER4. In one embodiment of the present invention, said other therapeutic agent comprises one or more tyrosine kinase inhibitors selected from cetuximab, gefitinib, imatinib, lapatinib and trastuzumab and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more aromatase inhibitors selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent contains one or more antimetabolites, which are antifolates selected from methotrexate, raltitrexed and pyrimidine analogues. In one embodiment of the present invention, said other therapeutic agent contains one or more antimetabolites that are pyrimidine analogues selected from 5-fluorouracil, capecitabine and gemcitabine. In one embodiment of the present invention, said other therapeutic agent contains one or more antimetabolites that are purine and/or adenosine analogs selected from mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine, fludarabine, and combinations thereof. In one embodiment of the present invention

- 33 043367 and said other therapeutic agent contains one or more antitumor antibiotics selected from anthracyclines, doxorubicin, daunorubicin, epirubicin and idarucibin, mitomycin-C, bleomycin, dactinomycin, plicamycin, streptozocin and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more platinum derivatives selected from cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more alkylating agents selected from estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolamide, nitrosoureas, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises nitrosoureas selected from carmustine, lomustine, thiotepa, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises antimitotic agents selected from vinca alkaloids and taxanes. In one embodiment of the present invention, said other therapeutic agent comprises one or more taxanes selected from paclitaxel, docetaxel, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more vinca alkaloids selected from vinblastine, vindesine, vinorelbine, vincristine, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more topoisomerase inhibitors that are epipodophyllotoxins. In one embodiment of the present invention, said other therapeutic agent comprises one or more epipodophyllotoxins selected from etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more serine/threonine kinase inhibitors selected from PDK 1 inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTORC1/PI3K inhibitors, STK 33 inhibitors, AKT, PLK 1 inhibitors, CDK inhibitors, Aurora kinase inhibitors and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more tyrosine kinase inhibitors, which are PTK2/FAK inhibitors. In one embodiment of the present invention, said other therapeutic agent comprises one or more protein-protein interaction inhibitors selected from IAP, Mcl-1, MDM2/MDMX, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more rapamycin analogs selected from everolimus, temsirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment of the present invention, said other therapeutic agent comprises one or more therapeutic agents selected from amifostine, anagrelide, clodronate, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer and combinations thereof . In one embodiment of the present invention, said other therapeutic agent comprises one or more therapeutic agents selected from 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4,3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10 -hydroxycamptothecin, 16azaepothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apamin, aranose, arglabin, arzoxifene, atamestane, atrasentan, auristatin RE, AVT-199 (venetoclax), AVT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD- 6244 (selumetinib), ARRY-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS- 703026 (primasertib), Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (afatinib, Tomtovoka), BIBF 11 20 (vargatefa), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycin acid, bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin, busulfan, BYL -719 , prodrugs CA-4, CA-4, CapCell, calcitriol, canertinib, canfosfamide, capecitabine, carboxyphthalatoplatin, CCl-779, CC-115, CC-223, CEP-701, CEP-751, SVT-1 cefixime, ceflatonin, ceftriaxone , celecoxib, celmoleuquin, cemadotin, CH4987655/RO-4987655, chlorotrianizene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI27, clofarabine, colchicine, combretastatin A4, COT inhibitors, CHS-828, CH-51327 99 , CLL-Thera, CMT-3 cryptophycin 52, STP-37, monoclonal antibodies CTLA-4, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepothilone B, dexamet azona , dexrazoxanet, diethylstilbestrol, diphlomothecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010, E-6201, edatrexate, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enza lutamide, elsamitrucine , epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-OSNZ, ethinylcytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, form stana, fotemustina, Galarubi

- 34 043367 cin, gallium maltolate, gefitinib, gemtuzumab, gimatecan, glufosfamide, GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879 , immunogen G17DT, GMK, GPX-100, gp100-peptide vaccines, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334 470, GSK -2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homogarringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibrutinib, ibritutomab, idatrexate, idenestrol, IDN-5109, inhibitors IGF-1R, IMC-1C11, IMC-A12 (cixutumumab), immunol, indisulam, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha2b, interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab, iproplatin, irofulvene, isohomohalichondrin -B, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, cahalide F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuprorelin, lexidronam, LGD -1550, linezolid, lutetium texafirin, lometrexol, losoxantrone, LU 223651, lurtothecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechlorethamine, MEK inhibitors, MEK-162, methyltestosterone, methylprednisolone, MEDI-573 , MEN-10755, MDX-H21O, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexafine gadolinium, MS-209, MS275, MX6, neridronate, neratinib , Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-027, OSI-906 (linsitinib) , 41BB antibodies, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF- 05197281, PF-05212384, PF-04691502, RNT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors, PG-TXL , PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054, RT-100, PWT-33597, PX-866, picoplatinum, pivaloyloxymethylbutyrate, pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprene ova acids, porphyromycin, prednisone, prednisolone, Quinnamed, Quino-Presistine, R115777, RAF-265, Rametron, Ranpirnase, RDEA-119/Bay 869766, RDEA-436, ANASOVENS of Rebecycin, inhibitors (RTK), Revimida, RG7 167, RG-7304 , RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan , R -flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim, satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101, semustine, seocalcitol, SM-11355, SN-38, SN4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tatsedinalin, talaporfin, tarceva, tariquitara , tasisulam, taxotere, taxoprexin, tazarotene, tegafur, temozolomide, tesmilifen, testosterone, testosterone propionate, tesmilifen, tetraplatin, tetrodotoxin, tezacytabine, thalidomide, theralux, terarubicin, thymalfasin, timectacin, tiazofurin, tipifarnib, tirap zamina, tocladesine, tomex, toremofin, trabectedin, TransMID-107, transretinoic acid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin, valrubicin, vatalanib, vincristine, vinflunine, virulysin, WX-UK1 , WX-554, Vectibixa, Xeloda, XELOX, XL-147, XL-228, XL-281, XL518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD -6474, ZD-4054, ZD-0473, ZD-6126, ZD9331, ZD1839, ZSTK-474, zoledronate, zosuquidar and combinations thereof.

In one embodiment of the present invention, said other therapeutic agent comprises a steroid, including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazole. In one embodiment of the present invention, said other therapeutic agent comprises an antiemetic agent. These antiemetics include, but are not limited to, 5-HT3 receptor agonists (such as dolasetron, granisetron, ondansetron, tropisetron, palonosetron and mirtazapine), dopamine agonists (such as domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine and metoclopramide), NK1 receptor antagonists (such as aprepitant and casopitant), antihistamines (such as cyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine), cannabinoids (such as cannabis, dronabinol, nabilone and sativex), benzodiazepines (such as midazolam and lorazepam), anticholinergics (such as hyoscine), trimethobenzamide, ginger, emetrol, propofol, peppermint, muscimol and ajgon.

In some embodiments of the present invention, said other therapeutic agent comprises an anticancer agent that includes a mitosis inhibitor. In one embodiment of the present invention, said mitosis inhibitor comprises a taxane. In one embodiment of the present invention, said mitosis inhibitor comprises a taxane selected from the group consisting of paclitaxel and docetaxel.

In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, and

- 35 043367 at least one anti -cancer agent, while the specified anti -cancer agent includes one or more of: activic, aclarubicin, acodazole, acronine, adeslezine, alvesileikin, ilitretinoin, allopurinol, altretine, ambomicin, amifostin, amino -zero -amphostine, aminoglutethemide, amzacarin, aminogluthemide, aminacrine, AMSACRINA, AMCSACRINA, AMCSACRINA, AMCSACRINA, AMCSACRINA, AMCSACRINA, AMCSARINA. anastrosola , anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azethep, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, bisnafide dimesylate, biselezin, bleomycin, brequinar, bropyrimine, busulfan, cactinomycin, calusterone, capecite bina, caracemide, carbetimer, carboplatin , carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, deazaguanine, deazaguanine me zylate, diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone , duazomycin, edatrexate, elfomithine, elsamitrucin, enloplatin, enpromat, epipropidine, epirubicin, erbulozole, ezorubicin, estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, fluorouracil , fluorcitabine, fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea, idarucibin, ifosfamide, ilmofosin, interleukin II (IL-2, including recombinant interleukin II or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta- Ia, interferon gamma-Ib, iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol, melengestrol acetate, melphalan, menogaryl, mercaptopurine, methotre xata, methoprine, meturedepa, mitindomide , mitocarcin, mitocromine, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin, ormaplatin, oxysuran, paclitaxel, pegaspargase, peliomycin, pentamustine, eplomycin, perf osfamide, pipobromane, piposulfan, piroxantrone hydrochloride, plicamycin , plomestan, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprin, rogletimide, safingol, semustine, simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine, spiroplatinum, streptonigrin, streptozocin, sulofenur, taliz icin, tamoxifen, tecogalan, tegafur, teloxantrone , temoporfin, teniposide, teroxiron, testolactone, thiamiprin, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone, triciribine, trimetrexate, triptorelin, tubulozol, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, wine Christina sulfate, vindesine, vinepidine , vinglycinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, zorubicin and combinations thereof.

Examples of suitable anticancer agents include those described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th Ed., edited by Laurence Brunton, Bruce Chabner, Bjorn Knollman, McGraw Hill Professional, 2010.

ONC201 In some illustrative embodiments of the present invention, the pharmaceutical composition comprises a salt (eg, mono- or di-salt) of imipridone, i.e. ONC201, or an analog thereof, and at least one other therapeutic agent, wherein said other therapeutic agent comprises an antiangiogenic agent, for example, bevacizumab. In one embodiment of the present invention, said antiangiogenic agent is selected from aflibercept, axitinib, angiostatin, endostatin, prolactin 16 kDa fragment, laminin peptides, fibronectin peptides, tissue metalloproteinase inhibitors (TIMP 1, 2, 3, 4), plasminogen activators, inhibitors (PAI -1, -2), tumor necrosis factor α, (in high doses, in vitro), TGF-e1, interferons (IFNα, -β, γ), chemokines ELR-CXC:, IL-12; SDF-1; MIG; platelet factor 4 (PF-4); IP-10, thrombospondin (TSP), SPARC, 2-methoxyestradiol, proliferin-associated protein, suramin, sorafenib, regorafenib, thalidomide, cortisone, linomide, fumagillin (AGM-1470; TNP-470), tamoxifen, retinoids, CM101, dexamethasone , leukemia inhibitory factor (LIF), Hedgehog inhibitor and combinations thereof.

The pharmaceutical combination may contain the first and second therapeutic agents in any desired proportions, provided that a synergistic effect or cooperative effect still occurs. Said synergistic pharmaceutical combination preferably contains the first and second therapeutic agents in a ratio of about 1:9 to about 9:1. In one embodiment of the present invention, the synergistic combination contains the first and second therapeutic agents in a ratio of about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1, from about 1:5 to about 5:1, from about 1:4 to about 4:1, from about 1:3 to about 3:1, or from about 1:2 to about 2:1. In one embodiment of the present invention, the synergistic pharmaceutical combination contains the first and second therapeutic agents in a ratio of approximately 1:1.

In one embodiment of the present invention, said second therapeutic agent is selected from allopurinol, arsenic trioxide, azacitidine, bortezomib, bevacizumab, capecitabine, carboplatin, celecoxib, chlorambucil, clofarabine, cytarabine, dacarbazine, daunorubicin HCl, docetaxel, doxorubicin HCl, floxuridine, heme citabine HCl, hydroxyurea, ifosfamide,

- 36 043367 imatinib mesylate, ixabepilone, lenalidomide, megestrol acetate, methotrexate, mitotane, mitoxantrone HCl, oxaliplatin, paclitaxel, pralatrexate, romidepsin, sorafenib, streptozocin, tamoxifen citrate, topotecan HCl, tretinoin, vandetanib, vismodegib, vorinostat and their combinations.

In one embodiment of the present invention, said second therapeutic agent comprises a small molecule multikinase inhibitor, i.e. sorafenib or regorafenib. In one embodiment of the present invention, said second therapeutic agent comprises an inhibitor of the Hedgehog signaling pathway, i.e. vismodegib. In one embodiment of the present invention, said second therapeutic agent comprises drugs selected from Table. 2 below.

table 2

Drug classes

Drug classes Examples Purine analogues Allopurinol, oxypurinol, clofarabine and tisopurine Pyrimidine analogues 5-fluorouracil, Floxuridine (FUDR), capecitabine, cytarabine, 6-azauracil (6-AU) and gemcitabine (Gemzar) Proteasome inhibitors bortezomib, carfilzomib, cediranib, disulfiram, epigallocatechin-3-gallate, salinosporamide A, ONCX 0912, CEP18770, MLN9708, epoxomicin and MG132 Antiangiogenic substances bevacizumab, aflibercept, sunitinib, sorafenib, pazopanib, vandetanib, cabozantinib, axitinib, ponatinib, regorafenib, ranibizumab, lapatinib and vandetanib Platinum-containing antitumor drugs Cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedolatin and triplatin COX-2 inhibitors Celecoxib, valdecoxib (Bextra), parecoxib (Dynastat), lumiracoxib, etoricoxib, and rofecoxib Nitrogen mustards Cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan, bendamustine and mustard gas Alkylating agents cyclophosphamide, mechlorethamine or mustine (HN2) (trade name Mustardgen), uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin and busulfan Anthracyclines daunorubicin (daunomycin), daunorubicin (liposomal), doxorubicin (adriamycin), doxorubicin (liposomal),

- 37 043367

epirubicin, idarucibin, valrubicin and mitoxantrone Taxanes paclitaxel (Taxol), docetaxel (Taxotere), and albumin-bound paclitaxel (Abraxane) Synthesis inhibitor nucleotides methotrexate, pralatrexate, hydroxyurea and 5fluorodeoxyuridine, 3,4dihydroxybenzylamine BC-α inhibitors imatinib, nilotinib, dasatinib, bosutinib and ponatinib Other arsenic trioxide, thalidomide, remilide and mitotane Inhibitors topoisomerases amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, Topotecan (Hycamtin), Irinotecan (CPT-11, Camptosar), Exatecan, Lurtotecan, ST 1481, CKD 602, ICRF-193 and genistein HDAC inhibitors Vorinostat (SAHA), Romidepsin (Istodax), Panobinostat (LBH589), Valproic acid (as MD valproate), Belinostat (PXD101), Mocetinostat (MGCD0103), Abexinostat (PCI-24781), Entinostat (MS-275), SB939, Resminostat (4SC-201), Givinostat, Quisinostat (JNJ26481585), CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, Sulforaphane, Kevetrin and ATRA Multitarget kinase inhibitors sorafenib, regorafenib and vandetanib Hormonal agents tamoxifen, toremifene, arimidex (anastrozole), aromazin (exemestane), Femara (letrozole) and fulvestrant (faslodex) Hedgehog signaling pathway inhibitors vismodegib, BMS-833923, IPI-926, LDE- 225, PF-04449913, LEQ 506 and TAK-441 Checkpoint inhibitors Opdivo (nivolumab), Durvalumab (Medi4736), Keytruda (pembrolizumab, MK3475), BGB-A317, AMP-224, PDR001, REGN 281atezolizumab (MPDL3280A), pidilizumab (BMS-936559, CT-011, ONO4538), avelumab (MSB001 0718 C ), Yervoy (ipilimumab), tremelimumab BCL2 inhibitors AT-101, Bcl-2/xL inhibitor, navitoclax (AVT-263), venetoclax (AVT-199), apogosipol, PTN1258, obatoclax, G3139

In one embodiment of the present invention, said second therapeutic agent comprises drugs that target tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. In one embodiment of the present invention, said second therapeutic agent comprises a recombinant TRAIL or an agonistic antibody that activates one or more TRAIL receptors. In one embodiment of the present invention, said second therapeutic agent comprises one or more antibodies or recombinants

- 38 043367 nant TRAIL, which activate signaling through DR4, DR5, or both. In one embodiment of the present invention, said second therapeutic agent comprises one or more of AMG-655, LBY-135, mapatumumab, lexatumumab, apo2L/TRAIL. In one embodiment of the present invention, said second therapeutic agent comprises an active agent selected from camptothecin, 5-FU, capecitabine, cisplatin, doxorubicin, irinotecan, paclitaxel, cisplatin, bortezomib, BH3I-2, rituximab, radiation, triterpenoids, sorafenib, gemcitabine, HDAC inhibitors, carboplatin, T-101 (gossypol derivative), ABT-263, ABT-737 and GX-15-070 (obatoclax), vorinostat, cetuximab, panitumumab, bevacizumab, ganitumab, interferon gamma, sorafenib, XIAP antagonists, Bcl antagonists -2 and Smac mimetics.

VI. Dose.

In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose of from about 40, 50, 60 or 100 mg to about 2000 mg; from about 4, 5, 6 or 10 mg to about 200 mg; or about 0.4, 0.5, 0.6, or 1 mg to about 20 mg, which weight may be calculated based on said compound in free base form. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 50 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg; from about 5 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg; or from about 0.5 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mg. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 40 mg to about 200, 300, 400, 500, 6θθ, 700, 800, 900, 1θθθ, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2θ0θ mg; from about 4 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg; or from about 0.4 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mg. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 60 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg; from about 6 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 20θ mg; or from about 0.6 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 100 mg to about 200, 300, 400, 500, 600, 700, 800, 900 , 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 19θ0 mg or 2000 mg; from about 10 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg; or from about 1 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 200 mg to about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg; from about 20 mg to about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg; or from about 2 mg to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19 or 20 mg based on the compound in the form of its free base. In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 400 mg to about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 190θ or 2000 mg; from about 40 mg to about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg; or from about 4 mg to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19 or 20 mg based on the compound in its free base form . In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 50 to about 60, 70, 80, 90 or 100 mg; from about 60 to about 70, 80, 90 or 100 mg; from about 70 to about 80, 90 or 100 mg, from about 80 to about 90 or 100 mg; from about 90 mg to about 100 mg; from about 5 mg to about 6, 7, 8, 9 or 10 mg; from about 6 to about 7, 8, 9 or 10 mg; from about 7 to about 8, 9 or 10 mg, from about 8 to about 9 or 10 mg; from about 9 mg to about 10 mg; from about 0.5 mg to about 0.6, 0.7, 0.8, 0.9 or 1 mg; from about 0.6 mg to about 0.7, 0.8, 0.9 or 1 mg; from about 0.7 to about 0.8, 0.9 or 1 mg, from about 0.8 to about 0.9 or 1 mg; or from about 0.9 mg to about 1 mg.

In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, at a dose of about 1 mg/kg to about 40 mg/kg; 0.1 mg/kg to approximately 4 mg/kg; or from 0.01 mg/kg to about 0.40 mg/kg. In one embodiment of the present invention, said pharmaceutical composition comprises imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from about 1, 2, 3, 4, 5, 6, 7, 8, or 9 mg/kg to about 10, 20, 30 or 40 mg/kg; from about 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mg/kg to about 20, 30 or 40 mg/kg; from about 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 mg/kg to about 30 or 40 mg/kg; from about 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 mg/kg to about 40 mg/kg; from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mg/kg to about 1, 2, 3 or 4 mg /kg; from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 mg/kg to about 2, 3 or 4 mg/kg; from about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 mg/kg to about 3 or 4 mg /kg; or from about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9 mg/kg to about 4 mg/kg kg; from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 mg/kg to about 0.10, 0.20, 0.30 or 0.40 mg/kg; from about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18 or from 0.19 mg/kg to about 0.20 , 0.30 or 0.40 mg/kg; from about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28 or from 0.29 mg/kg to about 0.30 or 0.40 mg/kg; or from about 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, or 0.39 mg/kg to about 0.40 mg/kg.

