Pharmaceutical compositions and uses thereof for the treatment of gliomas

Abstract

Imipyridones selectively modulate class A G protein-coupled receptors (GPCRs), such as the D2-like subfamily of dopamine receptors, and are useful for treating conditions and disorders requiring such modulation, such as cancer. Specifically, the cancer involves brain midline structures, cancers with histone H3 mutations, or both. In addition, methods are provided for identifying whether subjects with these conditions are likely to respond to a treatment regimen, such as imipyridone administration. Furthermore, methods are provided for assessing the effectiveness of a treatment regimen, such as administering imipyridone, monitoring, or providing a prognosis, for subjects with these conditions.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 63 / 370,520, filed August 5, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] ONC201 (7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one) is the original member of a class of anticancer compounds called imipridones, currently in Phase II clinical trials in multiple advanced cancers. Since its discovery as a p53-independent inducer of TRAIL gene transcription, preclinical studies have shown that it has antiproliferative and proapoptotic effects on a wide range of tumor cells, but not normal cells. Its mechanism of action involves PERK-independent activation of the integrated stress response, leading to tumor upregulation of DR5 and dual Akt / ERK inactivation, resulting in Foxo3a activation and upregulation of the death ligand TRAIL. ONC201 is orally active with infrequent dosing in animal models, produces sustained pharmacodynamic effects, and is not genotoxic. First-in-human ONC201 clinical trials in advanced, aggressive, refractory solid tumors confirmed that it was well tolerated.

[0003] The discovery of H3 K27M (a specific mutation in histone H3 protein) as an oncogenic mutation has been found in association with cancers involving midline brain structures, namely the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. Due to the location of these tumors, brain regions involved in important physiological functions, they have historically been inoperable (especially in the brainstem, where the pons is located). This meant that, until recently, midline gliomas, such as diffuse intrinsic pontine glioma (DIPG), were diagnosed solely on the basis of radiography. Advances in neurosurgical techniques and increased parental consent for postmortem tumor tissue collection have led to the availability of sufficient biopsies to enable systematic genomic evaluation of DIPG and other midline gliomas. Gliomas in the midline brain are among the most aggressive types of primary malignant brain cancer. This disease arises from glial cells, which form tissue that surrounds and protects other nerve cells found within the brain and spinal cord.

[0004] Standard treatment for midline gliomas involves neurosurgery, when possible, followed by fractionated external beam radiation therapy. Due to their location in the brain, invasiveness, and poor survival time, gliomas in the midline of the brain are considered some of the most deadly forms of cancer.

[0005] No medical therapy has been shown to extend survival in adult and pediatric patients with H3 K27M-mutant glioma. Standard-of-care DIPG treatment, 55 Gy of local radiation fractionated over 6 weeks, is associated with an overall survival of 9 to 11 months. Adult H3 K27M gliomas are often treated with the same regimen as glioblastoma, which involves radiation with concomitant and maintenance temozolomide. Despite its use to treat this newly defined disease in adults, the efficacy of this regimen has not been thoroughly evaluated in patients with adult mutant H3 K27M gliomas.

[0006] The function of histones is primarily protein-DNA and protein-protein interactions; they do not function as enzymes, which represents the majority of targeted cancer therapies (kinases, HDAC inhibitors, etc.). Therefore, there are no therapies that directly target the mutant H3 protein itself (such as in the case of mutant BRAF). Instead, therapeutic efforts have focused on targeting characteristics of tumor cells harboring the H3 K27M mutation, such as their epigenetic and transcriptional dependencies. Inhibition of proteins involved in epigenetics, such as histone deacetylases, histone demethylases, or bromodomains, has shown efficacy in preclinical models, but their ability to improve clinical outcomes has not been demonstrated.

[0007] A major challenge for effective H3 K27M glioma therapy is the need to penetrate the blood-brain barrier, a rare feature of current cancer therapies. This is further enhanced by the location of these tumors in midline brain structures, which have proven more difficult to penetrate than other brain locations. H3 K27M mutations also tend to occur in midline gliomas, where dopamine is present and DRD2 expression is widespread in the tumor environment.

[0008] The lack of treatment for H3 K27M gliomas leaves a large unmet medical need in terms of disease control, symptom relief, and survival. Patients with recurrent disease after radiation are left without treatment options with a demonstrated survival benefit. Summary of the Invention [Means for solving the problem]

[0009] In one aspect, provided herein is a compound of formula (10):

[0010] [ka]

[0011] wherein R and R are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, and acyl radicals. In one embodiment, when R is CHPh, R is not CH-(2-CH-Ph). In one embodiment, R is CHPh and R is CH-(2-CH-Ph) (ONC). In one embodiment, R is CHPh and R is CH-(2,4-diF-Ph) (ONC). In one embodiment, R is CHPh and R is CH-(4-CF-Ph) (ONC). In one embodiment, R1 is CH2Ph and R2 is CH2-(3,4-diF-Ph) (ONC213). In one embodiment, R1 is CH2-(3,4-di-Cl-Ph) and R2 is CH2-(4-CF3-Ph) (ONC234). In one embodiment, R1 is CH2-3-thienyl and R2 is CH2-(4-CF3-Ph) (ONC236).

[0012] In another aspect, provided herein is a method for treating or preventing cancer in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of compound (1) represented by the following formula:

[0013] [ka]

[0014] In another aspect, provided herein is a method for treating or preventing cancer in a subject in need thereof, comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (10) or an analog thereof, or a pharmaceutically acceptable salt thereof, wherein the cancer has a histone H3 mutation.

[0015] In another aspect, provided herein is a method for treating or preventing cancer in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (10) or an analog thereof, or a pharmaceutically acceptable salt thereof, wherein the cancer involves midline structures of the brain.