In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, at a dose ranging from about 37.5 mg/ ^m2 to about 1500 mg/ ^m2 ; from about 3.75 mg/m ² to about 150 mg/m ² ; or from about 0.4 mg/m ² to about 15 mg/m ² . In one embodiment of the present invention, said pharmaceutical composition contains imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging from 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215 , 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305,

310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410,415,

420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,525,

530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630,635,

640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740,745,

750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850,855,

860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960,965,

970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1 075, 1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145,1150,

1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235,1240,

1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325,1330,

1335, 1340, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415,1420,

1425, 1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495 mg/ ^m2 to about 1500 mg/ ^m2 ; from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,

114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149 mg/ ^m2 to about 150 mg/ ^m2 ; or from about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 , 8.5, 9, 9.5, 10, 10.5, 11, 111, 11.5, 12, 12.5, 13, 13.5, 14 or 14.5 mg/ ^m2 to about 15 mg / ^m2 .

VII. Dosage forms.

Suitable pharmaceutical compositions for use in the methods described herein can be formulated into a dosage form that can be administered to a patient. In one embodiment of the present invention, said pharmaceutical composition is in the form of an oral dosage form or a parenteral dosage form. In one embodiment of the present invention, said pharmaceutical composition is in the form of an oral dosage form. In one embodiment of the present invention, said oral dosage form is divided into several smaller doses that are administered to a subject over a predetermined period of time to reduce the toxicity of the administered therapeutic agent. In some embodiments of the present invention, said oral dosage form is administered as a tablet or capsule containing a controlled release formulation, which may include multiparticulates, granules, pellets, mini-tablets, or tablets. In one embodiment of the present invention, said pharmaceutical composition is in the form of a parenteral dosage form. In one embodiment of the present invention, said pharmaceutical composition is in the form of a parenteral dosage dosage form selected from the group consisting of intravenous (IV), subcutaneous (SC) and intramuscular (IM), rectal (R) and transdermal dosage dosage form. In one embodiment of the present invention, said pharmaceutical composition is in the form of a dosage form selected from the group consisting of sterile solutions, suspensions, suppositories, tablets and capsules. In one embodiment of the present invention, said composition is in the form of an oral dosage form selected from the group consisting of tablet, caplet, capsule, lozenge, syrup, liquid, suspension and elixir. In one embodiment of the present invention, the composition is in the form of an oral dosage form selected from the group consisting of tablets, hard capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solid and semi-solid forms.

In some embodiments of the present invention, suitable forms of pharmaceutical compositions for use in the methods described herein include dermatological compositions adapted for topical administration through the skin.

Dermatological compositions contain a cosmetically or pharmaceutically acceptable vehicle. These dermatological compositions for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In one embodiment, conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners, skin permeation enhancers, and the like may be needed or desired and therefore may be used. Examples of suitable enhancers include ethers such as diethylene glycol monoethyl ether (commercially available as Transcutol®) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and lecithin (US Pat. No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzyl alcohol and the like; polyethylene glycol and its esters, such as polyethylene glycol monolaurate; amides and other nitrogen-containing compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine and triethanolamine; terpenes; alkanones; and organic acids, in particular citric acid and succinic acid. Azone® and sulfoxides such as DMSO and C10MSO can also be used, but are less preferred.

In one embodiment of the present invention, said pharmaceutical composition is in the form of a dosage form selected from the group consisting of sustained release forms, controlled release forms, delayed release forms and response release forms.

VIII. Methods of application.

The compositions and methods described herein find use in the treatment of many disease states, including cancer (eg, colorectal cancer, brain cancer, and glioblastoma). In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as ocular melanoma, desmoplastic small round cell tumor, chondrosarcoma, leptomeningeal disease, diffuse large cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-associated cancers, AIDS-associated lymphoma, anal and rectal cancer, appendix cancer, astrocytomas and atypical teratoid/rhabdoid tumor. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as basal cell carcinoma, basal cell nevus syndrome, Gorlin syndrome, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, tumor brain, breast cancer, bronchial tumors, Burkitt's lymphoma and spinal cord tumors. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as carcinoid tumor, carcinoma of unknown primary site, atypical teratoid/rhabdoid tumor of the central nervous system, leptomeningeal disease, embryonal tumors of the central nervous system, central nervous system lymphoma. nervous system, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative diseases, colon cancer, colorectal cancer, craniopharyngioma and cutaneous T-cell lymphoma (including Sézary syndrome and mycosis fungoides (FM)). In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as germinal tumors of the central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing's sarcoma family, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer and ocular cancer, including intraocular melanoma and retinoblastoma. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, and glioma. In one embodiment of the present invention, the compositions and methods described herein are used to treat a cancer selected from the group consisting of hairy cell leukemia, head and neck cancer, hepatocellular cancer (liver cancer), histiocytosis, Hodgkin's lymphoma, and hypopharyngeal cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as Kaposi's sarcoma and kidney cancer (renal cell carcinoma). In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lung cancer, including non-small cell lung cancer and small cell lung cancer, non-Hodgkin's lymphoma and primary lymphoma of the central nervous system. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as Waldenström's macroglobulinemia (lymphoplasmacytic lymphoma), malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary site, multiple endocrine neoplasia syndrome, oral cancer, multiple myeloma/plasma cell neoplasm, fungoid mycosis, myelodysplastic syndromes, complex karyotype, blast leukemia, myelodysplastic/myeloproliferative neoplasms, multiple myeloma and myeloproliferative diseases. In one embodiment of the present invention, the compositions and methods described herein are used to treat cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as nasal and sinus cancer, nasopharyngeal cancer, and neuroblastoma. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian germ cell tumor, epithelial ovarian cancer and ovarian tumors with low malignant potential. In one embodiment of the present invention, the compositions and methods described herein are used to treat diseases such as pancreatic cancer, papillomatosis, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumors intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma and prostate cancer. In one embodiment of the present invention, the compositions and methods described herein are used for the treatment of cancer selected from the group consisting of colorectal cancer, renal cell carcinoma (kidney), renal pelvis and ureter cancer, respiratory tract carcinoma involving NUT -gene on chromosome 15, retinoblastoma and rhabdomyosarcoma. In one embodiment of the present invention, the compositions and methods described herein are used to treat high-grade prostate cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat moderate prostate cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat low-grade prostate cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat castration-resistant prostate cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat a tumor of the nervous system. In one embodiment of the present invention, the compositions and methods described herein are used to treat a tumor of the central nervous system. In one embodiment of the present invention, the compositions and methods described herein are used to treat a tumor of the peripheral nervous system. In one embodiment of the present invention, the compositions and methods described herein are used to treat paraganglioma. In one embodiment of the present invention, the compositions and methods described herein are used to treat pheochromocytoma.

In in vitro models, animal models and human clinical trials, compound (1) (ONC201) has broad spectrum anticancer activity, low toxicity including few if any side effects, low genotoxicity and high bioavailability including oral bioavailability . These features make ONC201 and its various analogues particularly suitable for pediatric patients. These attributes make ONC201 and various analogues particularly well suited for long-term therapy, for high-risk patients, and for providing durable responses or preventing relapse of stable disease or preventing recurrence of disease.

In one embodiment of the present invention, the compositions and methods described herein are used for the treatment of childhood cancer (solid tumors in children, sarcomas in children, Ewing's sarcomas in children, gliomas in children, cancer of the central nervous system in children, leukemia in children and lymphoma in children).

In one embodiment of the present invention, the compositions and methods described herein are used to treat a proliferative skin disorder such as psoriasis. In one embodiment of the present invention, the compositions and methods described herein

- 42 043367 document, used for the treatment of cancer selected from the group consisting of salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, eye cancer, skin carcinoma, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck with unknown primary localization and supratentorial primitive neuroectodermal tumors. In one embodiment of the present invention, the compositions and methods described herein are used to treat cancer selected from the group consisting of T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma renal pelvis and ureter and gestational trophoblastic tumor. In one embodiment of the present invention, the compositions and methods described herein are used to treat a cancer selected from the group consisting of carcinoma of unknown primary site, cancer of unknown primary site, uncommon childhood cancers, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer and uterine sarcoma. In one embodiment of the present invention, the compositions and methods described herein are used to treat cancer selected from the group consisting of vaginal and vulvar cancer. In one embodiment of the present invention, the compositions and methods described herein are used to treat a cancer selected from the group consisting of Wilms tumor and gynecological cancers.

In one embodiment of the present invention, the compositions and methods described herein are used as first-line therapy (sometimes referred to as primary therapy). In one embodiment of the present invention, the compositions and methods described herein are used as second-line therapy. In one embodiment of the present invention, the compositions and methods described herein are used as third-line therapy. In one embodiment of the present invention, the compositions and methods described herein are used as salvage therapy. The term rescue therapy as used herein refers to a therapeutic agent that can be administered in any regimen after the subject's initial treatment regimen has failed or after the subject has failed to respond to the initial treatment. In one embodiment of the present invention, the compositions and methods described herein are used as backup therapy. In one embodiment of backup therapy, the compositions are used as a backup agent to counteract the effects of the initial treatment. In one embodiment of salvage therapy, the compositions are used as a salvage agent to be administered to a subject who is already resistant to standard or initial treatment. In one embodiment of the present invention, the compositions and methods described herein are used as neoadjuvant therapy. In one embodiment of the present invention, said neoadjuvant therapy comprises administering to a subject one or more therapeutic agents described herein prior to primary or first line therapy. In one embodiment of the present invention, said neoadjuvant therapy reduces the size or extent of the cancer being treated before administering primary or first line therapy to the subject being treated. In one embodiment of the present invention, the compositions and methods described herein are used as adjuvant therapy. In one embodiment of the present invention, said adjuvant therapy comprises administering to a subject one or more therapeutic agents described herein, wherein said one or more therapeutic agents modify the action of other therapeutic agents that have already been administered to the subject, or are simultaneously administered to the subject, or are sequentially administered to the subject.

In one embodiment of the present invention, the compositions and methods described herein demonstrate a reduced likelihood of drug interactions. In one embodiment of the present invention, imipridone, such as ONC201, or an analogue thereof is removed from the patient's body before it can interact with another pharmaceutically active substance.

In one embodiment of the present invention, the compositions and methods described herein exhibit toxicity levels that facilitate combinations with other pharmaceutical agents.

The methods and compositions described herein are not limited to specific animal species. In one embodiment, the subject being treated in accordance with the methods and compositions described herein may or may not be a mammal. In one embodiment of the present invention, said mammalian subject includes, but is not limited to: a human; non-human primates; a rodent such as a mouse, rat or guinea pig; a domesticated animal such as a cat or dog; horse, cow, pig, sheep, goat, or rabbit. In one embodiment of the present invention, said non-mammalian subject includes, but is not limited to: a bird such as a duck, goose, chicken or turkey. In one embodiment of the present disclosure, said subject is a human. In one embodiment of the present invention, said subjects may be of any gender and any

- 43 043367 age. The compositions and methods can also be used to prevent cancer. The compositions and methods can also be used to stimulate the immune system.

The methods and compositions described herein are not limited to a particular age of the subject. In one embodiment of the present invention, the subject being treated in accordance with the methods and compositions described herein may be over 50 years of age, over 55 years of age, over 60 years of age, or over 65 years of age. . In one embodiment of the present invention, the subject being treated in accordance with the methods and compositions described herein may be under 50 years of age, under 55 years of age, under 60 years of age, or under 65 years of age. .

In one embodiment of the present invention, the subject being treated in accordance with the methods and compositions described herein may be a pediatric patient. In one embodiment of the present invention, said pediatric patient is under 18 years of age, under 17 years of age, under 16 years of age, under 15 years of age, under 14 years of age, under 13 years of age, under 12 years of age, under 11 years of age, under 10 years of age, under 9 years of age, under 8 years old, under 7 years old, under 6 years old, under 5 years old, under 4 years old, under 3 years old, under 2 years old, under 1 year old. In one embodiment of the present invention, said pediatric patient is less than 12 months, less than 11 months, less than 10 months, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, under 2 under, under 1 month. In one embodiment of the present invention, said pediatric patient is less than 4 weeks, less than 3 weeks, less than 2 weeks, less than 1 week. In one embodiment of the present invention, said pediatric patient is less than 7 days old, less than 6 days old, less than 5 days old, less than 4 days old, less than 3 days old, less than 2 days old, or less than 1 day old. In one embodiment of the present invention, said pediatric patient is a newborn. In one embodiment of the present invention, said pediatric patient is premature.

In one embodiment of the present invention, said patient weighs less than 45 kg, weighs less than 40 kg, weighs less than 35 kg, weighs less than 30 kg, weighs less than 25 kg, weighs less than 20 kg, weighs less than 15 kg, weighs less than 14 kg, weight less than 10 kg, weight less than 5 kg, weight less than 4 kg, weight less than 3 kg, weight less than 2 kg or less than 1 kg.

In one embodiment of the present invention, the subject has received at least one prior therapeutic agent. In one embodiment of the present invention, the subject has received at least two, at least three, or at least four prior therapeutic agents. In one embodiment of the present invention, said precursor therapeutic agent is ibrutinib, bortezomib, carfilzomib, temozolamide, bevacizumab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, or lenalidomide.

In one embodiment of the present invention, the subject is treated with radiation. In one embodiment of the present invention, the subject is treated surgically. In one embodiment of the present invention, the subject is treated with adoptive T-cell therapy.

In some embodiments, the cancer no longer responds to treatment with ibrutinib, bortezomib, carfilzomib, temozolamide, bevacizumab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, lenalidomide, radiation, surgery or combinations thereof.

In some embodiments of the present invention, the compositions and methods described herein have a dose-response relationship in cancer cells that is different from the dose-response relationship of the same compositions and methods in normal cells. The dose response of ONC201 on proliferation and cell death in normal and tumor cells was determined by measuring cell viability after treatment with ONC201 at various concentrations for 72 hours. Tumors tested included a human colon cancer cell line (HCT116), a breast tumor cell line (MDA-MB-231), a primary human glioblastoma cell line (U87). Normal cells tested included human foreskin fibroblasts (HFF), human fetal lung fibroblasts (MRC-5), and a human lung fibroblast cell line (WI-38). Doxorubicin was used as a positive control at 1 μg/ml on normal fibroblasts. The viability of normal cells tested was at least about 75% at a concentration of about 1-5 mg/ml ONC201, while the viability of tumor cells was significantly lower (eg, 50% or lower) at the same concentration of ONC201. In addition, when the concentration of ONC201 increased beyond about 5 mg/ml, the viability of tumor cells dropped to below 25%, while the viability of normal cells remained at about 75%. Cell viability assays on human fetal lung fibroblasts (MRC-5) were performed after 72 hours of treatment with compound (1) (5 μM) or DMSO and a recovery period in completely drug-free media after treatment. μ Cell recovery was observed with ONC201 but not with DMSO.

In one embodiment, the compositions and methods described herein are used in the treatment of cancer in a subject. In one embodiment, the compositions and methods described herein are used in the treatment of cancer in a human subject. In some embodiments, the method of treatment includes administering to a subject in need of such treatment a pharmaceutically effective amount of ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In one embodiment of the present invention, said method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent, including imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent wherein said first and said second therapeutic agents are administered simultaneously or sequentially. The second therapeutic agent may be any suitable therapeutic agent, including any pharmaceutically active agent described herein. The pharmaceutically acceptable salt of ONC201 includes the dihydrochloride salt given below:

It should be understood that the dihydrochloride salt of ONC201 or an analogue thereof (including, but not limited to, the compound of formula (10)) or an alternative di-salt thereof described herein may be replaced by ONC201 or an analogue thereof in a composition or dosage regimen, described in this document.

In some embodiments of the present invention, said method of treatment comprises administering a synergistic pharmaceutical combination, either simultaneously or sequentially, to a subject in need of such treatment, wherein said synergistic pharmaceutical combination comprises (i) a first therapeutic agent comprising imipridone, such as ONC201, or an analogue thereof or a pharmaceutically acceptable salt thereof; and (ii) a second therapeutic agent. In one embodiment of the present invention, said method of treatment comprises administering to a subject in need of such treatment, simultaneously or sequentially, a therapeutically synergistically effective amount of said first therapeutic agent in combination with said second therapeutic agent. In one embodiment, said method of treatment comprises administering to a subject in need of such treatment an effective amount of said first therapeutic agent in combination with an effective amount of said second therapeutic agent, said combination producing a synergistic effect in the in vivo treatment of a cancer sensitive to the combination, and wherein said first and second therapeutic agents are administered simultaneously or sequentially. In one embodiment of the present invention, said method of treatment comprises administering to a subject in need of such treatment an effective amount of said first therapeutic agent in combination with an effective amount of said second therapeutic agent, wherein said combination produces a synergistic effect in the in vivo treatment of minimal residual disease, sensitive to said combination, and wherein the first and second therapeutic agents are administered simultaneously or sequentially. In some embodiments, the second therapeutic agent may be administered before or prior to the first therapeutic agent.

In one embodiment, the method of treatment is directed to a cancer selected from the group consisting of solid tumors, liquid tumors, lymphomas, leukemias or myelomas.

In one embodiment, said method of treatment is directed at a solid tumor, wherein the solid tumor is selected from the group consisting of: cervical cancer, endometrial cancer, extracranial germ cell tumors; extragonadal germ cell tumors; germ cell tumor; gestational trophoblastic tumor; ovarian cancer, ovarian germ cell tumor, epithelial ovarian cancer and ovarian tumor with low malignant potential; penile cancer, prostate cancer; breast cancer in pregnant women; high grade prostate cancer; moderate prostate cancer; low grade prostate cancer; castration-resistant prostate cancer; breast cancer; bile duct cancer; extrahepatic bile duct cancer; gallbladder cancer; hepatocellular cancer (liver); kidney (renal cell) cancer; liver cancer, renal cell carcinoma (kidney), renal pelvis and ureter cancer; basal cell carcinoma; basal cell nevus syndrome, Gorlin syndrome, melanoma, Merkel cell carcinoma, papillomatosis, multiple endocrine neoplasia syndrome; pancreatic cancer, parathyroid cancer, ocular melanoma; eye cancer; retinoblastoma; malignant fibrous histiocytoma; Ewing's sarcoma family; desmoplastic small round cell tumor; chondrosarcoma, Kaposi's sarcoma, rhabdomyosarcoma; spinal cord tumors, leptomeningeal disease, embryonal tumors of the central nervous system, chordoma, embryonal tumors of the central nervous system, ependymoblastoma, ependymoma, neuroblastoma; tumors of the pineal gland parenchyma of intermediate differentiation, pineoblastoma; adrenocortical carcinoma; bone cancer, osteosarcoma; malignant fibrous histiocytoma of bone and osteosarcoma; osteosarcoma and malignant fibrous histiocytoma of bone; carcinoid tumor, carcinoma of unknown primary localization, bronchial tumors, lung cancer, pleuropulmonary blastoma; respiratory tract carcinoma involving the NUT gene on chromosome 15, astrocytomas, atypical teratoid/rhabdoid tumor; atypical teratoid/rhabdoid tumor of the central nervous system, craniopharyngioma, glioma, brain cancer, medulloblastoma, medulloepithelioma, supratentorial primitive neuroectodermal tumors; pituitary tumors; gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), bladder cancer, anal and rectal cancer, appendix cancer, esophageal cancer, hypopharyngeal cancer; cancer of the larynx, cancer of the lips and oral cavity, metastatic squamous cell cancer of the neck with an unknown primary site, cancer of the oral cavity, cancer of the nasal cavity and paranasal sinuses, cancer of the nasopharynx, cancer of the oral cavity, cancer of the lips and oral cavity, cancer of the oropharynx, cancer of the paranasal sinuses and nasal cavities, pharyngeal cancer; head and neck cancer and mesothelioma.

In one embodiment, the method of treatment is directed to a lymphoma selected from the group consisting of: diffuse large B-cell lymphoma, AIDS-associated lymphoma, Cutaneous T-cell lymphoma, Sézary syndrome, mycosis fungoides (FM); histiocytosis; Burkitt's lymphoma and lymphoma of the central nervous system; non-Hodgkin's lymphoma and primary lymphoma of the central nervous system, Hodgkin's lymphoma, Waldenström's macroglobulinemia; fungoid mycosis; primary lymphoma of the central nervous system; lymphoplasmacytic lymphoma and primary lymphoma of the central nervous system.

In one embodiment, the method of treatment is directed to non-Hodgkin's lymphoma (NHL) selected from the group consisting of mantle cell lymphoma, diffuse large cell B cell lymphoma, follicular lymphoma, marginal zone lymphoma, small cell lymphocytic lymphoma, lymphoplasmacytic NHL, Waldenström's macroglobulinemia, and cutaneous lymphomas.

In one embodiment, the method of treatment is directed to a leukemia selected from the group consisting of: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloproliferative diseases; hairy cell leukemia; acute myeloid leukemia (AML); chronic myelogenous leukemia (CML); and Langerhans cell histiocytosis.

In one embodiment of the present invention, the method of treatment is directed to an acute leukemia selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome and myeloproliferative disease.

In one embodiment, the method of treatment is directed to a myeloma selected from the group consisting of: IgA myeloma; IgG myelomas; IgM myelomas; IgD myelomas; IgE myelomas; light chain myeloma; nonsecreting myeloma; complex karyotype, blast leukemia, multiple myeloma/plasma cell neoplasm, multiple myeloma, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms and myeloproliferative diseases.

In one embodiment of the present invention, the method of treatment is directed at a tumor of the peripheral nervous system. In one embodiment of the present invention, said method of treatment is directed at a paraganglioma. In one embodiment of the present invention, the method of treatment is directed to pheochromocytoma.

In one embodiment of the present invention, treating cancer includes preventing the growth of a tumor in a subject suffering from cancer. In one embodiment of the present invention, treating cancer includes preventing the formation of cancer metastases in a subject with cancer. In some embodiments of the present invention, treating cancer includes targeting treatment of minimal residual disease in a subject with cancer known to have minimal residual disease or in a subject at risk of having minimal residual disease.

This may be indicated after the primary tumor has been treated with surgery and/or chemotherapy (radiation therapy) has been started or determined to be effective. Disseminated tumor cells may be dormant and often cannot be attacked by chemotherapy (radiation therapy). The patient treated in this manner appears to be in a state of healing and is referred to as having minimal residual disease. However, quiescent tumor cells have the potential to form metastases if they become metastatic cells due to a growth stimulus after a long period of dormancy.

The term minimal residual disease refers to the small amount of cancer cells that remains in a subject during treatment or after treatment while the subject is in remission (showing no symptoms or signs of disease). The methods described herein are preferably applied to the forms of the diseases listed herein, including the adult and pediatric forms of these diseases.

In one embodiment, the method of treatment is suitable for treating an autoimmune disease. Autoimmune diseases include, but are not limited to, alopecia areata, antiphospholipid, autoimmune hepatitis, celiac disease, type 1 diabetes, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, inflammatory myopathies, multiple sclerosis, primary biliary liver cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus and vitiligo.

In one embodiment, the treatment method of the present invention is suitable for the treatment of autoimmune and inflammatory disorders of the peripheral nervous system, such as amyotrophic lateral sclerosis (Lou Gehrig's disease), due to various causes, such as metabolic disorders, which include diabetes, vitamin B12 deficiency and folic acid, chemotherapy drugs and drugs used to treat HIV infection, poisons that cause damage to peripheral nerves, cancers that develop into peripheral neuropathies, as well as paraneoplastic syndromes, alcohol abuse, chronic kidney disease, injuries that cause compression of nerves and other lesions, infections such as Lyme disease, Guillain-Barré syndrome, connective tissue diseases, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, some inflammatory conditions such as sarcoidosis, celiac disease, hereditary diseases such as Charcot-Marie syndrome Tutus, Friedreich's ataxia, and/or idiopathies, where no specific cause is found, but inflammatory and/or autoimmune mechanisms are the cause of the disease.

In one embodiment of the present invention, the method of treatment is suitable for the treatment of autoimmune and inflammatory diseases with ocular manifestations. Such ocular manifestations include, but are not limited to, ocular cicatricial pemphigoid, Moraine's corneal ulcer, various forms of uveitis, rheumatoid arthritis, systemic lupus erythematosus, polyarthritis nodosa, relapsing polychondritis, Wegener's granulomatosis, scleroderma, Behçet's disease, Reiter's disease, inflammatory bowel disease ( ulcerative colitis and Crohn's disease) and ankylosing spondylitis, retinitis pigmentosa, macular degeneration, keratoconjunctivitis sicca, scleritis, episcleritis, keratitis, peripheral corneal ulceration, and less common diseases such as choroiditis, retinal vasculitis, episcleral nodules, retinal detachments, and /or macular edema.

In one embodiment of the present invention, the method of treatment is suitable for the treatment of acute allograft rejection in transplant patients. In one embodiment of the present invention, the method of treatment is suitable for the treatment of ischemic stroke. In one embodiment of the present invention, the method of treatment is useful for treating inflammatory diseases, including arthritis, psoriasis, asthma and colitis.

In one embodiment of the present invention, said therapeutic agent comprises a pharmaceutically acceptable mono-salt of ONC201 or an analogue thereof (eg, a compound of formula (10)). In one embodiment of the present invention, said therapeutic agent comprises a pharmaceutically acceptable di-salt of ONC201 or an analogue thereof (eg, a compound of formula (10)). As described herein, some analogues may be tri-salts. In one embodiment of the present invention, said first therapeutic agent comprises ONC201 or an analogue thereof (e.g., a compound of formula (10)) in the form of a pharmaceutically acceptable mono- or di-salt selected from the group consisting of hydrochloride, hydrobromide, bisulfate, sulfates, phosphates , fumarates, succinates, oxalates and lactates, bisulfates, hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate, maleate, fumarate sulfonate, methyl sulfonate, formate, acetate and carboxylate. In one embodiment of the present invention, said therapeutic agent comprises ONC201 or an analogue thereof in the form of a pharmaceutically acceptable mono- or di-salt selected from p-toluenesulfonate, benzenesulfonate, methanesulfonate, oxalate, succinate, tartrate, citrate, fumarate and maleate. In one embodiment of the present invention, said therapeutic agent comprises ONC201 or an analogue thereof in the form of a pharmaceutically acceptable mono- or di-salt containing a counterion selected from the group consisting of ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/or counterions , such as counterions methylamino, dimethylamino, diethylamino, triethylamino and combinations thereof. In one embodiment of the present invention, said therapeutic agent comprises a compound described herein in the form of a halide di-salt, such as a dihydrochloride or dihydrobromide salt.

In one embodiment of said method of treatment, said second therapeutic agent comprises an anti-cancer agent. In one embodiment of said method of treatment, the second therapeutic agent is selected from activicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, aspar gynase, asperlin , azacitidine,

- 47 043367 azetepa, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, bisnafide dimesylate, biselezin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, capecitabine, caracemid, carbetimer, carboplatin, carmustine , carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, deazaguanine, deazaguanine mesylate, diaziquone, docetaxel, doxorubicin, drol oxyphene, dromostanolone, duazomycin, edatrexate, elfomithine g emcitabine, hydroxyurea, idarucibin, ifosfamide , ilmofosin, interleukin II (IL-2, including recombinant interleukin II or rIL2), interferon a-2a, interferon a-2b, interferon a-n1, interferon a-n3, interferon β-Ia, interferon gamma-Ib, iproplatinum, irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol, melengestrol acetate, melphalan, menogaryl, mercaptopurine, methotrexate, methoprine, meturedepa, mitin domid, mitocarcin, mitocromine, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin, ormaplatin, oxysuran, paclitaxel, pegaspargase, peliomycin, pentamustine, eplomycin, perfosfamide, pipobromane, piposulfan, piroxantrone hydrochloride, plicami cin, plomestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprin, rogletimide, safingol, semustine, simtrazen, sparphosate, sparsomycin, spirogermanium, spiromustine, spiroplatinum, streptonigrin, streptozocin, sulofenur, thalisomycin, tamoxifen, tecogalan, tegafur, teloxantrone , temoporfin, teniposide, teroxiron, testolactone, thiamiprin, thioguanine, thiotepa, thiazofurin, tirapazamine, topotecan, toremifene, trestolone, triciribine, trimetrexate, triptorelin, tubulozol, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglicinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, zorubicin and their combinations.

In one embodiment of said method of treatment, the second therapeutic agent is selected from: hormone analogues and antihormones, aromatase inhibitors, LH-RH agonists and antagonists, growth factor inhibitors, anti-growth factor antibodies, anti-growth factor receptor antibodies, tyrosine kinase inhibitors; antimetabolites; antitumor antibiotics; platinum derivatives; alkylating agents; antimitotic agents; tubulin inhibitors; PARP inhibitors, topoisomerase inhibitors, serine/threonine kinase inhibitors, tyrosine kinase inhibitors, protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, IGF-1R inhibitors, ErbB receptor inhibitors, rapamycin analogs, amifostine, anagrelide, clodronate, filgrastin, interferon, interferon a , leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4,3-alethine, 131-1-TM601, 3CPA, 7-ethyl-10-hydroxycamptothecin a , 16-azaepothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG2037, AP-5280, apaziquone, apamin, aranose, arglabin, arzoxifene, atamestana, atrasentana, auristatin PE, AVT-199 (venetoclax), AVT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 ( selumetinib), ARRY704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib) , Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-439006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS754807, BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomtovoka), BIBF 1120 (vargatefa), BI 836 845 , BI 2536, BI 6727, BI 836845, BI 847325, BI 85352θ, BUB-022, bleomycin acid, bleomycin A, bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin, busulfan, BYL-719, CA-4 prodrug , CA-4, CAPCELL, CALCITIRILOLA, CAVONENIBA, KANFOSFAMIDA, CACTICITABIN, CARBOXIFTALATOPATIN, CCL-779, SS-115, SS-223, Ser-701, CER-751, SVT-1 of the Cefixima, Ceflatonin, Ceftrixon, Celecoxib, Celmoleykin, Celmadin, Ceemadotin , CH4987655/RO-4987655, chlorotrianizene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, combretastatin A4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera . oxaneta, diethylstilbestrol , diphlomothecan, didox, DMDC, dolastatin 10, doranidazole, DS7423, E7010, E-6201, edatrexate, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B , epratuzumab, ER- 86526, erlotinib, ET-18-OSNZ, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulinde, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, gal lily maltolate, gefitinib, gemtuzumab , gima

- 48 043367 tecan, glufosfamide, GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC0068, GDC-0349, GDC-0879, immunogen G17DT, GMK, GPX-100, gplOO -peptide vaccines, GSK5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homogarringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibritutomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1d1, IMC-A12 (cixutumumab), immunol, indisulam, interferon a-2a, interferon a-2b, pegylated interferon a-2b, interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF- 154, J-107088, conjugated estrogens, cahalide F, ketoconazole, KW-2170, KW2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuprorelin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone , LU 223651, lurtothecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechlorethamine, MEK inhibitors, MEK-162, methyltestosterone, methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexafine gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, ormiplatin, ortataxel, antibodies OX44, OSI027, OSI-906 (linsitinib), antibodies 4-1BB, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK -3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibodies, PEGpaclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF04691502, RNT-427, P-04 , PKC412 , P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors, PG-TXL, PG2, PLX4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054 , RT-100, PWT-33597, PX-866, picoplatinum, pivaloyloxymethylbutyrate, pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyprenic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramose trona, ranpirnase , RDEA-119/BAY 869766, RDEA-436, rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors, revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb, rinfabate , risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim , satraplatinum, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX101, semustine, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib , spiroplatinum, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tatsedinalin, thalaporfin, tarceva, tariquitar, tasisulam, taxotere, taxoprexin, tazarotene, tegafur, temozolomide, tesmylifen, dough sterone, testosterone propionate , tesmilifen, tetraplatin, tetrodotoxin, tezacitabine, thalidomide, teralux, terarubicin, thymalfasin, timektacin, tiazofurin, tipifarnib, tirapazamine, tocladesine, tomex, toremofin, trabectedin, TransMID-107, transretinoic acid, traszutumab, tremel imumab, tretinoin, triacetyluridine , triapine, triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidin, valrubicin, vatalanib, vincristine, vinflunine, virulysin, WX-UK1, WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281, XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD- 9331, ZD1839, ZSTK-474, zoledronate, zosuquidar and their combinations.

In one embodiment of said treatment method, said second therapeutic agent is selected from tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone, octre otida and their combinations. In one embodiment of said method of treatment, said second therapeutic agent is selected, without limitation, from the group consisting of LH-RH agonists and LH-RH antagonists. In one embodiment, the LH-RH agonist is selected from goserelin acetate, luprolide acetate, triptorelin pamoate, and combinations thereof. In one embodiment, said second therapeutic agent comprises an LH-RH antagonist selected from combinations of Degarelix, Cetrorelix, Abarelix, Ozarelix, combinations thereof with Degarelix. In one embodiment of said method of treatment, said second therapeutic agent comprises a growth factor inhibitor. In one embodiment of the present invention, said growth factor inhibitor is selected from the following inhibitors: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factors (IGF), human epidermal growth factor (HER), hepatocyte growth factor (HGF) and combinations thereof. In some embodiments, the human epidermal growth factor (HER) is selected from HER2, HER3, and HER4.

In one embodiment of said method of treatment, said second therapeutic agent comprises a tyrosine kinase inhibitor. In some embodiments of this method of treatment

- 49 043367 tyrosine kinase inhibitor selected from cetuximab, gefitinib, imatinib, lapatinib and trastuzumab and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises an aromatase inhibitor. In one embodiment of this treatment method, the aromatase inhibitor is selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof.

In one embodiment of said method of treatment, said second therapeutic agent comprises an antimetabolite. In one embodiment of this treatment method, the antimetabolite contains an antifolate. In one embodiment of this method of treatment, the antifolate is selected from methotrexate, raltitrex, pyrimidine analogues, and combinations thereof. In one embodiment of this treatment method, the antimetabolite contains a pyrimidine analogue. In one embodiment of said treatment method, said pyrimidine analogue is selected from 5-fluorouracil, capecitabine, gemcitabine, and a combination thereof. In one embodiment of this treatment method, the antimetabolite contains a purine analog or an adenosine analog. In some embodiments of the specified method of treatment, the specified purine analog or adenosine analog is selected mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine, fludarabine and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises an antitumor antibiotic. In one embodiment of the treatment method, said antitumor antibiotic is selected from anthracyclines, doxorubicin, daunorubicin, epirubicin and idarucibin, mitomycin-C, bleomycin, dactinomycin, plicamycin, streptozocin, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises a platinum derivative. In one embodiment of said treatment method, said platinum derivative is selected from the group consisting of cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises an alkylating agent. In one embodiment of the present invention, said alkylating agent is selected from estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolamide, nitrosoureas, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises nitrosourea. In one embodiment of said treatment method, said nitrosourea is selected from carmustine, lomustine, thiotepa, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises an antimitotic agent. In one embodiment of said method of treatment, said antimitotic agent is selected from vinca alkaloids and taxanes. In one embodiment of the method of treatment, said taxane is selected from paclitaxel, docetaxel, and a combination thereof. In one embodiment of said treatment method, said vinca alkaloids are selected from vinblastine, vindesine, vinorelbine, vincristine, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises a topoisomerase inhibitor. In one embodiment of said method of treatment, said topoisomerase is epipodophyllotoxin. In one embodiment of the treatment method, said topoisomerase inhibitor, which is an epipodophyllotoxin, is selected from etoposide, etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises a serine/threonine kinase inhibitor. In one embodiment of said treatment method, said serine/threonine kinase inhibitor is selected from PDK 1 inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTORC1/PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1 inhibitors, CDKs, Aurora kinase inhibitors and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises a tyrosine kinase inhibitor. In one embodiment of said method of treatment, said second therapeutic agent comprises a PTK2/FAK inhibitor. In one embodiment of said method of treatment, said second therapeutic agent comprises an inhibitor of protein-protein interactions. In one embodiment of said method of treatment, said protein-protein interaction inhibitor is selected from IAP, Mcl-1, MDM2/MDMX, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent comprises a rapamycin analogue. In one embodiment of said method of treatment, said rapamycin analogue is selected from the group consisting of everolimus, temsirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent is selected from amifostine, anagrelide, clodronate, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer and combinations thereof. In one embodiment of said method of treatment, said second therapeutic agent is selected from 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4,3-alethine, 131-1TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-azaepothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apamin, aranose, arglabin, arzoxifene, atamestane, atrasentan, and uristatin RE , AVT-199 (venetoclax), AVT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479

- 50 043367 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib), ARRY704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-439006, BAY 80-6946, BBR- 3464, BBR-3576, bevacizumab, BEZ-235, biricodar dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS7548 07 , BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomtovoka), BIBF 1120 (vargatefa), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 85352θ, BUB-022, bleomycin ic acid, bleomycin A , bleomycin B, brivanib, bryostatin-1, bortezomib, brostallicin, busulfan, BYL-719, prodrug CA-4, CA-4, CapCell, calcitriol, canertinib, canfosfamide, capecitabine, carboxyphthalatoplatinum, CCl-779, CC-115, CC -223, CEP-701, CEP-751, SVT-1 cefixime, ceflatonin, ceftriaxone, celecoxib, celmoleukin, cemadotin, CH4987655/RO-4987655, chlorotrianizene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, combretastatin A4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, STP-37, monoclonal antibodies CTLA-4, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diphlomothecan, didox, DMDC, dolastatin 10, doranidazole, DS74 23, E7010, E-6201, edatrexate, edotreotide, efaproxiral , eflornithine, EGFR inhibitors, EKB569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-OSNZ, ethinylcytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, ec zisulinda, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, gallium maltolate, gefitinib, gemtuzumab, gimatecan, glufosfamide, GCS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-098 0, GDC -0032, GDC0068, GDC-0349, GDC-0879, immunogen G17DT, GMK, GPX-100, gp100-peptide vaccines, GSK5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-212 6458 , GSK2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homogarringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibritutomab , idatrexate, idenestrol, IDN-5109, IGF- 1R, IMC-1d1, IMC-A12 (cixutumumab), immunol, indisulam, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha-2b, interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib , ipilimumab, iproplatin, irofulvene, isohomohalichondriin-B, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, cahalide F, ketoconazole, KW2170, KW-2450, lobaplatin, leflunomide, lenogra stim, leuprolide , leuprorelin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU 223651, lurtothecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechlorethamine, MEK inhibitors, MEK-162, methyl testosterone, methylprednisolone, MEDI- 573, MEN-10755, MDX-H21O, MDX-447, MDX-1379, MGV, midostaurin, minodronic acid, mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexafine gadolinium, MS-209, MS -275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibodies, OSI-02 7, OSI-906 (linsitinib), 4-1BB antibodies, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibodies, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, RNT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors, PG-TXL, PG2, PLX4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054, RT-100, PWT-33597, PX-866, picoplatinum, pivaloyloxymethylbutyrate, pixantrone, phenoxodiol O, PKI166 , plevitrexed, plicamycin, polyprenic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119/BAY 869766, RDEA-436, rebeccamycin analogues, receptor tyrosine kinase (RTK) inhibitors, Revimida , RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim, satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX101, semustine, seocalcitol, SM- 11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatinum, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103 , tacedinalin, thalaporfin, tarceva, tariquitara, tasisulam, taxotere, taxoprexin, tazarotene, tegafur, temozolomide, tesmilifen, testosterone, testosterone propionate, tesmilifen, tetraplatin, tetrodotoxin, tezacitabine, thalidomide, teralux, terarubicin, thymalfasin, thym ectacin, tiazofurin, tipifarnib, tirapazamine, tocladesine, tomex, toremofin, trabectedin, TransMID-107, transretinoic acid, traszutumab, tremelimumab, tretinoin, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD 258, tykerb/tyverb, urocidine, valrubicin, vatalan iba, vincristina , vinflunine, virulysin, WX-UK1, WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281, XL-518/R-7420/GDC-0973, XL-765, YM-511 , YM-598,

- 51 043367

ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronate, zosuquidar and their combinations.