[0016] One embodiment of the present disclosure includes a method of treating or preventing cancer in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition comprising compound (1) represented by the following formula, or a pharmaceutically acceptable salt thereof, according to a weekly regimen cycle of oral administration comprising two doses per week on two consecutive days, on days 1 and 2, followed by a rest period on days 3 through 7:

[0017] [ka]

[0018] In one embodiment, the weekly regimen cycle is repeated. In one embodiment, the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg. In one embodiment, the dose is 625 mg. In one embodiment, the selected dose is based on a mg / kg calculation based on the subject's weight (kg). In one embodiment, one or more subsequent doses decrease from the initial dose. In one embodiment, each dose is administered to the subject on an empty stomach without eating within two hours before or after each dose. In one embodiment, the weekly regimen is modified after one or more cycles to include a weekly dose on day 1, followed by rest periods on days 2 through 7. In one embodiment, the method further includes radiation treatment prior to the start of the weekly regimen cycle. In one embodiment, the radiation is 54-60 Gy at 1.8-2.2 Gy / dose. In one embodiment, the cancer involves midline structures of the brain. In one embodiment, the cancer has a histone H3 K27M mutation. In one embodiment, the cancer is a central nervous system tumor, brain tumor, glioma, peripheral nervous system tumor, pheochromocytoma, paraganglioma, adrenocortical carcinoma, adrenal tumor, or neuroendocrine tumor. In one embodiment, the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. In one embodiment, the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M. In one embodiment, DRD2 is overexpressed in the tissue, DRD5 is underexpressed in the tissue, or both. In one embodiment, the subject is a human. In one embodiment, the subject is a pediatric subject, and the dose is calculated based on the weight of the pediatric subject. In one embodiment, the dosage is increased in 125 mg increments. In one embodiment, administration begins 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after completion of radiation. In one embodiment, the administration comprises one or more doses of temozolomide. In one embodiment, the method further comprises administering one or more doses of bevacizumab. In one embodiment, the method further comprises one or more treatments of radiation after initiation of administration.

[0019] One embodiment of the present disclosure includes compounds for use in the preparation of a medicament or for use in the treatment of a medicament as outlined herein.

[0020] In one embodiment, the disclosure comprises compound (1) represented by the following formula, or a pharmaceutically acceptable salt thereof, for use in treating or preventing cancer in a subject in need thereof, in an amount according to a weekly regimen cycle of oral administration comprising twice-weekly doses on two consecutive days, on days 1 and 2, followed by a rest period on days 3 through 7:

[0021] [ka]

[0022] In one embodiment, the weekly regimen cycle is repeated. In one embodiment, the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg. In one embodiment, the dose is 625 mg. In one embodiment, the selected dose is based on a mg / kg calculation based on the subject's weight (kg). In one embodiment, one or more subsequent doses decrease from the initial dose. In one embodiment, each dose is administered to the subject on an empty stomach, with no food consumed within two hours before or after each dose. In one embodiment, the weekly regimen is modified after one or more cycles to include a weekly dose on day 1, followed by a rest period on days 2 through 7. In one embodiment, the use or compound for use is provided with radiation treatment prior to the start of the weekly regimen cycle. In one embodiment, the radiation is 54-60 Gy at 1.8-2.2 Gy / irradiation. In one embodiment, the cancer involves midline structures of the brain. In one embodiment, the cancer has a histone H3 K27M mutation. In one embodiment, the cancer is a central nervous system tumor, brain tumor, glioma, peripheral nervous system tumor, pheochromocytoma, paraganglioma, adrenocortical carcinoma, adrenal tumor, or neuroendocrine tumor. In one embodiment, the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. In one embodiment, the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M. In one embodiment, DRD2 is overexpressed in the tissue, DRD5 is underexpressed in the tissue, or both. In one embodiment, the subject is a human. In one embodiment, the subject is a pediatric subject, and the dose is calculated based on the weight of the pediatric subject. In one embodiment, the dosage is increased in 125 mg increments. In one embodiment, administration begins 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after completion of radiation. In one embodiment, the use or compound for use further comprises administration of one or more doses of temozolomide. In one embodiment, the use or compound for use further comprises administration of one or more doses of bevacizumab. In one embodiment, the use or compound for use further comprises one or more treatments of radiation after initiation of administration.

[0023] One embodiment of the present disclosure includes a pharmaceutical composition comprising compound (1) represented by the following formula, or a pharmaceutically acceptable salt thereof, the composition comprising a dose for twice-weekly administration on two consecutive days, days 1 and 2, followed by a rest period on days 3 through 7:

[0024] [ka]

[0025] In one embodiment, the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg. In one embodiment, the dose is 625 mg. In one embodiment, the selected dose is based on a mg / kg calculation based on the subject weight (kg). In one embodiment, the subject is a pediatric subject, and the dose is calculated based on the weight of the pediatric subject. In one embodiment, the dosage is increased in 125 mg increments.

[0026] One or more embodiments or aspects may be incorporated into different embodiments or aspects, even if not specifically described, i.e., all embodiments and aspects may be combined in any manner or combination.

[0027] The foregoing summary, as well as the following detailed description of embodiments of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. The drawings are as follows: [Brief explanation of the drawings]