In one embodiment of the present invention, said other therapeutic agent comprises a steroid, including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazole. In one embodiment, said other therapeutic agent comprises an antiemetic. These antiemetics include, but are not limited to, 5-HT3 receptor agonists (such as dolasetron, granisetron, ondansetron, tropisetron, palonosetron and mirtazapine), dopamine agonists (such as domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine and metoclopramide), NK1 receptor antagonists (such as aprepitant and casopitant), antihistamines (such as cyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine), cannabinoids (such as cannabis, dronabinol, nabilone and sativex), benzodiazepines (such as midazolam and lorazepam), anticholinergics (such as hyoscine), trimethobenzamide, ginger, emetrol, propofol, peppermint, muscimol and ajgon.

The pharmaceutical compositions can be administered to a subject via any suitable route of administration. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject orally, parenterally, transdermally or transmucosally. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject parenterally. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject via a parenteral route selected from the group consisting of intravenous (IV), subcutaneous (SC) and intramuscular (IM) routes. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject via a route of administration selected from the rectal and transdermal routes. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject in a dosage form selected from the group consisting of sterile solutions, suspensions, suppositories, tablets and capsules. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject in an oral dosage form selected from the group consisting of tablet, caplet, capsule, lozenge, syrup, liquid, suspension and elixir. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject in an oral dosage form selected from the group consisting of tablets, hard capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solid and semi-solid forms.

In one embodiment of the present invention, said pharmaceutical composition is administered to a subject in a dosage form selected from the group consisting of sustained release forms, controlled release forms, delayed release forms and response release forms.

In one embodiment of the present invention, said pharmaceutical composition is administered to a subject once daily. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject according to an infrequent dosing regimen (eg, administered once a week or less frequently). In one embodiment of the present invention, said pharmaceutical composition is administered to a subject on a frequent dosing schedule (eg, administered more than once a week). In one embodiment of the present invention, said pharmaceutical composition is administered to a subject once a week. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject once every four weeks. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject twice a week. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject once every two weeks. In one embodiment of the present invention, said pharmaceutical composition is administered to a subject once every three weeks. In one embodiment of the present invention, the pharmaceutical composition is administered to a subject in a repeating cycle of once a week, once every two weeks, once every three weeks, once every four weeks, or combinations thereof.

In one embodiment of the present invention, said method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent, including an imipridone-containing compound such as ONC201, or an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent, wherein said first therapeutic agent and said second therapeutic agent are administered simultaneously or sequentially; and further includes determining the expression of endoplasmic reticulum (ER) stress response genes in a biological sample. In one embodiment, said endoplasmic reticulum stress response gene is selected from the group that includes, but is not limited to, C/EBP homologous protein (CHOP), activating transcription factor 3 (ATF3), and both CHOP and ATF3. In one embodiment, said endoplasmic reticulum stress response gene is selected from the group that includes, but is not limited to: ATF3, transcription activating factor 3 (ATF3), CHOP, IRE1, immunoglobulin binding protein (BiP), eukaryotic trans-initiation factor 52 043367 lation 2A (EIF2a), X-box binding protein 1 (XBP1). The biological sample may be a tumor, peripheral blood mononuclear cells, or a skin biopsy. The specified biological sample can be obtained before, during or after administration of the drug. In one embodiment, the method of treatment further includes adjusting the dose of the first therapeutic agent to achieve an induction of about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450 , 475, 500, 525, 550, 575, 600 or more than 600% of one or more ER stress response genes. In one embodiment, the method of treatment further includes adjusting the dose of the first therapeutic agent to achieve an induction of from about 50% to about 100%, from about 100% to about 150%, from about 150% to about 200%, from about 200% to about 250%, from about 250% to about 300%, from about 300% to about 350%, from about 350% to about 400%, from about 400% to about 450%, from about 450% to about 500%, from about 500% to about 550%, about 550% to about 600%, or more than 600% of the ER stress response gene. In one embodiment, the method of treatment further includes adjusting the dose of the first therapeutic agent to achieve an induction of from about 50% to about 100%, from about 100% to about 200%, from about 200% to about 300%, from about 300% to about 400%, about 400% to about 500%, about 500% to about 600%, or more than 600% of the ER stress response gene.

In one embodiment of the present invention, said method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent, including an imipridone-containing compound such as ONC201, or an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent, wherein said first therapeutic agent and said second therapeutic agent are administered simultaneously or sequentially; and further includes determining the expression of proteasomal activity in the biological sample. In one embodiment, said proteasomal activity may be chymotrypsin-like, trypsin-like and/or caspase-like activity. In one embodiment of the present invention, said biological sample may be a tumor, peripheral blood mononuclear cells, or skin cells. The specified biological sample can be obtained before, during or after administration of the drug. In one embodiment, the method of treatment further includes adjusting the dose to achieve inhibition of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% proteasomal activity. In one embodiment, the method of treatment further includes adjusting the dose to achieve an inhibition of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, according to at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% proteasomal activity. In one embodiment, the method of treatment further includes adjusting the dose to achieve inhibition of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than 90% proteasomal activity.

In an aspect provided herein, methods of treatment are provided that comprise administering to a subject in need of such treatment a combination of a first therapeutic agent including imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof (eg, a di-salt or tri-salt). salt) and a second therapeutic agent, said method comprising:

(i) administering a first therapeutic agent to the subject;

(ii) waiting until a predetermined waiting time has elapsed after the time of administration of the first therapeutic agent to the subject; and/or until side effects are eliminated or are being eliminated; and (iii) administering a second therapeutic agent to the subject, wherein the predetermined wait time is selected to obtain a delayed therapeutic effect of the first therapeutic agent without increasing the risk of possible combined toxic effects of the first and second therapeutic agents. In one embodiment of the treatment method, said predetermined wait time is determined based on the rate of clearance of the first therapeutic agent compound or metabolite thereof. In one embodiment of this treatment method, a predetermined wait time is determined by quantifying kidney function and kidney parameters. In one embodiment of said treatment method, said predetermined wait time is determined using a renal function assay, wherein the assay is selected from the group consisting of a serum level of a compound of the first therapeutic agent or a metabolite thereof; the rate of elimination of the compound of the first therapeutic agent or its metabolite; 24-hour renal clearance of the first therapeutic agent compound or its metabolite.

In one embodiment of said treatment method, said predetermined wait time is substantially equal to the time required for systemic clearance of the first therapeutic compound

- 53 043367 tic agent or its metabolite from the body of the subject. In one embodiment of said method of treatment, said predetermined wait time is substantially equal to the time required for the kidneys to eliminate the compound of the first therapeutic agent or a metabolite thereof from the subject's body. In one embodiment of said method of treatment, said predetermined wait time is substantially equal to the time required for the liver to clear the first therapeutic agent compound or metabolite thereof from the subject's body. In one embodiment of this method of treatment, the predetermined wait time is substantially equal to the time required for overall clearance of the first therapeutic agent compound or metabolite thereof from the subject's body. In one embodiment of the treatment method, the predetermined wait time is approximately 4 hours. In other embodiments, the wait time is 1 day. In one embodiment, said wait time represents the period until the _{Cmax of} the compound of the first therapeutic agent has passed. In other embodiments, the wait time is a period after which most of the side effects are eliminated or eliminated. In one embodiment of the treatment method, the predetermined wait time is about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In one embodiment of the treatment method, the predetermined wait time is in the range of about 1 to 7 days, about 1 to 6 days, about 1 to 5 days, about 1 to 4 days, about 1 to 3 days, or about 1 to 2 days. In one embodiment, the wait time is up to 3 weeks. The above are considered therapeutic time periods.

When the order of administration is reversed, the time for administration of said first therapeutic agent occurs when the _Cmax of said second therapeutic agent (ie, the first of the drugs administered) has passed. In one embodiment, administration of said first therapeutic agent may occur after most or substantially all of the first drug administered has been removed from the body or the toxic effects of the first drug administered have been or are eliminated.

In one embodiment, the method of treatment further includes monitoring the level of the compound of the first therapeutic agent or its metabolite in the subject by assessing pharmacokinetic parameters. In some such embodiments, monitoring the level of a compound of the first therapeutic agent or a metabolite thereof in a subject by assessing pharmacokinetic parameters includes constructing a pharmacokinetic profile of the compound of the first therapeutic agent or a metabolite thereof for the subject using the concentration of the compound of the first therapeutic agent or metabolite thereof in at least two samples. , obtained from the specified subject at time points suitable for creating a pharmacokinetic profile. In one embodiment of a method that includes monitoring levels of a compound of a first therapeutic agent or a metabolite thereof in a subject using pharmacokinetic measures, samples are collected from the subject at the point of treatment or at the point of use through sample collection or self-sampling using point-of-care devices or point-of-use devices or into matrices suitable for storing samples prior to quantitation in the laboratory. In one embodiment of said treatment method, each of the point-of-care devices or point-of-use devices is capable of quantifying a compound of the first therapeutic agent or a metabolite thereof. In one embodiment of a method that includes monitoring levels of a compound of a first therapeutic agent or a metabolite thereof in a subject, one or more samples are collected from the subject at a point of treatment or point of use using a biopsy device for analysis by point-of-care devices or devices. at the point of use or for storing samples until quantitation in the laboratory. In one embodiment, the biopsy is performed after a time interval of 3-8 hours has passed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 3-24 hours has passed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 8-24 hours has passed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 2 days has elapsed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 3 days has elapsed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 4 days has elapsed after administration of the first therapeutic agent to the subject. In one embodiment, the biopsy is performed after a time interval of 1-7 days has passed after administration of the first therapeutic agent.

In one embodiment of the present invention, the pharmacokinetic profile includes pharmacokinetic parameters suitable for controlling the dosage of the first therapeutic agent to the subject being treated. In one embodiment of the present invention, the C _max of the first therapeutic agent after its administration to the specified subject is in the range of from about 1000 to 1500 ng/dL over the therapeutic period of time. In one embodiment

- 54 043367

_Cmax is less than 1500 and more than 85 ng/dL during the therapeutic period of time. In one embodiment of the present invention, the first therapeutic _Cmax after administration to the subject is in the range of about 1000 to 1500 ng/ml over the therapeutic period of time. In one embodiment, the _Cmax is less than 1500 ng/dL and more than 85 ng/dL during the therapeutic period of time.

In one embodiment, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma or serum) ( _Cmax ) of a subject after administration to the subject is from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090 , 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340 , 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490 ng/dL to approximately 1500 ng/dL; from about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 123, 123, 123, 123 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48 or 149 ng/dL to approximately 150 ng/dL; or from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng/dL to about 15 ng/dL.

In one embodiment, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma or serum) ( _Cmax ) of a subject after administration to the subject is from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090 , 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340 , 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng/ml to approximately 1500 ng/ml; from about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 123, 123, 123, 123 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 1 48 or 149 ng/ml to approximately 150 ng/ml; or from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng/ml to about 15 ng/ml.

In one embodiment, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma or serum) ( _Cmax ) of a subject after administration to the subject is selected from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090 , 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340 , 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, or 1490 ng/dL. In one embodiment, the C _max of the first therapeutic agent in the blood (whole blood, plasma or serum) (C _max ) of a subject upon administration to said subject is selected from about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 , 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 , 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL. In one embodiment, the Cmax of the first therapeutic agent after administration is selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng/dL.

In one embodiment, the Cmax of the first therapeutic agent after administration is selected from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 11 70, 1180 , 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430 , 1440, 1450, 1460, 1470, 1480 or 1490 ng/ml. In one embodiment, the _Cmax of the first therapeutic agent after administration is selected from about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117. 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148 or 149 ng/ml. In one embodiment, the C _max of the first therapeutic agent after administration is selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng/ml.

In one embodiment, the Cmax of the first therapeutic agent after administration is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265 , 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 505, 505, 505

515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725,

735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945,

955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135,

1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1 345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485, 1495 or 1500 ng/dL. In one embodiment, the C _max of the first therapeutic agent after administration is selected from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,

117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 1 41, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL. In one embodiment, the _Cmax of the first therapeutic agent after administration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 or 14.5 ng/dL .

In one embodiment, the C _max of the first therapeutic agent after administration is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285,

- 55 043367

295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505,

515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725,

735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945,

955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135,

1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1 345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485, 1495 or 1500 ng/ml. In one embodiment, the C _max of the first therapeutic agent after administration is selected from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,

117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 1 41, 142, 143, 144, 145, 146, 147, 148 or 149 ng/ml. In one embodiment, the _Cmax of the first therapeutic agent after administration is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 or 14.5 ng/ml .

In one embodiment, the _Cmax of the first therapeutic agent after administration ranges from 85 ng/dL to 1500 ng/dL; from about 8.5 ng/dL to 150 ng/dL; or from about 0.85 ng/dL to 15 ng/dL. In one embodiment, the Cmax of the first therapeutic agent in the blood (whole blood, plasma or serum) of the subject after administration is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185.

195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395,405,

415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615,625,

635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835,845,

855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045,1055,

1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225,1235,

1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1355, 1365, 1375, 1385, 1395, 1405,1415,

1425, 1435, 1445, 1455, 1465, 1475, 1485, or 1495 ng/dL to approximately 1500 ng/dL; from about 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 1 36, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL to approximately 150 ng/dL; or from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 , 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/dL to approximately 15 ng/dL.

In one embodiment, the Cmax of the first therapeutic agent after administration ranges from 85 ng/ml to 1500 ng/ml; from about 8.5 ng/ml to 150 ng/ml; or from about 0.85 ng/ml to 15 ng/ml. In one embodiment, the Cmax of the first therapeutic agent after administration is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265 , 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515 , 525,

535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745,

755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965,

975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145,

1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1 355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485 or 1495 ng/ml to about 1500 ng/ml; from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,

112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 1 36, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149 ng/ml to about 150 ng/ml; or from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5 , 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14 or 14.5 ng/ml to approximately 15 ng/ml.

In one embodiment of the method, said total drug exposure over time, measured as the area under the curve (AUC) of a plot of drug concentration in the blood (whole blood, plasma, or serum) of a subject after drug administration versus time after drug administration, ranges from about 150 ng/h/ml to about 8000 ng/h/ml; from about 15 ng/h/ml to about 800 ng/h/ml; from about 1.5 ng/h/ml to about 80 ng/h/ml. In one embodiment, the AUC is less than 8000 ng/h/ml and is 150 ng/h/ml or more. In one embodiment, the AUC is less than 800 ng/h/ml and is 15 ng/h/ml or more. In one embodiment, the AUC is less than 80 ng/h/ml and is 1.5 ng/h/ml or more.

In one embodiment, the total drug exposure over time AUC is from about 100 ng/hour/ml to about 8000 ng/hour/ml; from about 10 ng/h/ml to about 800

- 56 043367 ng/h/ml; or from about 1 ng/h/ml to about 80 ng/h/ml. In one embodiment, the general exposition of the AUC drug is from about 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7 600 or 7800 ng/h/ml to approx. 8000 ng/h/ml. In one embodiment, the total drug exposure over time AUC is from about 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760 or 780 ng/h/ml to approx. 800 ng/h/ml. In one embodiment, the total drug exposure over time AUC is from about 1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32. 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 ng/h/ml to approximately 80 ng/h/ml.

In one embodiment, the total drug exposure over time AUC is from about 100 ng/hour/ml to about 8000 ng/hour/ml; from about 10 ng/h/ml to about 800 ng/h/ml; or from about 1 ng/h/ml to about 80 ng/h/ml. In one embodiment, the total drug exposure over time AUC is from about 150 ng/h/ml to about 7800, 7600, 7400, 7200, 7000, 6800, 6600, 6400, 6200, 6000, 5800, 5600, 5400, 5200, 5000, 4800, 4600, 4400, 4200, 4000, 3800, 3600, 3400, 3200, 3000, 2800, 2600, 2400, 2200, 2000, 1800, 1600, 1400, 1200, 1 000, 800, 600, 400 or 200 ng /h/ml. In one embodiment, the total drug exposure over time AUC is from about 15 ng/hour/ml to about 780, 760, 740, 720, 700, 680, 660, 640, 620, 600, 580, 560, 540, 520, 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 80, 60, 40 or 20 ng /h/ml. In one embodiment, the total drug exposure over time AUC is from about 1.5 ng/h/ml to about 78, 76, 74, 72, 70, 68, 66, 64, 62, 60, 58, 56, 54. 52, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 8, 6, 4 or 2 ng/h/ml. In one embodiment, the total drug exposure over time AUC is from about 100 ng/h/ml to about 200 ng/h/ml; from about 10 ng/h/ml to about 20 ng/h/ml; or from about 1 ng/h/ml to about 2 ng/h/ml.

In one embodiment, the overall exposition of the drug from the time of the AUC was selected from approximately 100, 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 46000, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800 and 8000 ng/h /ml. In one embodiment, the total drug exposure over time AUC is selected from about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 4600, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780 and 8000 ng/h /ml. In one embodiment, the total drug exposure over time AUC is selected from about 1, 15, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32. 34, 36, 38, 40, 42, 44, 460, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 8000 ng/h /ml.

In another aspect provided herein are methods of treating or using a composition for treating a disease state, comprising administering to a subject in need of such treatment a combination of a first therapeutic agent and a second therapeutic agent, said method comprising:

(i) administering to said subject a first therapeutic agent containing imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof;

(ii) monitoring the level of the compound of the first therapeutic agent or its metabolite in the subject using pharmacokinetic assessment; and (iii) administering a second therapeutic agent depending on the level of said first therapeutic agent in the subject. In one embodiment, the monitoring step includes constructing a pharmacokinetic profile of the compound of the first therapeutic agent or a metabolite thereof for a subject using concentrations of the compound of the first therapeutic agent or metabolite thereof in a plurality of samples obtained from the subject at time points suitable for constructing the pharmacokinetic profile. In some embodiments, at least two point-of-care or point-of-use samples are collected by sampling or self-sampling into point-of-care or point-of-use devices or matrices suitable for sample storage prior to quantitation of the compound or its metabolite in the laboratory. In one embodiment, each of the point-of-care devices or point-of-use devices is capable of quantifying a compound or a metabolite thereof.

In one embodiment of the present invention, the pharmacokinetic profile includes pharmacokinetic parameters suitable for controlling the dosage of the compound or salt thereof in a subject. In one embodiment of the present invention, said samples include from 2 to 12 samples. In one embodiment of the present invention, said samples are collected over a period of up to 8 hours, up to 24 hours, up to 48 hours, or up to 72 hours. In one embodiment of the method, said pharmacokinetic parameters include at least one parameter selected from the group consisting of from AUC, AUCinf, Tmax, Cmax, time above threshold, steady-state concentration, speed

- 57 043367 absorption, rate of elimination, rate of distribution, terminal T-1/2 or parameters taken from non-compartmental pharmacokinetic (PK) or compartmental PK analysis, including compartmental PK analysis based on a physiological model. In one embodiment, the method of treatment further includes generating a report including an assessment of the subject's pharmacokinetic parameters. In one embodiment of the present invention, the report contains dosage recommendations based on an assessment of the subject's pharmacokinetic parameters. In one embodiment of the present invention, a dose reduction of ONC201 or an analogue thereof or a pharmaceutically acceptable salt thereof is recommended to reduce the risk of toxicity based on one or more pharmacokinetic parameters. In one embodiment of the present invention, a dose reduction of a compound or a salt thereof is recommended based on time above a threshold value, where said threshold value is the drug concentration at which toxicity occurs, or one or more of AUC, AUC _inf , mean residence time (MRT ), exponents defining the pharmacokinetic profile, volume of distribution at steady state (Vss), volume of distribution during the terminal phase (Vz), or a combination of a group of pharmacokinetic variables to adequately describe the specified pharmacokinetic profile. In one embodiment of the present invention, dosage adjustment of a compound or salt thereof is recommended to improve efficacy based on one or more pharmacokinetic parameters. In one embodiment of the present invention, increasing the dose of a compound or a salt thereof is recommended based on one or more of AUC, AUC _inf , MRT, exponents defining the pharmacokinetic profile, volume of distribution at steady state (Vss), volume of distribution during the terminal phase (Vz) or a combination of a group of pharmacokinetic variables to adequately describe the pharmacokinetic profile. In one embodiment, the dose of the specified compound or salt thereof is adjusted within the range of 5 to 25% of the desired target value. In one embodiment of the method, each of the samples is placed in a point-of-care device or point-of-use device to determine the concentration of the compound or its metabolite, wherein the point-of-care device or point-of-use device contains a lateral flow strip having such a design and a composition such that application of one or more samples to the test strip causes a fraction of the drug in the sample to bind to a component of the test strip such that the result is a detectable signal proportional to the concentration of drug in the applied sample. In one embodiment, said samples are placed in matrices suitable for sample storage prior to quantitation in the laboratory. In one embodiment of the present invention, these samples are stored in the form of dried blood spots. In one embodiment, the drug concentration is measured using ELISA, LC-MS-MS, LC-UV, or LC-MS. In one embodiment, said pharmacokinetic parameters include at least one of steady state concentration, absorption, and terminal half-life T _1/2 . In one embodiment, at least one of the samples is whole blood.

IX. Multimodal therapeutic modalities.

In one aspect provided herein are multimodal therapeutic methods in which the administration of imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof to a subject in need of such treatment is supplemented with another therapeutic modality. In one embodiment, the multimodal therapeutic method comprises administering to a subject a pharmaceutical composition containing imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof, in combination with radiation therapy or after it has been determined that radiation therapy has been ineffective. In one embodiment, said multimodal therapeutic method comprises administering to a subject a pharmaceutical composition containing imipridone, such as ONC201, or an analogue thereof, or a pharmaceutically acceptable salt thereof, in combination with radiation therapy, wherein said pharmaceutical composition containing imipridone, such as ONC201, or an analogue thereof or a pharmaceutically acceptable salt thereof, and radiation therapy is administered simultaneously or sequentially, in any order. In one embodiment, the multimodal therapeutic method comprises administering to a subject a pharmaceutical composition containing ipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof, in combination with radiation therapy sequentially. In one embodiment, said multimodal therapeutic method comprises administering to a subject in need of such treatment a pharmaceutical composition containing imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof, concurrently with radiation therapy. In one embodiment, said multimodal therapeutic method is used to treat cancer. In one embodiment, the multimodal therapeutic method comprises administering to a subject suffering from cancer in need of such treatment a pharmaceutical composition containing imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof, and irradiating the cancer cells with a beam of radiation. In one embodiment, said multimodal therapeutic method uses conformal radiation therapy (CRT) techniques to deliver the histogram

- 58 043367 dose-volume (VDV) administered to a subject suffering from cancer. In one embodiment, the multimodal therapeutic method uses intensity modulated radiation therapy (IMRT) to deliver radiation to cancer cells. In one embodiment, the multimodal therapeutic method employs techniques to compensate for the movement of tumors in a subject during treatment (eg, when radiation doses must be administered to a tumor in the thoracic region that moves when the patient breathes). For example, this multimodal therapeutic method uses four-dimensional computed tomography (4D CT) scanning techniques to adjust the delivered radiation field to compensate for tumor movement during the respiratory cycle.

Any suitable type of radiation can be used with a multimodal therapeutic modality, including fractionable gamma radiation, IMRT (intensity modulated radiation therapy), gamma knife, proton therapy, and brachytherapy. Radiation therapy and administration of imipridone, such as ONC201, or an analogue thereof or a pharmaceutically acceptable salt thereof can be used to treat brain tumors such as glioblastoma, or a disease in which metastases from lung cancer develop in the brain. The multimodal therapeutic method can be used to treat lung cancer, pancreatic cancer, colorectal cancer, breast cancer, sarcoma, prostate cancer, gynecological malignancies and lymphoma. Gamma Knife is often used to treat brain metastases. In one embodiment, the multimodal therapeutic method includes the use of proton therapy to treat cancer, including brain tumors, prostate cancer, and any tumor in close proximity to vital organs where it is important to minimize toxicity to adjacent normal tissue .

In one embodiment, said multimodal therapeutic method comprises administering to a subject suffering from cancer in need of such treatment a pharmaceutical composition comprising imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof, in combination with an adaptive cell therapy (e.g., CAR-T (JCAR 14, 15, 16, 17, KTE-G19 or CTL019), other T cells (AFM13) or NK natural killer cells (CDNO-109 or NK-92)) either simultaneously or in combination.

In one embodiment, said multimodal therapeutic modality eliminates minimal residual disease without contributing to the toxicity resulting from treatment with imipridone, such as ONC201, an analogue thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, said multimodal therapeutic method improves the prognosis and/or reduces unwanted side effects associated with the disease state or condition of the subject being treated.

X. Additional imipridone derivatives, analogues and salts.

In one aspect, compounds are provided that are analogues of compounds of formula (10) and methods for their preparation. Those skilled in the art will understand that the general principles and concepts described herein with respect to the ONC201 compounds of formula (10) and salts thereof, including the principles and concepts relating to methods and pharmaceutical compositions, apply equally to the analogues described below and their salts.

In one embodiment of the present invention, said analogues have the structure of compound (25):

O

(25) where Y is NR4 or O, and where R _b R ₂ , R ₃ and R 4 are independently H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy , aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R _b R ₂ , R ₃ and R 4 are optionally substituted. In one embodiment, some or all of the hydrogen atoms in R _b R ₂ , R ₃ and R 4 are substituted with deuterium. In other embodiments of the present invention, R _b R ₂ , R 3 , and R 4 are independently selected from the group consisting of H, C ₁ _ ₄ alkyl, C ₁ _ ₄ alkylphenyl, C ₁ _ ₄ alkylphenyl ketone, C ₁ _ ₄ benzylpiperazine, and C ₁ _ ₄ alkylthienyl, wherein _{C1_4} alkyl, _{C1_4 alkylphenyl} , _{C1_4 alkylphenyl ketone} and _{C1_4 benzylpiperazine} are optionally substituted with _{C1_4 alkyl} , hydroxyl or halogen. In other embodiments, R _b R ₂ , R ₃ , and R 4 are independently selected from the group consisting of H, CH ₃ , CH ₂ Ph, CH 2-((2Cl)-Ph), CH 2-(2-thienyl), CH ₂ CH ₂ Ph, CH ₂ CH ₂ (4-N-benzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH2CHOHPh and (CH2)3CO-4F -Ph.

- 59 043367

In one embodiment of the present invention, these analogs have the structure of compound (26):

(26)

D where R1 and _R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio , arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R ₁ and R ₂ are independently selected from the group consisting of H, C ₁ . ₄ alkyl, C ₁ . ₄ alkylphenyls, C ₁ . ₄ alkyl phenyl ketones, C ₁ . ₄ benzylpiperazine and C ₁ . ₄ alkylthienyl, where C ₁ . ₄ alkyl, C ₁ . ₄ alkylphenyl, C ₁ . ₄ alkyl phenyl ketone and C ₁ . ₄ benzylpiperazine is optionally substituted with C ₁ . ₄ alkyl, C ₁ . ₄ alkoxyl, hydroxyl, perhalogenated _C1 . ₄ alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, CH ₃ , CH ₂ Ph, CH ₂ -((2-Cl)-Ph), CH2-(2-thienyl), CH ₂ CH ₂ Ph, CH ₂ CH ₂ (4^-benzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment, R2 is selected from the group consisting of H, _CH3 , _CH2Ph , _CH2 ((2-Cl)-Ph), _CH2- ( ₂ -thienyl), _CH2CH2Ph , _CH2CH2 (4-N-benzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )Ph), CH2CHOHPh and (CH2)3CO-4F-Ph.

In one embodiment, R ₁ is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH3, _-NO2 , -OCH3, -CHH2 , -СХ2Н, -СХ3, -СШШШ3), -СН(СХз)2, -С(СХз)з, -СрХ2р+1, -ОСХ ₃ or -OS _р Х _2р+1 , where р is an integer from 2 up to 20 and where X is a halogen, including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, _R2 is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: _-CH3 , _-NO2 , -OCH3, -CHH2 , -СХ2Н, -СХ3, -СН2(СХ3), -СН(СХ3)2, -С(СХ3)3, -СрХ2р+1, -OCХ3 or -OCрХ2р+1, where p is an integer from 2 to 20 and where X is halogen.

In one embodiment of the present invention, R1 is hydrogen. In one embodiment of the present invention, R ₁ represents a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF ₃ and -OCH ₃ . In one embodiment of the present invention, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF ₃ and -OCH ₃ .

In one embodiment of the present invention, these analogs have the structure of compound (27):

where R ₂ represents H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxy- 60 043367 carbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio , arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R1 is selected from the group consisting of H, C ₁ . ₄ alkyl, C ₁ . ₄ alkylphenyls, C ₁ . ₄ alkyl phenyl ketones, C ₁ . ₄ benzylpiperazine and C ₁ . ₄ alkylthienyl, where C ₁ . ₄ alkyl, C ₁ . ₄ alkylphenyl, C ₁ . ₄ alkyl phenyl ketone and C ₁ . ₄ benzylpiperazine is optionally substituted with C ₁ . ₄ alkyl, C ₁ . ₄ alkoxyl, hydroxyl, perhalogenated C1. ₄ alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, CH ₃ , CH ₂ Ph, CH ₂ -((2-Cl)-Ph), CH 2 -(2-thienyl), CH ₂ CH ₂ Ph, CH ₂ CH ₂ (4-Nbenzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph.

In one embodiment, R ₁ is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH ₃ , -NO2 , -OCH ₃ , - СХН ₂ , -СХ ₂ Н, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -С _р Х _2р+1 , -OCХ ₃ or -OS _p X _2p+1 where p is an integer from 2 to 20 and where X is a halogen including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment of the present invention, R ₁ is hydrogen. In one embodiment of the present invention, R ₁ represents a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with _C1-4 alkyl, C1-4 alkyl, hydroxyl, perhalogenated _C1-4 alkyl, or halogen.

In one embodiment of the present invention, these analogs have the structure of compound (28):

(28) where R ₁ and R ₂ independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R ₁ and R ₂ are independently selected from the group consisting of H, C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone, C _1-4 benzylpiperazine and C _1-4 alkylthienyl, wherein _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone and _C1-4 benzylpiperazine are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, perhalogenated _C1-4 alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, CH ₃ , CH ₂ Ph, CH ₂ -((2-Cl)-Ph), CH2-(2-thienyl), CH ₂ CH ₂ Ph, CH2-( 2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH ₂ CHOHPh, CH ₂ CH ₂ (4-N-benzylpiperazine) and (CH ₂ ) ₃ CO-4F-Ph . In one embodiment, R2 is selected from the group consisting of H, _CH3 , _CH2Ph , _CH2 ((2-Cl)-Ph), CH2-( ₂ -thienyl), _CH2CH2Ph , CH2CH2(4- N-benzylpiperazine), CH2-(2,4-di F-Ph), ^2-((2-^3)Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment of the present invention, when R1 is CH ₂ Ph, R2 is non-CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R ₁ is CH ₂ Ph and R ₂ is CH ₂ -(2,4-di F-Ph). In one embodiment of the present invention, R ₁ is CH ₂ Ph and R2 is CH ₂ -(4-CH ₃ -Ph).

In one embodiment, R ₁ is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH3, -NO2, -OCH3, -CHH2, -СХ2Н, -СХ3, -СН2(СХ3), -СН(СХ3)2, -С(СХ3)3, -СрХ2р+1, -OCХ ₃ or -OC _р Х _2р+1 , where р is an integer from 2 to 20 and where X is a halogen, including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, _R2 is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: _-CH3 , _-NO2 , -OCH3, -CHH2 , -СХ2Н, -СХ3, -СН2(СХ3), -СН(СХ3)2, -С(СХ3)3, -СрХ2р+1, -OCХ3 or -OCрХ2р+1, where p is an integer from 2 to 20 and where X is halogen.

In one embodiment of the present invention, R1 is hydrogen. In one embodiment of the present invention, R ₁ represents a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R2 is a substituted or unsubstituted

- 61 043367 substituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH3, _-CF3 and _-OCH3 . In one embodiment of the present invention, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxyl, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF ₃ and -OCH ₃ .

In one embodiment of the present invention, these analogs have the structure of compound (29):

(29) where R ₁ and R ₂ independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R ₁ and R ₂ are independently selected from the group consisting of H, C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone, C _1-4 benzylpiperazine and C _1-4 alkylthienyl, wherein C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone and C _1-4 benzylpiperazine are optionally substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, CH ₃ , CH ₂ Ph, CH ₂ -((2-Cl)-Ph), CH2-(2-thienyl), CH ₂ CH ₂ Ph, CH2CH2 (4 -N-benzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment, R2 _is selected from the group consisting of H, _CH3 , _CH2Ph , _CH2 ((2-Cl)-Ph), CH2-( ₂ -thienyl), _CH2CH2Ph , _CH2CH2 (4-N-benzylpiperazine), CH2-(2,4-di F-Ph), CH2-((2-CH ₃ )Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment of the present invention, when R1 is CH ₂ Ph, R2 is non-CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R ₁ is CH ₂ Ph and R ₂ is CH2-(2,4-di F-Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(4-CH ₃ -Ph).

In one embodiment, R ₁ is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH ₃ , -NO2 , -OCH ₃ , - СХН ₂ , -СХ ₂ Н, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -С _р Х _2р+1 , -OCХ ₃ or -OS _p X _2p+1 where p is an integer from 2 to 20 and where X is a halogen including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, _R2 is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: _-CH3 , _-NO2 , -OCH3, -CHH2 , -СХ2Н, -СХ3, -СН2(СХ3), -СН(СХ3)2, -С(СХ3)3, -СрХ2р+1, -OCХ3 or -OCрХ2р+1, where p is an integer from 2 to 20 and where X is halogen.

In one embodiment of the present invention, R ₁ is H. In one embodiment of the present invention, R ₁ is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF ₃ and -OCH ₃ . In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment of the present invention, R ₂ is

- 62 043367 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxyl, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH3, -CF3 and -OCH3.

In one embodiment of the present invention, said analogues have the structure of compound (30):

O

(30) where R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R ₁ and R ₂ are independently selected from the group consisting of H, C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone, C _1-4 benzylpiperazine and C _1-4 alkylthienyl, wherein C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone and C _1-4 benzylpiperazine are optionally substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, _CH3 , CH2Ph, CH2-((2-Cl)-Ph), CH2-(2-thienyl), C^C^Ph, CH2CH2 (4-N- benzylpiperazine), CH2-(2,4-di F-Ph), CH ₂ -((2-CH3)-Pb), C^CHOHPh and (CH2) ₃ CO-4F-Ph. In one embodiment, R2 is selected from the group consisting of H, _CH3 , CH2Ph, CH2((2-Cl)-Ph), CH2-(2-thienyl), CH2CH2Ph _{, CH2CH2} (4-N-benzylpininerazine) , CH2-(2,4-di F-Ph), CH2-((2-CH ₃ )Ph), CH2CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment of the present invention, when R1 is CH2Ph, R2 is non-CH2-(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH2Ph and R2 is CH2-(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH2Ph and R2 is CH2-(2,4-di F-Ph). In one embodiment of the present invention, R1 is CH2Ph and R2 is CH ₂ -(4-CH ₃ -Ph).

In one embodiment, R ₁ is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH ₃ , -NO2, -OCH3, -CHN ₂ , -СХ ₂ Н, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -CpX _2p+1 , -OCX ₃ or -OS _р Х _{2р +1} where p is an integer from 2 to 20 and where X is a halogen including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, R2 is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH ₃ , -NO2, -OCH3, -CHH ₂ , -СХ ₂ Н, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -С _р Х _2р+1 , -OCХ ₃ or -OS _р Х _{2р +1} , where p is an integer from 2 to 20 and where X is a halogen.

In one embodiment of the present invention, R1 is H. In one embodiment of the present invention, _R1 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF 3 and -OCH ₃ . In one embodiment of the present invention, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment of the present invention, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH ₃ , -CF 3 and -OCH ₃ .

In one embodiment of the present invention, said analogues have the structure of compound (31):

where R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, mercapto, alkylthio, arylthio , alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocycle. In one embodiment of the present invention, R1 and R2 are independently selected from the group consisting of H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzylpiperazine and _C1-4 alkylthienyl, wherein C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone and C _1-4 benzylpiperazine are optionally substituted with O^alkyl, O^alkoxy, hydroxyl, perhalogenated _C1-4 alkyl or halogen. In one embodiment, R1 is selected from the group consisting of H, CH3, CH2Ph, CH2-((2-Cl)-Ph), CH2-(2-thienyl), CH ₂ CH ₂ Ph, CH2CH2 (4-N-benzylpiperazine ), CH2-(2,4-di F-Ph), CH ₂ -((2-CH ₃ )-Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment, R2 is selected from the group consisting of H, CH3, _CH2Ph , _CH2 ((2-Cl)-Ph), CH2-( ₂ -thienyl), _CH2CH2Ph , CH2CH-benzylpiperazine) , CH2-(2,4-di F-Ph), CH2-((2-CH3)Ph), CH ₂ CHOHPh and (CH ₂ ) ₃ CO-4F-Ph. In one embodiment of the present invention, when R1 is CH ₂ Ph, R2 is non-CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(2-CH ₃ -Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH2-(2,4-di F-Ph). In one embodiment of the present invention, R1 is CH ₂ Ph and R2 is CH ₂ -(4-CH ₃ -Ph).

In one embodiment, R1 is benzyl, optionally substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH ₃ , -NO ₂ , -OCH3 , -CHH ₂ , -СХ ₂ Н, -СХ3, -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -С _р Х _2р+1 , -OCХ3 or -OS _р Х _{2р +1} where p is an integer from 2 to 20 and where X is a halogen including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, R ₂ is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta, and/or para positions of the benzyl ring: -CH3, -NO2, -OCH3, -CHH2, - CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C( _CX3 ) ₃ , -CpX2p+1, -OCX3 or -OCpX^, where p is an integer from 2 to 20 and where X represents halogen.