[0028] [Figure 1] Antagonism of dopamine receptors (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5) by ONC201. [Figure 2] Tumor type sensitivity of the Genomic Program for Drug Sensitivity in Cancer (GDSC) cell line collection. Mean sensitivity was determined by the mean estimated IC50 value from cell viability assays performed 72 hours after treatment. Numbers above the bars indicate the number of cell lines per tumor type. [Figure 3] GBM cell lines with higher DRD2 or lower DRD5 expression are more responsive to ONC201. (A) Inhibition of NCI60 GBM cell lines as a function of ONC201 concentration. (B) Log ONC201 GI50(M) vs. DRD2 expression for each GBM cell line. R2=0.8707. (C) Low DRD5 expression significantly correlates with improved ONC201 efficacy in the NCI60 panel of cancer cell lines. [Figure 4] ONC206 and ONC212 demonstrated anti-cancer efficacy across a variety of tumor types in the NCI60 cancer cell line panel. ONC203 is an inactive negative control. [Figure 5] Bone cancer is more responsive to ONC206 than to ONC201. [Figure 6] Ewing's sarcoma is the most ONC206-responsive bone cancer subtype. [Figure 7] The anticancer efficacy of ONC206 was in the nanomolar range in 14 of 16 Ewing's sarcoma cell lines, demonstrating superior efficacy compared to ONC201 in all cell lines. [Figure 8] Leukemia is more responsive to ONC212 than to ONC201. [Figure 9] ONC212 exhibits anti-cancer efficacy in the nanomolar range (and superior efficacy compared to ONC201) in 55 leukemia cell lines, regardless of subtype. [Figure 10] Anticancer efficacy of ONC212 in acute myeloid leukemia (AML) cell lines. (A) Comparison of cell viability of MV411 AML cells treated with ONC212 or cytarabine. (B) Comparison of cell viability of MOLM14, MV411 AML cells, MRC5 lung fibroblasts, and Hs27a bone marrow cells treated with ONC212. (C) Viability of MOLM14 and MV411 AML cells treated with ONC212 (250 nM) for 4, 8, 24, 48, 72, and 96 hours. [Figure 11]Efficacy of ONC212 in an ONC201-resistant AML xenograft model (MV411 AML cells (5x10) implanted subcutaneously into the flanks 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. * denotes p<0.05 vs. vehicle. [Figure 12] Efficacy of ONC206 in a Ewing sarcoma xenograft model (MHH-ES-1 Ewing sarcoma cells (5x10) implanted subcutaneously in the flanks of athymic nude mice). ONC206 (PO) and methotrexate (IV) were administered on days 1 and 13 as indicated. Tumor volume (A) and body weight (B) (n=4) were measured on the indicated days. [Figure 13] ONC213 has similar in vitro anticancer potency as ONC212 in HCT116 / RPMI8226 cancer cells, but its in vitro toxicity against normal cells is reduced compared to ONC212. [Figure 14] Immune induction correlates with tumor regression in glioblastoma. [Figure 15] A 74-year-old woman with recurrent H3 K27M glioblastoma. MRI at 8 weeks into first treatment shows complete resolution of tumor lesions. [Figure 16] A 10-year-old girl with H3 K27M diffuse intrinsic pontine glioma has improvement in facial paralysis and lesion shrinkage after 16 doses. [Figure 17] A 3-year-old girl with an H3 K27M diffuse intrinsic pontine glioma. MRI at 6 weeks into first treatment shows stable tumor lesions. [Figure 18] Progression-free survival in patients with recurrent high-grade gliomas present at baseline by MRI before starting ONC201 therapy. The cohort is divided into two groups: one with known H3 K27M mutation (red curve) and the other with wild-type or unknown H3 status (blue curve). [Figure 19]Waterfall plot for adult patients with recurrent H3 K27M mutant glioma treated with ONC201. Change in tumor size calculated as the change during best treatment in the sum of the product of the perpendicular diameters of all measurable enhancing lesions compared to baseline. Progressive disease by PD-RANO, stable disease by SD-RANO, partial response by PR-RANO, not evaluable for response (multifocal lesions <1 cm) by NE-RANO. [Figure 20] (A) Gadolinium-enhanced MRI of two lesions at baseline and 72 weeks after starting ONC201 in a patient with recurrent H3 K27M-mutant midline thalamic glioma receiving ONC201 at 625 mg every 3 weeks. (B) Overall tumor size relative to baseline is shown for the duration of ONC201 administration. [Figure 21] Gadolinium-enhanced MRI at baseline and 8 weeks after starting ONC201 (625 mg once weekly) in a patient with recurrent H3 K27M mutant glioma. [Figure 22] Gadolinium-enhanced MRI at baseline and 15 weeks after starting ONC201 (625 mg once weekly) in a patient with recurrent H3 K27M mutant glioma. [Figure 23] 1 is a graphical representation of the results of testing the K27M mutant compared to wild type with compounds of the present disclosure. [Figure 24] 1 shows the results of an ONC201 washout experiment in U110 glioblastoma cells. DETAILED DESCRIPTION OF THE INVENTION

[0029] Scientific and technical terms used herein are intended to have the meanings commonly understood by one of ordinary skill in the art. Such terms may be used, for example, as described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; rdEd.,2001, FMAsubel,Ed.,Short Protocols in Molecular Biology,Current Protocols;5th Ed.,2002, B.Alberts et al.,Molecular Biology of the Cell,4 th Ed., Garland, 2002, Nelson and Cox, Lehninger Principles of Biochemistry, 4 th Ed., WHFreeman&Company, 2004, Engelke, DR, 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, et al. al.,Manipulating the Mouse Embryo:A Laboratory Manual,3 rd edition, Cold Spring Harbor Laboratory Press; 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 U.S. Patent No. 8,673,923. The contents of each of the above references are incorporated herein by reference in their entirety.

[0030] The term "substituted" means that one or more hydrogens on the specified atom are replaced with a selection from the specified group, provided that the replacement does not exceed the normal valence of the specified atom and that the replacement results in a stable compound. When the substituent is keto (i.e., =0), two hydrogens on the atom are replaced. Keto substituents do not occur on aromatic moieties. Ring double bonds are double bonds formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N).

[0031] Variables (e.g., R 4 When R occurs more than one time, its definition on each occurrence is independent of its definition at every other occurrence. Thus, for example, R 4 When indicated to be substituted with a moiety, the group may be substituted with up to three R 4 and R in each occurrence may be optionally replaced by a moiety. 4 is R 4 Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0032] Subscripted numerical ranges for atoms or chemical moieties (e.g., C 1~6 ), this will be understood to include each number within the range, and all intermediate ranges. For example, "C 1~6 "Alkyl" is meant to include alkyl groups having 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 carbons.

[0033] "Alkyl" includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, C 1~6Alkyl includes C1, C2, C3, C4, C5, and C6 alkyl groups. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, s-pentyl, neopentyl, and n-hexyl. In certain cases, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain). In other cases, a straight chain or branched chain alkyl has 4 or fewer carbon atoms. Likewise, cycloalkyls can have from 3 to 8 carbon atoms in their ring structure. In some cases, cycloalkyls have 5 or 6 carbons in the ring structure. Most preferred are C 1~6 Alkyl, in particular ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl, and cyclopropylmethyl.

[0034] The term "substituted alkyl" refers to alkyl substituted with one, two, or three substituents selected from halogen, -OH, alkoxy, -NH, -N(CH), -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C-C)alkyl, -C(=O)NH, -SONH, -C(=NH)NH, and -NO, preferably alkyl containing one or two substituents selected from halogen, -OH, alkoxy, -NH, trifluoromethyl, -N(CH), and -C(=O)OH, more preferably halogen, alkoxy, and -OH. Examples of substituted alkyl include 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

[0035] Unless the number of carbons is otherwise specified, a "lower alkyl" is an alkyl group having 1 to 6 carbon atoms in its backbone, preferably 1 to 4. "Lower alkenyl" and "lower alkynyl" have chain lengths of 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.

[0036] "Alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double bond. 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 certain cases, a straight-chain or branched-chain alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups can have from 3-8 carbon atoms in their ring structure, and in some cases, cycloalkenyl groups have 5-6 carbons in the ring structure. The terms "C2-C6" and "C3-C6" include alkenyl groups containing 2-6 carbon atoms and 3-6 carbon atoms, respectively.

[0037] "Alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), branched-chain alkynyl groups, and cycloalkyl- or cycloalkenyl-substituted alkynyl groups. In certain cases, a straight-chain or branched-chain alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The terms "C2-C6" and "C3-C6" include alkynyl groups containing 2 to 6 carbon atoms and 3 to 6 carbon atoms, respectively.

[0038] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some cases, the cycloalkyl group is saturated or partially unsaturated. In other cases, the cycloalkyl group is fused to an aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. Examples of cycloalkyl groups include, but are not limited to, the following moieties:

[0039] [ka]

[0040] Monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic 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, both of which refer to a non-aromatic carbocycle, as defined herein, having at least one carbon-carbon double bond or one carbon-carbon triple bond.

[0041] The term "cycloalkylalkyl" refers to an alkyl group substituted with a cycloalkyl group. Examples of cycloalkylalkyl groups include cyclopropylalkyl and cyclohexylalkyl.

[0042] The term "heterocycloalkyl" refers to a non-aromatic heterocycle in which one or more of the ring-forming atoms is a heteroatom, such as an O, N, or S atom. Heterocycloalkyl groups include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) ring systems and spirocycles. Examples of heterocycloalkyl groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, and imidazolidinyl. The definition of heterocycloalkyl can also include moieties having one or more aromatic rings fused (i.e., having a common bond) to a non-aromatic heterocyclic ring, such as quinolyl, isoquinolyl, and benzo derivatives of heterocycles. Heterocycloalkyl groups having one or more fused aromatic rings are bonded through either an aromatic or non-aromatic moiety. The definition of heterocycloalkyl also includes moieties in which one or more ring-forming atoms can be replaced by one or two oxo or sulfido groups. In some cases, heterocycloalkyl groups have 1 to about 20 carbon atoms, and in further cases, about 3 to about 20 carbon atoms. In some cases, heterocycloalkyl groups contain 3 to about 20, 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some cases, heterocycloalkyl groups have 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. In some cases, heterocycloalkyl groups contain zero to three double bonds. In some cases, heterocycloalkyl groups contain zero to two triple bonds.

[0043] The term "heterocycloalkylalkyl" refers to an alkyl group substituted by a heterocycloalkyl. Examples of heterocycloalkylalkyl include morpholinoalkyl and piperazinylalkyl.

[0044] The term "aryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, etc. In some cases, aryl groups have from 6 to about 20 carbon atoms.

[0045] The term "arylalkyl" refers to an alkyl group substituted with an aryl group. Examples of arylalkyl groups include benzyl and phenylethyl.

[0046] The term "heteroaryl" refers to an aromatic heterocycle having at least one heteroatom ring member, such as an O, S, or N atom. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3, or 4 fused rings) systems. Ring-forming N atoms in heteroaryl groups can also be oxidized to form N-oxo moieties. 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, heteroaryl groups have 1 to about 20 carbon atoms, and in some cases, about 3 to 20 carbon atoms. In some cases, heteroaryl groups contain 3 to about 14, 3 to about 7, or 5 or 6 ring-forming atoms. In some cases, heteroaryl groups have 1 to about 4, 1 to about 3, or 1 or 2 heteroatoms.

[0047] A "heteroarylalkyl" group refers to an alkyl group substituted with a heteroaryl group. An example of a heteroarylalkyl group is pyridylmethyl.

[0048] The term "halo" or "halogen" refers 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 hydrogens are replaced by halogens. The term "haloalkyl" refers to an alkyl group having halogens replacing hydrogens on one or more carbons of the hydrocarbon backbone. C1-C6 haloalkyl includes straight or branched alkyls having up to six skeletal carbon atoms, where halogens replace hydrogens on one or more skeletal carbons.

[0049] The terms "alkoxy" or "alkoxyl" include substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently bonded to an oxygen atom. C1-C6 alkoxy refers to a moiety having 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. (C1-C3)alkoxy, particularly ethoxy and methoxy, are preferred. Examples of substituted alkoxy groups include halogenated alkoxy groups.

[0050] The term "hydroxy" or "hydroxyl" refers to -OH or -O - The group includes a group having the formula:

[0051] The term "pharmaceutically acceptable salt" refers to a derivative of a compound modified by converting an existing acid or base moiety into its salt form. Non-limiting examples of pharmaceutically acceptable salts include mineral or organic acid salts of basic residues such as amines, and alkali 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. Pharmaceutically acceptable salts can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, Journal of Pharmaceutical Science, 66, 2 (1977), and P. Stahl and C. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2. nd Revised ed., Weinheim / Zuerich: Wiley-VCH / VHCA (2011), each of which is incorporated herein by reference in its entirety.

[0052] Examples of suitable inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid, while examples of suitable organic acids include carboxylic, sulfo, or sulfonic acids, 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 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.