In one embodiment of the present invention, R1 is H. In one embodiment of the present invention, R1 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen.

In one embodiment of the present invention, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment of the present invention, said arylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, perhalogenated C _1-4 alkyl, or halogen. In one embodiment of the present invention, said arylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH3, -CF3 and -OCH3. In one embodiment of the present invention, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment of the present invention, R ₂ represents a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with C _1-4 alkyl, C _1-4 alkoxyl, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment of the present invention, said heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halogen, -CH3, -CF3 and -OCH3.

In one embodiment of the present invention, the compounds represented by formula (100) are provided:

- 64043367% (100), where R1 and R2 are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio and acyl radicals. In one embodiment, R1 is CH2Ph and R2 is CH2-( ₂ - _CH3 -Ph), which is a linear isomer of ONC201 (i.e., TIC-10) o

which does not have anticancer activity (Jacob et al., Angew. Chem. Int. Ed., (2014) 53:6628; Wagner et al., Oncotarget (2015) 5(24):12728), but, as shown in the examples , TIC-10 is a CXCR7 agonist. CXCR7 agonists can be used to regenerate the liver and prevent or treat liver fibrosis (Nature (2014) 505:97).

In one embodiment of the present invention, R1 and R2 are independently selected from the group consisting of H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzylpiperazine, _C1-4 alkylthienyl, _{C1 -4} alkylpyridinyl, C _1-4 alkylisoxazolidinyl, C _1-4 alkylmorpholinyl, C _1-4 alkylthiazolyl and C _1-4 alkylpyrazinyl, where C _1-4 alkyl, C _1-4 alkylphenyl, C _1-4 alkylphenyl ketone, C _{1- 4} benzyl-piperazine, C _1-4 alkylthienyl, C _1-4 alkylpyridinyl, C _1-4 alkylisoxazolidinyl, C _1-4 alkylmorpholinyl, C _1-4 alkylthiazolyl and C _1-4 alkylpyrazinyl are optionally substituted with C _1-4 alkyl, C _{1 -4} alkoxy, hydroxyl, perhalogenated C _1-4 alkyl or halogen. In one embodiment, R1 and/or R2 is substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment of the present invention, said heteroarylalkyl is selected from _C1-4 alkylpyrrolyl, _C1-4 alkylfuryl, _C1-4 alkylpyridyl, _C1-4 alkyl-1,2,4-thiadiazolyl, _C1-4 alkylpyrimidyl, _{C1 -4} alkylthienyl, C _1-4 alkyl isothiazolyl, C _1-4 alkyl imidazolyl, C _1-4 alkyl tetrazolyl, C _1-4 alkyl pyrazinyl, C _1-4 alkyl pyrimidyl, C _1-4 alkyl quinolyl, C _1-4 alkyl isoquinolyl, C _1-4 alkylthiophenyl, _C1-4 alkylbenzothienyl, _C1-4 alkylisobenzofuryl, _C1-4 alkylpyrazolyl, _C1-4 alkylindolyl, _C1-4 alkylpurinyl, _C1-4 alkylcarbazolyl, _C1-4 alkylbenzimidazolyl and _C1-4 alkylisoxazolyl.

In one embodiment, R1 and/or R2 is benzyl, optionally substituted with one or more of the following substituents on the benzyl ring: X, _-CH3 , -NO2, _-OCH3 , -CN, -CHH2, -CH2H, _C2- C ₄ -alkyl, -СХ ₃ , -СН ₂ (СХ ₃ ), -СН(СХ ₃ ) ₂ , -С(СХ ₃ ) ₃ , -CpX _2p+1 , -OCX ₃ , -OCpH _2p+1 , - OCpX _2p+1 , ORm, SRm, NRmRn, NR ^m C(O)R ⁿ , SORm, SO2R ^m , C(O)R ^m and C(O)OR ^m ; Rm and ^Rn are independently selected from H or C1-C4alkyl; and where p is an integer from 2 to 20 and X is a halogen, including F, Cl, Br or I; preferably F, Cl or Br; more preferably F or Cl.

XI. Examples.

It should be understood that the description and examples given below are for illustrative purposes only and do not limit the scope of the present invention. The following examples are intended to illustrate the embodiments described and are not intended to be limiting.

Additional compounds other than those described below can be prepared using the reaction schemes described above or appropriate variations or modifications thereof.

Example 1. Synthesis of 2-chlorobenzylamino-2-imidazoline hydroiodide.

To a stirred solution of 2-methylthio-2-imidazoline hydroiodide (244 mg, 1.00 mmol) in dry dioxane (2.0 mL) was added 2-chlorobenzylamine (141 mg, 1.0 mmol). This reaction mixture was stirred for 90 minutes at a temperature of 70°C in an argon atmosphere. The solution was cooled to room temperature, filtered on a glass filter funnel, washed with cold dioxane (2 ml) and dried in vacuum. A white solid, 4-HI (R2=2-chlorobenzyl) (242 mg, 72%) was obtained and used without further purification.

Example 2. Synthesis of 2-chlorobenzylamino-2-imidazoline.

To a stirred solution of 2-chlorobenzylamino-2-imidazoline hydroiodide (242 mg, 0.72 mmol) in water (3 ml) was added 1.0 N. sodium hydroxide (2 ml) at 7°C. This reaction mixture was stirred for 30 minutes at a temperature of 7°C in an argon atmosphere. Methylene chloride (5 ml) was then added and the resulting mixture was stirred for another 5 minutes. The reaction mixture was extracted with methylene chloride (2x2.5 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The resulting free base (150 mg, 100%) was obtained as a viscous liquid and used for the next reaction without any further purification. MS (IER) 210 (M+N).

- 65 043367

Example 3 Synthesis of methyl 1-benzyl-4-oxo-3-piperidinecarboxylate (compound (6)).

Triethylamine (6 mL) was added to stirred methyl 1-benzyl-4-oxo-3-piperidinecarboxylate hydrochloride (5.7 g, 20 mmol) in ethyl acetate (50 mL) at 7°C. The reaction mixture was stirred for 30 min at 7°C in an argon atmosphere. The reaction mixture was extracted with ethyl acetate (2x50 ml), washed with water (50 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The resulting free base residue (5, R ₁ =benzyl) as a viscous oil was used for the next reaction without any further purification. MS (IER) 248 (M+N).

Example 4 Synthesis of ONC202 (compound (14))

To a solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mmol), methyl 1-benzyl-4-oxo-3-piperidinecarboxylate (5, R ₁ =benzyl) (195 mg, 0.79 mmol) in 1- butanol (2 ml), PPTS (10 mg) was added and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed at 125 to 130°C for 2 hours. Solvents were removed in vacuo, extracted with ethyl acetate (10 ml), washed with saturated sodium bicarbonate solution (2x10 ml) and water (10 ml). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The crude free base was purified by RP HPLC (10-40% acetonitrile/water) to give trifluoroacetic acid (TFA) salt ONC902 as a white solid (228 mg, 50% yield). MS (IER): 407 (M+N).

The same method was used, using different benzylamines, to obtain various analogues, for example, ONC203, 204, 205, 206, 912, 210, 211, 212, 213, 214, 217, 218, 219, 220, 221, 222, 223 , 224, 225 and 226.

Example 5 Synthesis of ONC207 (compound (19))

To a suspension of 60% sodium hydride (3.5 g, 88 mmol) in dry toluene (50 ml) was added dropwise dimethyl carbonate (4.32 g, 48.0 mmol) over 0.5 h at room temperature under nitrogen. After adding a few drops of methanol, 1-tert-butoxycarbonyl-4-piperidone (4.8 g, 24 mmol) dissolved in dry toluene (20 ml) was added dropwise to the reaction mixture with stirring at 80 °C for 1 h. The reaction mixture was stirred for 3 hours at the same temperature and then cooled to 0°C (ice bath), and adjusted to pH 6-6.5 with acetic acid. The resulting cold mixture was diluted with water (10 ml), and the pH was adjusted to 8 with 5% sodium hydroxide solution. The toluene layer was separated, and the aqueous layer was extracted with toluene (20 ml). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The compound was dried in vacuo to give methyl 1-tert-butoxycarbonyl-4-oxo-3-piperidinecarboxylate (5.0 g, 80%). The resulting compound was used in the next reaction without any further purification.

PPTS (10 ,0 mg), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed at 125 to 130°C for 2 hours. Solvents were removed in vacuo, extracted with ethyl acetate (10 ml), washed with saturated sodium bicarbonate solution (2x10 ml) and water (10 ml). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The crude free base was digested with 10% trifluoroacetic acid in dichloromethane, purified by RP HPLC (10-40% acetonitrile/water) to give TFA salt ONC907 (262 mg, 50%) as a white solid. MS (IER) 297 (M+N).

Example 6 Synthesis of ONC209 (compound (21)).

A mixture of ONC907 (100 mg, 0.2 mmol), phenylethyl bromide (55.0 mg, 0.28 mmol) and potassium carbonate (150 mg, 1.0 mmol) in N,N-dimethylformamide (3 ml) was heated to 70° C for 12 hours. Solvents were removed in vacuo, extracted with ethyl acetate (10 ml), washed with water (5 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude free base was purified by RP HPLC (10-40% acetonitrile/water) to give TFA salt ONC209 (62 mg, 50%) as a white solid. MS (IER) 401 (M+N).

The same method, starting with different halides, was used to prepare ONC215 and 214. Compounds 227, 228, 229, 230, 231, 232, 233, 234, 235 and 236 were prepared using a similar method from Examples 1 and 5, starting with a different benzylamine. The intermediate where R1 is H was then treated with various halides as above.

Compound ONC216 was prepared from ONC215 by treatment with TFA.

Compound (72) was prepared by reacting the NH compound precursor obtained by analogy with Example 5 and treating it with styrene oxide.

Example 7 Synthesis of ONC208 (compound (20)).

To a solution of 2-methylbenzylamino-2-imidazoline (190.0 mg, 1.0 mmol), methyl 1-methyl-4-oxo-3piperidinecarboxylate (185.0 mg, 1.0 mmol) in 1-butanol (2, 0 ml) PPTS (10.0 mg) was added and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed at 125 to 130°C for 2 hours. Solvents were removed in vacuo, extracted 66 043367 diluted with ethyl acetate (10 ml), washed with saturated sodium bicarbonate solution (2x10 ml) and water (10 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated. The crude free base was purified by HPLC using 10-40% acetonitrile and water to give TFA salt ONC908 (270.0 mg, 50%) as a white solid. MS (IER) 311 (M+N).

Example 8 Synthesis of ONC201 (compound (1)).

Compound (3) (239.7 g, 0.845 mol, 1.6 equiv) was added portionwise to a stirred 800 mL saturated solution of NaHCO ₃ in a 2 L round bottom flask. N-butanol (500 ml) was added to the resulting mixture, the mixture was stirred for 30 minutes and then transferred to a separatory funnel. The organic phase containing compound (4) was separated and transferred to a 2 L three-neck round bottom flask equipped with a mechanical stirrer, N ₂ inlet valve, thermocouple, condenser and Dean-Stark trap. Compound (5) (100 g, 0.528 mol, 1 eq) and pyridinium p-toluenesulfonate (PPTS) (6.63 g, 0.026 mol, 5 mol%) were added to the contents of the flask. The resulting mixture was refluxed for 6 hours. The water in the reaction mixture was separated, if necessary, using a Dean-Stark trap. The temperature for obtaining condensate when heated with reflux was increased from 93 to 118°C. The progress of the reaction was monitored by HPLC. When the peak area of compound (1) by HPLC analysis remained constant over the reaction time, the reaction was stopped.

Example 9. Synthesis of di-salt ONC201 (compound (1)-2HCl).

Without isolating compound (1), the reaction mixture obtained in Example 8 was washed with water (500 ml) and diluted with methyl tert-butyl ether (MTBE) (800 ml). The organic phase was washed with water (50 0 mlx2) and transferred to a 3 L three-neck round bottom flask equipped with a mechanical stirrer, N ₂ inlet valve, thermocouple, condenser and Dean-Stark trap. While stirring the reaction mixture, 1N was added dropwise. HCl in dioxane-MTBE solution (4N HCl in dioxane: 300 ml, 1.2 mol, 2.27 eq; MTBE: 1200 ml) until no more solids precipitated from the reaction mixture when HCl was added to the reaction mixture. The reaction mixture was refluxed at 60-65°C for 2 hours. Water was separated using a Dean-Stark trap as necessary. As it cooled to room temperature, the solid was filtered through a glass filter funnel and washed with n-butanol-MTBE (1:2, 600 ml) and MTBE (600 ml), respectively. The solid was dried in a vacuum oven at 65°C overnight (16 hours) to obtain 200 g of a yellow solid.

The above solid (200 g) was added to a 2 L three-neck round bottom flask equipped with a mechanical stirrer, _N2 inlet valve, thermocouple and condenser, followed by ethanol (1000 ml). The mixture was refluxed at 78°C for 2 hours. As it cooled to room temperature, the solid was filtered through a glass funnel and washed with ethanol (2 00 mlx3). The wet solid was dried in a vacuum oven at 85°C for 3 days until the residual solvent content satisfied the requirements. 120 g of compound (2) were obtained as a white solid in 49% yield and HPLC purity of 99.7%.

Example 10. Activity of imipridones.

A number of imipridones were prepared based on the syntheses described herein. For each of these compounds, the viability of human cancer cells was measured 72 hours after treatment with the indicated compound. The change in activity (relative to ONC201) was determined; the data are presented in table. 3.

- 67 043367

Table 3

Relative activity of ONC201 analogues

No. Identifier ator R1 r ₂ Relative activity* 1 ONC201 _CH2Ph CH ₂ - ((2-CH ₃ ) -Ph) No data 14 ONC202 _CH2Ph CH ₂ (2-Cl-Ph) IN 15 ONC203 _CH2Ph CH ₂ -(2-thienyl) WITH 16 ONC204 _CH2Ph CH ₂ CH ₂ Ph CH ₂ CH ₂ (4-N-benzyl- WITH 17 ONC205 _CH2Ph piperazine) WITH 18 ONC206 _CH2Ph CH ₂ -(2,4-di F-Ph) A 19 ONC207 H CH ₂ -( (2-CH ₃ )-Ph) IN 20 ONC208 CH ₃ CH ₂ -( (2-CH ₃ )-Ph) IN 21 ONC209 CH ₂ CH ₂ Ph CH ₂ - ((2-CH ₃ ) -Ph) IN 32 ONC215 ( _CH2 )3-NH-BOC CH ₂ - ((2-CH ₃ ) -Ph) IN 33 ONC216 (ch ₂ ) ₃ -nh ₂ CH ₂ - ((2-CH ₃ ) -Ph) IN 41 ONC210 _CH2Ph CH ₂ -(3,5-di F-Ph) IN 51 ONC211 _CH2Ph CH ₂ -(3,4-di Cl-Ph) IN 52 ONC212 _CH2Ph CH ₂ - (4-CF ₃ -Ph) A 53 ONC213 _CH2Ph CH ₂ -(3,4-di F-Ph) A 54 ONC214 cd ₂ c ₆ d ₅ CH ₂ -( (2-CH ₃ )-Ph) IN 43 ONC217 _CH2Ph CH ₂ (2-F-Ph) WITH 55 ONC218 _CH2Ph CH ₂ (2-CH ₃ , 4-F-Ph) A 56 ONC219 _CH2Ph CH ₂ - (2,4-di Cl-Ph) A 57 ONC220 _CH2Ph CH ₂ - ( (4-OSH ₃ ) -Ph) CH ₂ -(3- A 35 ONC222 _CH2Ph isoxalidinyl) CH ₂ CH ₂ - (4- WITH 36 ONC224 _CH2Ph morpholinyl) A 38 ONC221 H CH ₂ - (4-CF ₃ -Ph) A 72 ONC225 _CH2Ph CH ₂ -(2-F, 4-CF ₃ -Ph) A 37 ONC223 _CH2Ph CH ₂ -(4-CH ₃ -Ph) A 34 ONC226 _CH2Ph CH ₂ - (3-pyridinyl) A 77 ONC231 CH ₂ -3-pyridyl CH ₂ - (4-CF ₃ -Ph) A 78 ONC232 CH ₂ -4-methyl-2thiazolyl CH ₂ - (4-CF ₃ -Ph) IN 79 ONC233 CH ₂ -2pyrazinyl CH ₂ - (4-CF ₃ -Ph) IN 81 ONC234 CH ₂ -(3, 4-di Cl-Ph) CH ₂ - (4-CF ₃ -Ph) A 83 ONC236 CH ₂ -3-thienyl CH ₂ - (4-CF ₃ -Ph) A 84 ONC237 CH ₂ CH(OH)Ph CH ₂ - (4-CF ₃ -Ph) WITH 73 ONC227 CH ₂ - (4-CF ₃ -Ph) CH ₂ - (4-CF ₃ -Ph) IN 74 ONC228 CH ₂ -(4-F-Ph) CH ₂ - (4-CF ₃ -Ph) A 75 ONC229 CH ₂ -(4-OSH ₃ Ph) CH ₂ - (4-CF ₃ -Ph) IN 76 ONC230 4-F-Ph-4oxobutyl CH ₂ - (4-CF ₃ -Ph) A

*Regarding ONC201 activity; A denotes a 2-fold increase in activity compared to ONC201; B indicates activity that is within 2-fold of ONC201; and C denotes a 2-fold decrease in activity from ONC201.

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IC _50s were determined for ONC201 and ONC212 (5 nM - 5 μM, 72 h) when treated with several acute myeloid leukemia (AML) cell lines (n=3), the data are shown below in table. A.

Table A

ONC212

Cellular AML line ONC201 1C ₅₀ (µM) ONC212 1C ₅₀ (µM) MV411 3.25 0.01 HL60 >5 0.21 MOLM14 3, 92 0.01

Cell viability of MV411 AML cells treated with ONC212 and cytarabine (5 nM - 5 μM, 24 h) was measured (n=3) (Fig. 29A). In addition, the cell viability of MOLM14, MV411 AML, MCR5 lung fibroblasts, and Hs27a bone marrow cells treated with ONC212 (5 nM - 5 μM, 72 h) was measured (n=3) (Figure 29B). The viability of MOLM14 cells and MV411 AML cells treated with ONC212 (250 nM) was determined at 4, 8, 24, 48, 72, and 96 h. ONC212 media was replaced with fresh medium at these time points, and cell viability was determined at 96 h. for all samples. (n=2) (Fig. 29C).

Additionally, a single dose of compound (52) (ONC212) administered orally or intraperitoneally to mice bearing a human colon cancer xenograft resulted in a significant reduction in tumor volume compared to vehicle-treated control cohorts (FIG. 24). Compound (52) has a wide therapeutic range as it is well tolerated at doses in mice up to at least 225 mg/kg.

Additionally, ONC212 demonstrated efficacy in an ONC201-resistant AML xenograft model (FIG. 30). MV411 AML cells (5x10 ⁶ ) were implanted subcutaneously in the flank of athymic nude mice. ONC212 and ONC201 were administered orally (PO) as indicated. Tumor volume (A and B) and body weight (C) (n=10) were measured on the indicated days. * represents p < 0.05 relative to vehicle.