[0053] The term "antibody" encompasses the structure that constitutes the natural biological form of an antibody. In most mammals, including humans and mice, this form is a tetramer, consisting of two identical pairs of immunoglobulin chains, each pair having one light chain and one heavy chain, and each light chain containing immunoglobulin domain V. L and C L Each heavy chain comprises an immunoglobulin domain V H , Cγ1, Cγ2, and Cγ3. Each pair contains a light chain variable region and a heavy chain variable region (V L and V H ) are together responsible for binding to antigens, and the constant region (C L , Cγ1, Cγ2, and Cγ3, especially Cγ2 and Cγ3, are responsible for antibody effector functions. In some mammals, such as camels and llamas, full-length antibodies can consist of only two heavy chains, each containing immunoglobulin domain V. H, Cγ2, and Cγ3. 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 include full-length antibodies, antibody fragments, and V H , Cγ1, Cγ2, Cγ3, V L , and C L The immunoglobulin domains may have several structural forms, including individual immunoglobulin domains comprising:

[0054] Based on the heavy chain constant domain amino acid sequence, intact antibodies can be assigned to different "classes." There are five major classes (isotypes) of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses," e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different antibody classes are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0055] The terms "antibody" or "antigen-binding fragment" refer to intact molecules and functional fragments thereof, such as Fab, scFv-Fc bivalent molecules, F(ab')2, and Fv, respectively, that are capable of specifically interacting with a desired target. In some cases, antigen-binding fragments include: (1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain, whereby two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, wherein F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single chain antibodies ("SCAs"), genetically engineered molecules containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule; and (6) scFv-Fc, produced by fusing a single-chain Fv (scFv) with an immunoglobulin (Ig), such as IgG, and the hinge region from the Fc region.

[0056] In one embodiment, the antibodies provided herein are monoclonal antibodies. In one embodiment, the antigen-binding fragments provided herein are single-chain Fvs (scFvs), diabodies, tandem scFvs, scFv-Fc bivalent molecules, Fabs, Fab's, Fvs, F(ab')2, or antigen-binding scaffolds (e.g., affibodies, monobodies, anticalins, DARPins, Knottins).

[0057] The terms "bind," "binding," or grammatical equivalents refer, directly or indirectly, to compositions that have affinity for one another. "Specific binding" refers to the selective binding between two molecules. For example, specific binding occurs between an antibody and an antigen. Typically, the dissociation constant (K D ) is about 1 × 10 -5 Less than M or about 1 x 10 -6 M or 1 x 10 -7Specific binding can be distinguished from nonspecific binding when the binding affinity is less than M. Specific binding can be detected, for example, by ELISA, immunoprecipitation, coprecipitation, with or without chemical cross-linking, and two-hybrid assays. The use of appropriate controls can 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 two or more binding sites as the strength of binding of a ligand at one designated binding site. Noncovalent binding of a ligand to an antibody or other molecule is generally not as strong as covalent binding, but "high affinity" ligands can have binding affinity of 10 or more, as determined by inhibition ELISA. 4 M -1 Over, typically 10 5 ~10 11 M -1 The affinity constant (K a ) or by an equivalent technique such as a Scatchard plot, or K a The reciprocal of K d / binds to antibodies or other molecules with comparable affinities as determined using dissociation constants.

[0058] The term "selective" with respect to binding, inhibition, stimulation, or modulation refers to preferential binding, inhibition, stimulation, or modulation, respectively, of a first activity over a second activity (e.g., preferential binding of one receptor over another, preferential inhibition of another receptor, or preferential inhibition of a mutant over a wild-type, or vice versa). In some cases, binding has greater than 2-fold, greater than 5-fold, greater than 10-fold, greater than 50-fold, greater than 100-fold, or greater than 1000-fold selectivity for a desired molecular target or pathway over an undesired molecular target or pathway. In some cases, a compound binds to a first molecular target or affects a pathway at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold more selectively than a second target or pathway under the same conditions. In preferred embodiments, binding to the D2-like family of dopamine receptors or members thereof is understood to be selective for the D1-like family of dopamine receptors or members thereof by one of the aforementioned amounts. The in vitro or in vivo activity of a molecular target or pathway may be measured by any suitable reproducible means.

[0059] The term "modulate" refers to "stimulating" or "inhibiting" 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 the molecular 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 about 90%, at least about 95%, at least about 98%, or about 99% or more relative to the activity under the same conditions but in the absence of the composition. In another example, a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the target or pathway by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold relative to the activity under the same conditions but in the absence of the composition. The activity of a molecular target or pathway can be measured by any reproducible means. For example, the activity of a molecular target or pathway can be measured in vitro or in vivo by a suitable assay known in the art for measuring that activity, and a control sample (untreated with the composition) can be assigned a relative activity value of 100%.

[0060] In one embodiment, the antibody, antigen-binding fragment, or affinity tag binds to its target with a K in the range of 0.1 nM to 10 mM, 0.1 nM to 1 mM, or 0.1 nM. D In one embodiment, it binds to its target with a K of 0.1-2 nM, 0.1-1 nM, 0.05-1 nM, 0.1-0.5 nM, or 0.1-0.2 nM. D In one embodiment, it binds to its target directly, in one embodiment, it binds to its target indirectly, e.g., a secondary antibody binds to the antibody bound to the target.

[0061] The term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent, such as a nucleic acid probe or antibody, to facilitate detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0062] The term "probe" refers to a synthetic or biologically produced nucleic acid containing a specific nucleotide sequence that hybridizes to a target nucleic acid sequence under stringent conditions. 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 prepared with a marker group or "detectable label" for detection. The marker group may be attached at the 5' end, 3' end, internally, or a combination thereof. That is, one probe may be attached to multiple markers. A preferred group is a distinguishing label such as a fluorophore. A labeled probe may also contain multiple different nucleic acid sequences, each labeled with one or more markers. Each marker may be the same or different. It may be advantageous to label different probes (e.g., nucleic acid sequences) with different markers. This can be achieved by having a single distinguishing group on each probe. For example, probe A is attached to group X and probe B is attached to group Y. Alternatively, probe A is attached to groups X and Y, while probe B is attached to groups Z and W. Alternatively, probe A binds to group X and group Y, while probe B binds to group Y and group Z. All of the above probes "A" and "B" will be distinguishable and uniquely labeled.

[0063] A "tissue sample" refers to a collection of similar cells obtained from a subject's or patient's tissue, preferably containing nucleated cells with chromosomal material. The four major human tissues are (1) epithelium, (2) connective tissue, including blood vessels, bone, and cartilage, (3) muscle tissue, and (4) nervous tissue. The source of a tissue sample can be fresh, frozen, and / or preserved organ or tissue samples or biopsies or aspirates; blood or blood components; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or solid tissue, such as from cells from a subject's pregnancy or development. A tissue sample can be primary or cultured cells or cell lines. A tissue sample may contain compounds not naturally mixed with the native tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, or antibiotics. A tissue sample "section" refers to a single portion or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from the tissue sample. Multiple sections of a tissue sample can be taken and subjected to analysis. "Cell line" refers to a permanently established cell culture that will grow if given appropriate fresh medium and space.

[0064] Detection Method In various aspects, provided herein are methods for detecting or measuring a target receptor (e.g., a dopamine receptor or a GPCR) in a biological sample. The target is detected by contacting the sample with a target detection reagent, such as an antibody or fragment thereof, and a labeling reagent. The presence or absence of the target is detected by the presence or absence of the labeling reagent. In some cases, the sample is contacted with the target detection and labeling reagent simultaneously, e.g., the detection reagent is a primary antibody and the labeling reagent is a fluorescent dye conjugated thereto. Alternatively, the biological sample is contacted with the target detection and labeling reagent sequentially, e.g., the detection reagent is a primary antibody and the labeling reagent comprises a secondary antibody. For example, the sample is incubated with the detection reagent, in some cases the 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 the sample is further contacted with a labeled reagent that specifically binds to the detection reagent bound to the target, the sample can optionally be washed one or more times to remove unbound labeled reagent. The presence or absence of the target in the sample is then determined by detecting the labeled reagent.

[0065] Methods described herein are provided for detecting multiple targets in a sample, which are identified by contacting the biological sample with an additional detection reagent, followed by contacting the sample with an additional labeled reagent specific for the additional detection reagent, using the methods described.

[0066] A detection moiety, i.e., a detectable label, is a substance used to facilitate target identification and / or quantification. Detection moieties can be directly observed or measured, or indirectly observed or measured. Non-limiting examples of detection moieties include radioactive labels that can be measured with a radiation counter; pigments, dyes, or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent moieties, where the output signal is generated by excitation of an appropriate molecular adduct and can be visualized by excitation with light absorbed by the dye or measured with a standard fluorometer or imaging system. Detection moieties can be luminescent substances such as fluorescers or fluorogens, bioluminescent substances, chemiluminescent substances where the output signal is generated by chemical modification of the signal compound, metal-containing substances, or enzymes where enzyme-dependent secondary generation of a signal occurs, such as the formation of a colored product from a colorless substrate. Detection moieties can also take the form of chemical or biochemical, or inert particles, including colloidal gold, microparticles, quantum dots, or inorganic crystals such as nanocrystals or fluorophores. The term "detection moiety" or "detectable label" can also refer to a "tag" or hapten that can selectively bind to a labeled molecule, such that the labeled molecule, when subsequently added, is used to generate a detectable signal. For example, biotin, iminobiotin, or desthiobiotin can be used as a tag, and then an avidin or streptavidin conjugate of horseradish peroxidase (HRP) can be used to bind to the tag, followed by detection of the presence of HRP using a chromogenic (e.g., tetramethylbenzidine) or fluorogenic substrate, such as Amplex Red or Amplex Gold (Molecular Probes, Inc.). Similarly, the tag can be a hapten or antigen (e.g., digoxigenin), and can be bound to the tag using an enzymatically, fluorescently, or radioactively labeled antibody. Numerous labels are known to those skilled in the art; non-limiting examples include particles, fluorescent dyes, haptens, enzymes, and their chromogenic, fluorescent, and chemiluminescent substrates.

[0067] Fluorophores are chemical groups that exhibit an absorption maximum above 280 nm and retain their spectral properties when covalently attached to a labeling reagent. Fluorophores include pyrene, anthracene, naphthalene, acridine, stilbene, indole or benzindole, oxazole or benzoxazole, thiazole or benzothiazole, porphyrin, cyanine, perylene, 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), carbocyanine, carbostyryl, salicylate, anthranilate, azulene, pyridine, quinoline, borapolyazaindacene, xanthene, oxazine or benzoxazine, carbazine, phenalenone, coumarin, benzofuran, and benzphenalenone, as well as their derivatives. Oxazines include resorufins, aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

[0068] For xanthene fluorophores, the fluorophore can be fluorescein, rhodol, or rhodamine. Fluoresceins include benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins. Similarly, rhodols include seminaphthorhodafluors. Alternatively, the fluorophore is a xanthene attached to the 9-position of the xanthene via a single covalent bond. Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one, 6-amino-3H-xanthen-3-one, or 6-amino-3H-xanthen-3-imine. Fluorophores include xanthenes (rhodols, rhodamines, fluoresceins, and their derivatives), coumarins, cyanines, pyrenes, oxazines, and borapolyazaindacenes. In addition, the fluorophore can be a sulfonated xanthene, a fluorinated xanthene, a sulfonated coumarin, a fluorinated coumarin, and a sulfonated cyanine. The choice of fluorophore in a labeling reagent determines the absorption and fluorescence emission properties of the labeling reagent. The physical properties of the fluorophore label, including spectral properties (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, can all be used to distinguish one fluorophore from another.

[0069] Typically, a fluorophore contains one or more aromatic or heteroaromatic rings that are optionally substituted with one or more substituents including halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzo, or other substituents typically found in fluorophores known in the art.