The efficacy of ONC212 in AML was assessed in vitro and was 400-fold higher compared to ONC201 (Table A). ONC212 was also effective in AML cells resistant to cytarabine, which is the standard of care (Fig. 29A). Despite the sustained increase in efficacy, ONC212 maintained a wide therapeutic window in vitro and was nontoxic to normal cells at effective concentrations (Figure 29B). An 8-hour exposure to ONC212 at 250 nM was sufficient to cause a sustained decrease in the viability of MOLM14 and MV411 AML cells (Figure 29C). ONC201 required at least 24-48 hours to be effective.

The efficacy of ONC212 was determined in a leukemia xenograft model with MV411 AML cells resistant to cytarabine, which is the standard of care (FIG. 30). 50 mg/kg ONC212 significantly reduced leukemia xenograft tumor growth when administered weekly orally, whereas ONC201 was not effective in this model at similar doses (Figure 30A). Interestingly, biweekly dosing of ONC212 at 25 mg/kg and weekly/biweekly at 5 mg/kg were not effective (Figure 30B). None of these ONC212 administration regimens were associated with body weight loss (Figure 30C) or macroscopic observations.

mg/kg ONC212 represents the maximum dose causing no detectable adverse health effects (NOAEL) in non-NLP oral single dose studies in mice and rats, which is also the effective dose in mouse xenograft studies. ONC212 is approximately 10 times more toxic than ONC201 (maximum dose not causing detectable harm was 225 mg/kg in a non-NLP single oral dose study in rats).

ONC206

ONC206 demonstrated efficacy in a xenograft model of Ewing's sarcoma (Fig.

31). Ewing's sarcoma cells MHH-ES-1 (5x10 ⁶ ) were implanted subcutaneously into the flank of an athymic naked

- 69 043367 mice. ONC206 (PO) and methotrexate (IV) were administered on day 1 and day 13 as indicated. Tumor volume (FIG. 31A) and body weight (FIG. 31B) (n=4) were measured on the indicated days.

Additionally, IC50 for ONC201 and ONC206 (5 nM - 5 µM, 72 h) were determined when treating several cell lines (n=3), the data are shown in the table below. IN.

Table B

Cell line ONC201 1C ₅₀ (µM) ONC2 0 6 1С ₅₀ (µM) MV411 (AML) 3.25 0.2 K562 (HML) >5 0.22 MOLM14 (OML) 3, 92 0.27 MHH-ES-1 (sarcoma Ewing) 5, 65 0.61 HFF (normal fibroblasts) >5

ONC206 showed up to a 20-fold improvement in in vitro activity compared to ONC201 without any toxicity to normal cells at therapeutic doses (Table B). ONC206 had only twofold increased toxicity (maximum dose not causing a detectable adverse health effect of 125 mg/kg) compared to ONC201 (maximum dose not causing a detectable adverse health effect of 225 mg/kg) in a single oral dose study in rats not conducted as part of NLP. In vivo efficacy in Ewing's sarcoma model without toxicity (Fig. 31). The efficacy of ONC206 was comparable to methotrexate chemotherapy, but chemotherapy was associated with weight loss.

ONC213

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that ONC213 selectively interacts with DRD2/3 and GPR132/91 (Fig. 32). Dual targeting of DRD2/3 and GPR132/91 represents a novel strategy to provide anticancer efficacy in the absence of toxicity. ONC213 is a DRD2/3 inhibitor and GPR132/91 agonist. The activity against DRD2/3 of ONC213 is greater than that of ONC201, but less than that of ONC206. The effectiveness of ONC213 against GPR132 is lower than that of ONC212. Specifically, ONC213 demonstrated in vitro anticancer activity in HCT116/RPMI8226 cancer cells similar to ONC212, but in vitro toxicity to normal cells was reduced compared to ONC212 (Figure 33). The safety profile of ONC213 was confirmed in an IVD study in mice, with a maximum dose causing no detectable adverse health effects of 75 mg/kg, three times that of ONC212 (25 mg/kg). The agonistic activity of ONC213 against GPR91 opens up the possibility of using this compound in immunology, immune oncology and for hematopoietic applications (Nature Immunology 9:1261 (2008); J Leukoc Biol. 85(5):837 (May 2009)).

ONC237

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that ONC237 selectively interacts with DRD5 and GPR132 (Fig. 34). ONC237 is a GPR132 agonist and DRD5 antagonist and has reduced antitumor efficacy (IC50 31.2 μM) compared to ONC201. These data indicate that combining GPR132 agonist activity with DRD5 (D1-like dopamine receptor) antagonist activity results in poor anticancer activity compared to ONC213, which combines GPR132 agonist activity with DRD5 antagonist activity.

- 70 043367 regarding DRD2/3.

ONC236

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that ONC236 is a highly selective agonist of GPR132 (Fig. 35). ONC236 has antitumor efficacy (IC50 8 8 nm) that is comparable to that of ONC212 (10 nm), and better than that of ONC206/ONC201, the completeness of response is better than ONC201, but not than ONC212 in HCT116 cells.

ONC234

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that ONC234 is a broad-spectrum small molecule that selectively interacts with GPCRs (Figs. 36 and 38). ONC234 has activity at several GPCRs, including antagonist activity at the adrenergic, histamine, serotonin, CHRM, CCR, DRD2/5 receptors, as well as CXCR7 agonist activity. ONC236 has antitumor efficacy (IC ₅₀ 234 nM) comparable to that of ONC206, response completeness is the same as ONC212 and better than ONC201 in HCT116 cells.

Linear isomer ONC201 (TIC-10)

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that the linear isomer of ONC201 (TIC-10) is a CXCR7 agonist (Fig. 37). CXCR7 agonists can be used to regenerate the liver and prevent or treat fibrosis such as liver fibrosis (Nature 505:97 (2014)). Fibrosis is the formation of excess coarse fibrous connective tissue in an organ or tissue, including as a result of wound healing. Examples of fibrosis include pulmonary fibrosis, including cystic fibrosis and idiopathic pulmonary fibrosis; radiation damage to the lungs after cancer treatment; liver fibrosis (cirrhosis); cardiac fibrosis, including atrial fibrosis, endomyocardial fibrosis and old myocardial infarction; glial scar; arthrofibrosis; Crohn's disease; Dupuytren's contracture; keloid scars; mediastinal fibrosis; myelofibrosis; Peyronie's disease; nephrogenic systemic fibrosis; progressive massive fibrosis; retroperitoneal fibrosis; scleroderma/systemic sclerosis and adhesive capsulitis.

Example 11 Antagonism of GPCR ONC201.

ONC201 was assessed in a whole cell using a functional G protein-coupled receptor (GPCR) beta-arrestin activity assay, which directly measures dopamine receptor activity by detecting the interaction of beta-arrestin with an activated GPCR that can serve as a reporter. For each dopamine receptor (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5), cell lines overexpressing reporter constructs were recovered from freezer stocks. Cells were seeded in a total volume of 20 μl in 384-well white-walled microplates and incubated at 37°C prior to antagonist assay, followed by agonist administration at an EC concentration _{of 80} . Intermediate dilution of sample stock solutions was carried out to obtain 5 samples in assay buffer. 3.5 μl of 5x sample was added to the cells and incubated at 37°C or room temperature for 30 min. The carrier concentration was 1%. Cells were added with 5 μl of 6x EC80 agonist in assay buffer and incubated at 37°C or room temperature for 90 or 180 minutes before analysis. % antagonism was calculated by

- 71 043367 the following formula: % antagonism=100%x(1-(average RLU value of the analyzed sample - average RLU value of the media control)/(average RLU value of the EC80 control average RLU value of the media control).

Example 12 Selective antagonism of DRD2 by ONC201.

ONC201 is a first-in-class small molecule identified in a phenotypic screen for p53-independent inducers of tumor-selective proapoptotic pathways. Orally administered ONC201 is being evaluated as a novel therapeutic agent in five early-phase clinical trials in select advanced cancers due to its demonstrated efficacy in aggressive and refractory tumors and excellent safety.

This example reports the prediction and confirmation of selective direct molecular interactions between ONC201 and specific receptors of the dopamine receptor family. Experimental GPCR profiling showed that ONC201 selectively antagonizes the D2-like dopamine receptor subfamily, but not the D1-like dopamine receptor subfamily. Reporter assays in a heterologous expression system showed that ONC201 selectively antagonizes both the short and long isoforms of DRD2 and DRD3, with lower activity against DRD4 and no antagonism of DRD1 or DRD5. Increased prolactin secretion is a clinical criterion for DRD2 antagonism by several psychiatric drugs that potentially target this receptor. ELISA analysis of peripheral blood from ONC201-treated patients in a first-in-human study with advanced solid tumors showed that 10/11 patients reported detectable prolactin induction (mean 2-fold).

Using the TCGA database, the D2-like dopamine receptor subfamily, particularly DRD2, was found to be predominant and selectively overexpressed in several malignancies. Preclinical reports indicate that inhibition of DRD2 provides antitumor efficacy without killing normal cells through induction of ATF4/CHOP and inhibition of Akt and ERK signaling, all of which are properties possessed by ONC201.

Ways.

ONC201 dihydrochloride was obtained from Oncoceutics. Kinome kinase inhibition assays were performed as described (see Anastassiadis et al., Nat Biotech 29:1039 (2011)). GPCR arrestin recruitment and cAMP modulation reporter assay were performed as described (see McGuinness et al., Journal of Biomolecular Screening 14:49 (2009)). Path-HunterTM (DiscoveRx) β-arrestin cells expressing one of several target GPCRs were plated on 384-well white-bottom assay plates (Corning 3570), 5000 cells per well in a 20 μl volume in the appropriate cell seeding reagent. Cells were incubated at 37°C with 5% CO ₂ for 18-24 hours. Samples were prepared in buffer containing 0.05% fatty acid-free BSA (Sigma). To test the agonist regimen, samples (5 μl) were added to pre-seeded cells and incubated for 90 min at 37°C with 5% CO ₂ . For antagonist mode tests, samples (5 µl) were added to pre-seeded cells and incubated for 30 min at 37°C with 5% CO ₂ followed by the addition of agonist EC ₈₀ (5 µl) for 90 min at 37°C at 5% CO ₂ . For the Shield assay, samples (5 µl) were added to pre-seeded cells and incubated for 30 min at 37°C with 5% CO ₂ followed by the addition of serially diluted agonist (5 µl) for 90 min at 37°C at 5 % CO ₂ . In parallel, control wells were tested to determine the maximum and minimum response for each analytical mode. Arrestin recruitment was measured by adding 15 μl of PathHunter detection reagent, incubating for 1–2 h at room temperature, and reading on a Perkin Elmer Envision plate reader. For agonist and antagonist tests, data were normalized to percent efficacy using appropriate controls and fitted with a dose-response sigmoid (variable slope), Y=bottom+(top-bottom)/(1+10 ^Λ ((LogEC50- X)*HillSlope)), where X is the logarithmic concentration of the compound. For Schild analysis, data were normalized by percent efficiency using appropriate controls and fitted to the Gaddum/Schild EC50 shift using a global fit where Y=bottom+(top)/(1+10 ^Λ ((LogEC-X )*HillSlope)), Antag=1+(B/(10 ^l (-1*pA2))) ^l SchildSlope and LogEC=Log (EC50*Antag). EC ₅₀ /IC ₅₀ analysis was performed using CBIS data analysis software (Cheminnovation) and Schild analysis was performed in GraphPad Prism 6.0.5.

Results.

ONC201 is a small molecule in phase II clinical trials for selected advanced cancers that was discovered in a phenotypic screen for p53-independent inducers of the pro-apoptotic TRAIL pathway. Although the contribution of ONC201-induced ATF4/CHOP up-regulation and inactivation of Akt/ERK signaling (Allen et al., Science translational medicine 5, 171ra117-171ra117 (2013)) to the anticancer activity of this compound has been characterized, but the molecular target for binding remains unknown.

In vitro profiling of GPCR activity using a heterologous reporter gene assay with arrestin recruitment, a hallmark of GPCR activation, revealed that ONC201 selectively antagonizes the D2-like (DRD2/3/4) subfamily of dopamine receptors, but not the D1-like (DRD1/5) subfamily ) dopamine receptors (Fig. 4B and 5A). No antagonism of adrenergic receptors, alpha receptors, or other GPCRs was observed under the conditions under which they were evaluated. Among the DRD2 family, ONC201 antagonized both the short and long isoforms of DRD2 and DRD3, with lower efficiency for DRD4. Further characterization of ONC201-mediated antagonism of arrestin recruitment to DRD2L was assessed using the Gaddum/Schild EC ₅₀ shift assay, which determined a dissociation constant of 2.9 μM for ONC201, equivalent to the effective dose in a large number of human cancer cells (Fig. 4C). Supporting results were obtained for cAMP modulation in response to ONC201, another measure of DRD2L activation (Fig. 4D). The ability of dopamine to inversely antagonize dose-dependently up to 100 μM ONC201 suggests direct competitive antagonism of DRD2L ( Figures 5B and 5C ). Consistent with the specificity of ONC201 predicted by BANDIT, there were no significant interactions between ONC201 and nuclear hormone receptors, kinome, or other FDA-approved drug targets (Figures 5D and 5E) . Interestingly, the biologically inactive constitutional isomer ONC201 (Wagner et al., Oncotarget 5:12728 (2014)) did not inhibit DRD2L, suggesting that antagonism of this receptor may be related to its biological activity (Figure 5F). In summary, these studies demonstrate that ONC201 selectively antagonizes the D2-like dopamine receptor subfamily, which appears to be a promising therapeutic target in oncology, and that ONC201 is the first compound to utilize this treatment paradigm in several ongoing phase II clinical studies.

Example 13. Mapping of DRD2 epitopes using Shotgun Mutagenesis technology.

Shotgun Mutagenesis uses high-throughput cellular expression technology to express and analyze large libraries of mutated target proteins in eukaryotic cells. Each residue in the protein is individually mutated to alanine or another specific residue to analyze changes in function. Proteins are expressed in standard mammalian cell lines, so even complex proteins that require eukaryotic translational or post-translational processing can be displayed.

First, conditions for screening ONC201, a DRD2 antagonist, against wild-type DRD2 were evaluated and identified using a Shotgun Mutagenesis screening assay. An Ala DRA2 scan library was then prepared and residues critical for ONC201 binding were mapped to single amino acid resolution using Shotgun Mutagenesis technology.

DRD2 Shotgun Mutagenesis Library:

Parent plasmid: DRD2.

Library size: 442 mutant clones (full protein).

Mutation strategy: Alanine scanning mutagenesis.

Cell type: HEK-293T.

Screening analysis: Calcium flux.

Epitope tag: C-terminal V5/HIS6.

Parental construct: DNA encoding full-length human DRD2 (Code: NP_000786.1; MDPLNLSWYD DDLERQNWSR PFNGSDGKAD RPHYNYYATL LTLLIAVIVF GNVLVCMAVS REKALQTTTN YLIVSLAVAD LLVATLVMPW WYLEWGEW KFSRIHCDIF VTLDVMMCTA SILNLCAISI DRYTAVAMPM LY NTRYSSKR RVTVMISIVW VLSFTISCPL LFGLNNADQN ECIIANPAFV VYSSIVSFYV PFIVTLLVYI KIYIVLRRRR KRVNTKRSSR AFRAHLRAPL KGNCTHPEDM KLCTVIMKSN GSFPVNRRRV EAARRAQELE MEMLSSTSPP ERTRYSPIPP SHHQLTLPDP SHHGLHSTPD SPAKPEKNGH AKDHP KIAKI FEIQTMPNGK TRTSLKTMSR RKLSQQKEKK ATQMLAIVLG VFIICWLPFF ITHILNIHCD CNIPPVLYSA FTWLGYVNSA VNPIIYTTFN IEFRKAFLKI LHC (SEQ ID NO: 1) was subcloned into a mammalian high expression vector. This parent construct was sequence verified and then confirmed for mammalian cell expression by detecting calcium flux in response to dopamine. Received DNA from plasmid preparations was suitable for high-throughput processing.

Assay performance: The DRD2-specific calcium flux assay was successfully optimized for DRD2 expressed in human cells. An agonist dose-response analysis was used to determine the appropriate dopamine concentration to use in optimizing DRD2-specific calcium flux inhibition by the antagonist ONC201. Subsequent dose-response inhibition assays identified a concentration of ONC201 that inhibited the dopamine response by >95%.

Optimizing calcium flux assays: receptor activity assays. DRD2 activity was assessed using a published GPCR assay (Greene, T.A. et al., (2011) PLoS One 6, e20123). Briefly

- 73 043367 ce, HEK-293T cells were transfected with expression constructs for wild-type DRD2 or negative control GPCR in a 384-well format. After 22 h, calcium flux experiments were performed over a range of dopamine concentrations (300 pM-100 nM) using a Flexstation II-384 fluorescence reader (Molecular Devices). Data sets were analyzed and presented as percentages by signal baseline using Prism 5.0 software (GraphPad Software, Inc).

For cells expressing DRD2 but not the control GPCR, addition of dopamine resulted in an increase in cellular calcium flux, measured as increased fluorescence. A dose response plot of fluorescence peak heights as a function of dopamine concentration demonstrated a strong dopamine-induced calcium flux ( _EC50 =0.45 nM) in cells expressing DRD2 but not the control GPCR. This showed that calcium flux assay could be used to test ONC201 inhibition.

Optimization of the DRD2 calcium flux inhibition assay.

After determining the EC ₅₀ for dopamine in a calcium flux assay, inhibition of DRD2-specific calcium flux by ONC201 was examined at several concentrations of dopamine. Using 1 nM dopamine (>2-fold higher than EC ₅₀ of dopamine) with a range of ONC201 concentrations (1 nM to 100 μM), inhibition of dopamine-induced DRD2 calcium flux by ONC201 at the highest concentrations was observed (Figure 9A). with complete inhibition at 100 µM ONC201 (IC ₅₀ =21.5 µM). The inhibition of calcium flux by 100 μM ONC201 was not the result of broad GPCR inhibition or a nonspecific effect on cells, since ONC201 did not affect the calcium flux activity of cells expressing the control GPCR (Fig. 9B).

Analysis of a series of replicate values obtained for inhibition of DRD2 calcium flux by 100 μM ONC201 showed a significant study, with a Z' value of 0.61. The Z' value is a measure of assay quality calculated from the means and standard deviations obtained for replicate calcium flux determinations with or without ONC201.

Comparison of DRD2 inhibitors.

Inhibition of DRD2 by ONC201 was compared with inhibition of DRD2 by spironer and domperidone (Fig. 10), which were described to inhibit DRD2 at concentrations below the 100 μM required for ONC201 inhibition. These antagonists were screened at concentrations ranging from 100 pM to 1 μM, and in both cases complete inhibition of dopamine-induced calcium flux was observed, with spiperone IC ₅₀ = 19 nM and domperidone IC ₅₀ = 47 nM. These values are consistent with previously obtained characteristics and demonstrate that the relatively high IC ₅₀ value obtained for ONC201 (21.5 μM) is not the result of using a calcium flux assay to measure DRD2 activity.

Optimal screening conditions were determined for inhibition of DRD2-related calcium flux in response to dopamine by ONC201. These conditions allow a robust response to dopamine to be obtained, and this response is reduced by >95% when ONC201 is added to 100 μM, and the assay shows low variability between replicates. These data indicate that the selected conditions are suitable for successful high-throughput screening. Further screening of the DRD2 mutation library was performed at a dopamine concentration of 1 nM and an ONC201 concentration of 100 μM.

Screening the DRD2 alanine scanning library for dopamine response.