[0070] Preferably, the detection moiety is a fluorescent dye. Examples of fluorescent dyes include 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 dye family, N-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)caproyl) (NBD), BODIPY™, dipyrromethene boron difluoride, and Oregon Green, MITOTRACKER™ Red, DiOC7(3), DiIC18, phycoerythrin, phycobiliproteins BPE (240 kDa), RPE (240 kDa), CPC (264 kDa), APC (104 kDa), 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, therefore, are also preferred chromophores.

[0071] In addition to fluorophores, enzymes also find use as detectable moieties. Enzymes are desirable detectable moieties because amplification of the detectable signal can be achieved, resulting in increased assay sensitivity. Enzymes themselves do not produce a detectable response, but rather cleave the substrate upon contact with an appropriate substrate, such that the converted substrate generates a fluorescent, colorimetric, or luminescent signal. Enzymes amplify the detectable signal because a single enzyme on a labeling reagent can result in multiple substrates being converted to detectable signals. This is advantageous when low amounts of target are present in a sample, or when no fluorophore is available that provides a signal comparable to or stronger than that of the enzyme. However, fluorophores are preferred because they do not require additional assay steps, thereby shortening the overall time to complete the assay. Enzyme substrates are selected to produce a desired measurable product, such as colorimetric, fluorescent, or chemiluminescent. Such substrates are widely used in the art.

[0072] A preferred colorimetric or fluorogenic substrate and enzyme combination uses an oxidoreductase, such as horseradish peroxidase, and substrates such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole-e (AEC), which produce distinguishing colors (brown and red, respectively). Other non-limiting examples of colorimetric oxidoreductase substrates that produce detectable products include 2,2-azino-bis(3-ethylbenzothiaz-oline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, and 4-chloro-1-naphthol. Non-limiting examples of fluorogenic substrates include homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplexe Red reagent and its variants, and reduced dihydroxanthenes, including dihydrofluorescein, and dihydrorhodamines, including dihydrorhodamine 123. Tyramide peroxidase substrates represent a class of peroxidase substrates that may be intrinsically detectable prior to the action of the enzyme but are "locked in place" by the action of peroxidase in a process described as tyramide signal amplification (TSA). These substrates are widely utilized to label targets in samples that are cells, tissues, or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning, and fluorometry.

[0073] Additional colorimetric (and in some cases, fluorogenic) substrate and enzyme combinations include a phosphatase enzyme, such as acid phosphatase, alkaline phosphatase, or a recombinant version of such a phosphatase, combined 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 in combination with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP), fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF97, ELF39, or related phosphates.

[0074] Glycosidases, particularly β-galactosidase, β-glucuronidase, and β-glucosidase, are other suitable enzymes. Suitable colorimetric substrates include 5-bromo4-chloro-3-indolyl β-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, such as o-nitrophenyl β-D-galactopyranoside (ONPG) and p-nitrophenyl β-D-galactopyranoside. Preferred substrates include resorufin β-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants, 4-methylumbelliferyl β-D-galactopyranoside, carboxyumbelliferyl β-D-galactopyranoside, and fluorinated coumarin β-D-galactopyranoside. Other enzymes include hydrolases, such as cholinesterases and peptidases, oxidases, such as glucose oxidase, and cytochrome oxidases and reductases for which suitable substrates are known.

[0075] Chemiluminescence-generating enzymes and their substrates are preferred for some assays. These include, for example, natural and recombinant luciferase and aequorin. For luciferase or aequorin, exemplary substrates are luciferin, ATP, Ca ++ and coelenterazine. Chemiluminescent substrates for phosphatases, glycosidases, and oxidases are also useful, such as those containing stable dioxetanes, luminol, isoluminol, and acridinium esters.

[0076] In addition to enzymes, haptens such as biotin are useful detectable moieties. Biotin is present in enzyme systems that can further amplify the detectable signal and can function as a tag in affinity chromatography for isolation purposes. For detection, an enzyme conjugate with affinity for biotin, such as avidin-HRP, is used. A peroxidase substrate is then added to generate a detectable signal. Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, or nucleotides.

[0077] In some cases, the detectable moiety is a fluorescent protein. Examples include green fluorescent protein (GFP), phycobiliproteins and their derivatives, luciferase, or aequorin. Fluorescent proteins, especially phycobiliproteins, are particularly useful for creating tandem dye-labeled labeling reagents. Tandem dyes contain a fluorescent protein and a fluorophore to obtain a larger Stokes shift, which is further shifted from the absorption spectrum of the fluorescent protein. This is particularly advantageous for detecting low-abundance targets in samples where the emitted fluorescence is maximally optimized; in other words, where the fluorescent protein reabsorbs little or no emitted light. The fluorescent protein and fluorophore function as an energy transfer pair, where the fluorescent protein emits at a wavelength absorbed by the fluorophore, and the fluorophore then emits at a wavelength farther from the fluorescent protein than would be possible with the fluorescent protein alone. A particularly useful combination is a phycobiliprotein and a sulforhodamine fluorophore, a sulfonated cyanine fluorophore, or a sulfonated xanthene derivative. Alternatively, the fluorophore is the energy donor and the fluorescent protein is the energy acceptor.

[0078] Label-dependent methods for visualizing detection moieties In some cases, the sample is illuminated with a wavelength of light selected to give a detectable optical response and observed with a means for detecting the response. Devices useful for illuminating fluorescent compounds include handheld ultraviolet lamps, mercury arc lamps, xenon lamps, lasers, and laser diodes. These illumination sources are optically integrated into laser scanners, fluorescence microplate readers, or standards or microfluorometers. The degree or position of the signal compared to a standard or expected response indicates whether and to what extent the sample possesses a given characteristic or desired target.

[0079] The optical response is detected by visual inspection or by using one of the following devices: a CCD camera, a video camera, photographic film, a laser scanning device, a fluorometer, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, a fluorescence microplate reader, or by means for amplifying the signal, such as a photomultiplier tube. When samples are examined using a flow cytometer, the examination optionally includes sorting portions thereof according to their fluorescent response.