The library of alanine-scanning DRD2 mutations (and with alanines replaced by serines) was 442 clones, covering residues 2-444 of the DRD2 protein, representing 100% of the target residues. The DRD2 mutation library was first screened using a calcium flux assay with dopamine (1 nM) in the absence of ONC201 to identify residues whose mutation reduced dopamine-induced calcium flux. Twenty-eight amino acid residues were identified that were critical for dopamine-induced DRD2 flux (Fig. 11).

The residues identified from the analysis are given in Table. 4 and shown in FIG. 11. Clones were considered calcium flux deficient if they exhibited flux values less than 2 standard deviations below the average calcium flux value (AV - 2SD) for the entire library.

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Table 4

DRD2 residues, for

calcium

Screening of the DRD2 alanine scanning library for inhibition of dopamine-induced signaling by ONC201 to identify residues required for ONC201 inhibition.

To identify residues important for DRD2 inhibition by ONC201, a library of DRD2 alanine scanning mutations was screened by calcium flux assay for the ability to respond to dopamine in the presence of an inhibitory concentration of ONC201 using dopamine at 1 nM and ONC201 at 100 μM. Eight residues critical for the inhibitory activity of ONC201 were identified (Fig. 12). All residues identified during this screen showed high calcium flux with dopamine only (Table 5). Clones were considered critical for inhibition by ONC201 at 100 μM if they exhibited flux values more than 2 standard deviations above the mean calcium flux (AV+2SD) for the entire library. In table Figure 5 also shows for these critical clones the calcium flux values obtained in similar experiments performed using 250 µM ONC201 or without ONC201 (1 nM dopamine), and in addition the % conservation of critical residues across 5 DRD receptors, and the residues detected in each receptor.

Table 5

DRD2 residues critical for inhibition of dopamine-induced calcium flux

Calcium flux as % of flux with WT DRD2 (100) Saving DRD % DRD Mutation ONC201 100 µM ONC201 250 µM Dopamine 1nM 1 2 3 4 5 I397A 122 89 105 20 R I T A R E95A 97 39 123 100 E E E E E V91A 94 58 119 40 TO V V F TO Y192A 85 eleven 64 60 S Y Y Y S V19 6A 79 22 119 40 I V V WITH I A177S 77 26 85 40 A A T V D T165A 67 28 92 20 L T A A L L81A 63 20 83 100 L L L L L

Since the average inhibition at 100 μM ONC201 across the entire library was approximately 75%, a screen was also performed at 250 μM ONC201 to determine whether the critical residues would be the same at higher levels of inhibition. Under this condition, dopamine-induced calcium flux was inhibited by approximately 93%, and the previously identified residues V91, E95, and 1397 were also critical for inhibition at 250 μM ONC201 (Table 5), using the same criteria for flux values, by more than 2 standard deviations above the average calcium flux (AV+2SD) for the library.

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Conclusions.

In an initial screening of a library of alanine-scanning DRD2 mutations using a dopamine-induced calcium flux assay, 28 mutations were found to significantly reduce calcium flux, thereby identifying residues critical for DRD2 function. As shown in a similar analysis of the CXCR4 GPCR, critical residues were distributed throughout the protein, in the predicted dopamine binding pocket, transmembrane regions, and in the exposed cytoplasmic portion of DRD2. These 28 residues are critical for dopamine binding, signal transduction across transmembrane domains, or G protein binding. From detailed analysis comparable to that performed for CXCR4, as well as structural analysis of the DRD3-ethyllopride structure (Chien et al., 2010), a specific function can be assigned to each critical residue of DRD2.

To identify residues important for DRD2 inhibition by ONC201, a library of DRD2 alanine scanning mutations was screened using a calcium flux assay with dopamine and 100 μM ONC201. From this screen, 8 residues were identified as critical for ONC201 inhibition of DRD2-dependent dopamine-induced calcium flux L81, V91, E95, T165, A177, Y192, V196 and 1397. Residues V91, E95 and I397 were also identified as critical for resistance to inhibition of DRD2 by 250 μM ONC201, indicating that they are key residues interacting with ONC201. These residues define a relatively large ligand binding site, which is not unexpected due to the larger size of ONC201 compared to dopamine and ethiclopride. The location of these residues is generally consistent with a role in mediating ONC201 inhibition of DRD2-dependent dopamine-induced calcium flux. Residues critical for inhibition of the GPCR taste receptor by probenecid were identified previously (Greene et al., 2011), and the location of these residues was consistent with a noncompetitive mechanism of inhibition. In contrast, the residues identified here for DRD2 are consistent with competitive inhibition at the dopamine binding site by ONC201. When modeled on the structure of the homologous receptor DRD3, the majority of identified residues surround the binding pockets containing the cocrystallized antagonist ethicloprid, with 5 of the 8 identified residues being conserved between DRD2 and DRD3. Two of the residues appear to be more distant from the putative binding site (A177 and L81) and may influence ONC201 binding in a more allosteric manner. Additional residues that contribute to ONC201 inhibition can be identified using DRD2 agonists whose structures are different from dopamine.

Example 14. Determination of rate constants of association and dissociation of unlabeled dihydrochloride ONC201 on the human D2S receptor.

In this example, the kon/koff rate constants of unlabeled ONC201 dihydrochloride at the D2S receptor were determined. The kon/koff rate constants were estimated by competitive ligand binding according to the method described in: M.R. Dowling & S.J. Charlton (2006) Brit. J. Pharmacol. 148:927937 and H.J. Motulsky & L.C. Mahan (1984) Mol. Pharmacol. 25:1-9. In this method, the kon/koff rate constants of unlabeled test compounds were calculated from the Ki value (competitive binding) and its effect on the radioligand binding kinetics (competitive kinetics).

First, the IC ₅₀ and Ki values of ONC201 dihydrochloride were determined and suitable concentrations of compounds were selected for the competitive binding kinetics experiment. The rate constants kon and koff of the radioligand ([ ³ H]methylspiperone) were then determined. The rate constants kon and koff of unlabeled ONC201 dihydrochloride were then determined. ONC201 dihydrochloride was tested at 8 concentrations in duplicate (n=2) in a competitive binding assay and IC ₅₀ and Ki values were determined.

The reference compound, (+)butaclamol, and the test compound, ONC201-2HCl, successfully competed for [ ^3H ]methylspiperone, with _IC50 values of 2.5 nM and 21 μM, respectively. Previously, the compound ONC201-2HCL gave a similar IC ₅₀ value of 16 μM. For the competitive binding assay, the following 6 concentrations of ONC201-2HCL were selected: 5/10/20/40/60/80 μM.

The kinetics of binding of [ ³ H]methylspiperone to the D2S receptor was determined. To do this, [ ^3H ]methylspiperone (at a single concentration of 0.3 nM) was incubated with D2S receptor membranes for 12 different incubation periods to measure the rate of association. Nonspecific binding was measured using butaclamol (10 μM) for each incubation period. Dissociation was initiated by adding excess butaclamol (10 μM) after 60 min of incubation of [ ³ H]methylspiperone (0.3 nM) with D2S receptor membranes, and signal reduction was measured after 12 different incubation periods. The experiment was carried out in triplicate (n=3), the incubation time was adjusted to 0/30/60/80/120/180/240/300/360/420/480 min and 24 h for association kinetics and 2/5/8/ 10/15/20/25/30/40/60/120/180 min for dissociation kinetics.

[ ³ H]Methylspiperone showed a kon value of 2.3 x 10 ⁸ M^min ^-1 and a koff value of 0.009506 min ^-1 (and therefore a t1/2 value of 73 min) at the D2S receptor. _Kdi calculated from the association/dissociation experiment (0.04 nM) is in the same range

- 76 043367 compared to the Kd observed in the saturation experiment (0.15 nM), thereby validating the experiment.

The effect of unlabeled ONC201-2HCl at six concentrations on the association kinetics of [ ³ H]methylspiperone (0.3 nM) was investigated. Nonspecific binding was measured using butaclamol (10 μM). The same 12 incubations were used as above: 2/5/8/10/15/20/25/30/40/60/120/180 min.

Measurement in the absence of compound was performed as a negative control.

ONC201-2HC1 showed a kon value of 4.1x105 M ^-1 min ^-1 and a koff value of 1.32 min ^-1 (and therefore a t1/2 value of 0.53 min) at the D2S receptor. The Ki calculated from the association/dissociation experiment (3.2 μm) is in the same range compared to the Ki observed in the saturation experiment (7 μm), thereby validating the experiment. In conclusion, ONC201-2HCl exhibits much slower association and much faster dissociation compared to [ ^3H ]methylspiperone.

Example 15. Bactericidal activity of imipridones.

Materials and methods.

Test material: dihydrochloride ONC201; Control: microcrystalline cellulose.

Method: harmonized microbiological study of EP/USP non-sterile drugs (currently valid United States Pharmacopeia (USP) <61>/<62>).

Results.

Table 6

Verification of inoculum recovery regulation and microorganism quantification

Dilution 1:300 with TSB Mod Number of indicator organisms EU Sa Ra Bs Ca (TSA) /But (TSA) SA (SDA) /But (SDA) Inoculum 27 31 28 52 48 21 52 20 434019 No data 0 24 48 51 18 46 19

Table 7

Testing for specified microorganisms

Sample BTGN EU Ra Sa Sa dilution 1:300 with TSB Mod R R R F R

P=satisfactory F=unsatisfactory NA=not applicable;

Ec=Escherichia coli ATCC# 8739; Pa=Pseudomonas aeruginosa ATCC# 9027; Sa=Staphylococcus aureus ATCC# 6538; Bs=Bacillus subtilis ATCC# 6633; Ca=Candida albicans ATCC# 10231; Ab=Aspergillus brasiliensis ATCC# 16404; BTGN=bile-resistant gram-negative bacteria; Cs=Clostridium spp.; TSA=trypticase soy agar; SDA=Sabouraud dextrose agar.

ONC201 dihydrochloride, when tested at a 1:300 dilution with TSB Mod, did not meet USP (United States Pharmacopeia) <61>/<62> requirements.

Analysis for microbiological purity. Inhibition was observed for Staphylococcus aureus for USP <61>/<62>. Therefore, it can be assumed that the failure to isolate the inoculated microorganism is due to the bactericidal activity of ONC201 dihydrochloride, and, therefore, the specified compound is unlikely to be contaminated with inhibited particles of the microorganism.

The minimum inhibitory concentration (MIC) of the six imipridones against wild-type methicillin-resistant Staphylococcus aureus was then determined.

Materials and methods.

Connections.

ONC201 and ONC206 were pre-solubilized at 40 mM in DMSO. ONC212, ONC207 and ONC213 were solubilized at 20 mg/ml in DMSO and the linear isomer of ONC201 (TIC-10) was solubilized at 10 mg/ml in DMSO. Methicillin and/or vancomycin were evaluated in parallel as antibiotics (positive control) and were purchased from Sigma-Aldrich and solubilized in deionized _H2O at a concentration of 10 mg/ml.

Bacteria.

Bacterial strains used in these assays were obtained from the American Type Culture Collection (ATCC). All bacterial strains were propagated in accordance with the recommendations

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ATSS. Each strain was stored frozen in glycerol at −80°C and 10 μl of frozen stock was used to inoculate each culture for these assays. The strains with their classification and properties are given in Table. 8 below.

Table 8

Staphylococcus aureus strains and their characteristics

ATCC No. Classification Properties Environments for quantification 29213 Grample flax cocci QC wild type strain Trypticase-soy broth (TSB) 33591 Nosocomial methicillin-resistant Nutritious broth 700699 Nosocomial, multiple drug resistance (MDR), reduced vancomycin susceptibility Brain heart broth extract + 0.004 g/l vancomycin

Determination of minimum inhibitory concentration (MIC).

The susceptibility of bacterial organisms to the test compounds was assessed by determining the MIC of each compound using a liquid dilution method in accordance with methods recommended by the Clinical and Laboratory Standards Institute CLSI. All microbial strains were obtained from American Type Culture Collections (ATCC) and cultured according to the supplier's recommendations. The assessment of the susceptibility of each organism to the test compounds included antibiotic/antibiotics (positive control). For each organism, a standardized inoculum was prepared by direct suspension of freshly plated colonies in the appropriate media as indicated in Table 1. 8, up to an optical density of 625 nm (OD ₆₂₅ ) 0.1 (equivalent to McFarland standard 0.5). The suspended inoculum was diluted to a concentration of approximately 1 x 10 ⁶ colony forming units per milliliter (CFU/ml) and 100 μl was placed in triplicate into wells of a 96-well plate containing 100 μl of test compound serially diluted 2-fold in the appropriate broth. 100 μL of inoculum was also added in triplicate to wells containing 100 μL of two-fold serial dilutions of the antibiotic (positive control) and to wells containing 100 μL of medium alone. This dilution scheme yielded final concentrations for each microbial organism of 5 x 10 ⁵ CFU/ml estimated. The concentration of the test compound was varied from 100 (stringent test) to 0.2 μM (lax test) using a two-fold dilution scheme. The plates were incubated for 24 or 48 hours (Staphylococcus aureus 700699) at 37°C, microbiological growth at each compound concentration was determined by measuring absorbance at 625 nm on a Molecular Devices SpectraMax Plus-384 plate and visually by assigning +/- ratings to the plate. regarding bacterial growth. The MIC for each compound was determined as the lowest dilution of the compound that completely inhibited microbial growth.

Results.

Six (6) imipridones were evaluated for their ability to inhibit the growth of three strains of Staphylococcus aureus. ONC201, ONC207, and the linear isomer of ONC201 (TIC-10) are inactive against all three strains up to a concentration of 100 μg/ml. Relative to the wild-type Staphylococcus aureus strain (ATCC 29213), the MICs of ONC206, ONC212, and ONC213 were 6.25 μg/ml, 3.13 μg/ml, and 25 μg/ml, respectively. Relative to Staphylococcus aureus strain (ATCC 33591), the MICs of ONC206, ONC212, and ONC213 were 12.5 μg/ml, 3.13 μg/ml, and 3.13 μg/ml, respectively. Against multidrug-resistant Staphylococcus aureus (ATCC 700699), activity was similar for all three compounds, with an MIC of 12.5 μg/ml. Vancomycin, a positive control compound, was active at the expected concentration, methicillin was inactive up to a concentration of 100 μg/ml against two methicillin-resistant strains of bacteria. The data is presented in table. 9.

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Table 9

MIC determinations for 6 imipridones for three strains of staphylococcus aureus Staphylococcus aureus compounds

Staphylococcus aureus Staphylococcus aureus e ATCC 700699 (48

ATCC 29213 ATCC 33591 (µg/ml) hours)

Visa Visa Visa

MICgg MICgg MICgg ELN MICgg MICgg MICgg ELN MICg ₀ MIC95 MICgg ILYA

ONC201 O ABOUT ABOUT >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 ONC206 6.25 6.25 6.25 6.25 12.5 25 >100 12.5 12.5 12.5 25 12.5 ONC207 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 ONC212 3, 13 3.13 3, 13 3.12 5 3.13 6.25 100 3.12 5 6.25 12.5 12.5 12.5 ONC213 12.5 12.5 25 25 3.13 6.25 100 3.12 5 6.25 12.5 12.5 12.5 TIC-10 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 Methicillin — — — — >100 >100 >100 >100 >100 >100 >100 >100 Vankomitsi n 3, 13 3.13 6.25 3.12 5 0.39 0.39 0.78 0.39 1 12.5 25 25 25

Discussion.

Six (6) imipridones were evaluated for activity against 3 strains of Staphylococcus aureus.

ONC201, ONC207, and TIC-10 are inactive against all three strains. ONC206, ONC212, and ONC213 had different activities, ranging from 3.13 μg/ml to 25 μg/ml against all three bacterial strains. With respect to vancomycin, the activity of these three imipridones was comparable or 2-8 times less against strain 29213. All three of these imipridones showed 10-30 times less activity compared to vancomycin against strain 33591, and the activity for all three compounds was 2 times higher than the activity of vancomycin against strain 700699.

These experiments were repeated with additional impyridones and additional bacteria, including both gram-positive and gram-negative bacteria, such as those shown in table. 10.

Table 10

Gram+/Organism Status gram Nosocomial bacterium.

Enterococcus faecium infection in wounds, endocarditis, + genitourinary tract infections

Staphylocossus

Bacteremia, endocarditis + aureus

Pneumonia, infections

Klebsiella pneumonia genitourinary tract, upper respiratory tract infections

Infections in intensive care patients and burn patients, also Acinetobacter observed in multidisciplinary baumannii, hospitals and nursing homes

Pseudomonas

Pneumonia, cystic fibrosis aeruginosa

Urinary tract infections, Enterobacter cloacae respiratory tract infections

Example 16: Case study of ONC201 treatment of a subject with recurrent glioblastoma.

This example describes the specific case of a 22-year-old woman with recurrent glioblastoma (unmethylated MGMT, H3.3 K27M mutant) treated with 625 mg ONC201 once every three weeks. In fig. 28 (A) shows tumor size relative to baseline (%) of the subject's total tumor burden. One cycle was 3 weeks. (B) Contrast-enhanced MRI scan: baseline, 21, 27, and 36 weeks after ONC201 initiation of one of the 2 malignant lesions in

-

Claims (8)

subject with 625 mg ONC201 every three weeks. One skilled in the art will appreciate that changes may be made to the exemplary embodiments presented and described above without deviating from the general concept of the present invention. Thus, it is to be understood that the present invention is not limited to the exemplary embodiments shown and described, but is intended to cover modifications within the scope of the present invention as defined by the claims. For example, specific features of the exemplary embodiments may or may not be part of the claimed invention, and features of the described embodiments may be combined. Unless specifically stated herein, the indefinite and definite singular form is not limited to one element, but should instead be read to mean at least one. It should be understood that the drawings and descriptions of the present invention may be simplified in order to focus attention on elements necessary for a clear understanding, while omitting, for purposes of clarity, other elements which, in the judgment of those skilled in the art, may also form part of of the present invention. However, because such elements are well known in the art and because they do not necessarily contribute to a better understanding of the invention, a description of such elements is not provided herein. Moreover, to the extent that the method is independent of the specific order of the steps described herein, the specific order of the steps should not be construed as limiting the scope of the claims. The claims relating to the method of the present invention are not limited to performing its steps in the order described, and one skilled in the art will readily understand that said steps may be varied and still remain within the scope of the present invention. All references, including publications, patent applications and patents set forth herein, are incorporated herein by reference to the same extent as if each reference had been individually and specifically incorporated by reference and fully incorporated herein. CLAIM 1. A method of treating or preventing cancer of the central nervous system in a subject in need thereof, comprising administering to the subject in need of such treatment a therapeutically effective amount of a compound of formula (1) or a pharmaceutically acceptable salt thereof, wherein the central nervous system cancer has a histone H3 mutation. 2. The method according to claim 1, characterized in that the cancer is a glioma. 3. The method according to claim 1, characterized in that said cancer is selected from the group consisting of meningioma, ependymoma, neuroblastoma or diffuse brainstem glioma. 4. The method according to claim 1, characterized in that said cancer is a brain tumor. 5. Method according to any one of claims 1 to 4, characterized in that said histone H3 mutation is the H3.3 K27M mutation. 6. The method according to any one of claims 1 to 5, characterized in that said cancer has an epigenetically silenced unmethylated O(6)-methylguanine-DNA methyltransferase (MGMT) gene. 7. Method according to any one of claims 1 to 6, characterized in that the subject is a human. 8. Method according to any one of claims 1 to 7, characterized in that the pharmaceutically acceptable salt is a dihydrochloride salt. -