[0080] When an indirectly detectable label is used, irradiation typically involves adding a reagent to generate a detectable signal, such as a colorimetric enzyme substrate. Radioisotopes are also considered indirectly detectable when no additional reagents are required, but rather the radioisotope is exposed to X-ray film or other mechanism to record and measure the signal. This is true for some chemiluminescent signals observed after exposure to film.

[0081] I. ONC201 (Compound (1)), its salts and synthesis thereof

[0082] Provided herein are ONC201 (compound (1)) represented by the following formula, its analogs, and pharmaceutically acceptable salts thereof, as well as syntheses thereof.

[0083] [ka]

[0084] In in vitro models, animal models, and human clinical trials, ONC201 has broad anticancer activity, low toxicity with few, if any, adverse effects, low genotoxicity, and high bioavailability, including oral. These characteristics make ONC201 and various analogs well suited for a variety of applications. ONC201 can be synthesized as shown in Scheme 1.

[0085] [ka]

[0086] The synthesis of ONC201 dihydrochloride begins with the commercially available intermediate N-benzyl-3-carbomethoxy-4-piperidone hydrochloride, compound (3). In one embodiment, compound (3) is neutralized with a base (Step 1) to provide compound (4), the free base. For example, compound (3) is neutralized with an inorganic base to provide compound (4). Alternatively, it is neutralized with an organic base to provide compound (4). In one embodiment, compound (3) is neutralized in the presence of an alcohol (e.g., n-butanol). In one embodiment, compound (3) is neutralized in the presence of at least one organic solvent (e.g., n-butanol, ethyl acetate, or both). In one embodiment, it is neutralized in the presence of a base and at least one organic solvent (e.g., NaHCO and n-butanol). In one embodiment, compound (3) is neutralized in the presence of n-butanol and triethylamine (EtN).

[0087] In one embodiment, the synthesis involves reacting compound 4 with 5 (Step 2) to produce intermediate compound 1. In one embodiment, Step 2 involves heating compound 4 with 5. In one embodiment, Step 2 involves heating compound 4 with 5 at reflux in the presence of a solvent. In one embodiment, Step 2 involves using a Dean-Stark trap to remove water and / or methanol (MeOH) formed during the reaction.

[0088] In one embodiment, ONC201 dihydrochloride is synthesized (Step 3). In one embodiment, Step 3 comprises treating ONC201 with HCl in dioxane. In one embodiment, Step 3 comprises treating ONC201 with 4N HCl in dioxane. In one embodiment, the synthesis optionally comprises recrystallizing the ONC201 dihydrochloride. Preferably, ONC201 dihydrochloride is synthesized as shown in Scheme 2.

[0089] [ka]

[0090] II. TNF-Related Apoptosis-Inducing Ligand ("TRAIL")

[0091] TRAIL protein can be assayed in a sample obtained from a subject to detect TRAIL expression induced by the compounds described herein and their salts. Immunoassays can be used, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, enzyme-linked immunofiltration assay (ELIFA), immunoblot, immunoprecipitation, fluorescent immunoassay (FIA), immunohistochemistry, immunocytochemistry, luminescent immunoassay (LIA), and radioimmunoassay. Qualitative and / or quantitative results can be obtained. Suitable methods for qualitative and quantitative assays are described in Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Breitling & 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; 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, MG, 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: morphologic-immunophenotypic correlation, Taylor & Francis, 2006, Crowther, JR, The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000, Wild, D., The Immunoassay Handbook, 3. rd Edition, Elsevier Science, 2005, and Sambrook & Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3 rd ed., 2001.

[0092] An assay for analyzing samples for TRAIL to detect the effect of a pharmaceutical composition is described in US Pat. No. 8,673,923, which is incorporated herein by reference in its entirety.

[0093] In one embodiment, the TRAIL assay is used to monitor a subject. For example, samples are obtained from a subject before treatment with an agent and at one or more times during and / or after treatment to assess the effectiveness of the treatment. In another example, samples are obtained from a subject at various time points to assess the course or progression of a disease or cure. In one embodiment, death receptors from circulating tumor cells are assayed to determine whether the treatments described herein increase the amount or type of death receptors.

[0094] Cancers that may be treated using the methods and compositions described herein are characterized by abnormal cell proliferation, including pre-neoplastic hyperproliferation, carcinoma in situ, tumors, and metastases. The methods and compositions described herein can be used for prevention and amelioration of signs or symptoms of cancer. "Treating" cancer in a subject includes preventing, inhibiting, or ameliorating cancer in a subject, such as slowing the progression of cancer or reducing or ameliorating signs or symptoms of cancer. Examples of cancers that may be treated using the methods and compositions described herein include breast cancer, CNS cancer, colon cancer, ovarian cancer, prostate cancer, leukemia, lung cancer, and lymphoma.

[0095] III. Compounds of formula (10) and salts thereof

[0096] In one aspect, provided herein are compounds and salts of formula 10, and methods for making them. Those skilled in the art will understand that the general principles and concepts described herein in conjunction with ONC201 (compound 1) and its salts, including principles and concepts related to methods and pharmaceutical compositions, apply equally to compounds of formula 10 and their salts.

[0097] In one embodiment, provided herein is a compound of formula (10):

[0098] [ka]

[0099] wherein R and R are independently selected from H, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R is CHPh and R is CH-(2-CH-Ph) (ONC). In one embodiment, R is CHPh and R is CH-(2,4-diF-Ph) (ONC). In one embodiment, R is CHPh and R is CH-(4-CF-Ph) (ONC). In one embodiment, R is CHPh and R is CH-(3,4-diF-Ph) (ONC). In one embodiment, R1 is CH2-(3,4-di-Cl-Ph) and R2 is CH2-(4-CF3-Ph)(ONC234). In one embodiment, R1 is CH2-3-thienyl and R2 is CH2-(4-CF3-Ph)(ONC236).

[0100] In one embodiment, R1 and R2 are H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl 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 independently selected from alkylpyrazinyl, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzyl-piperazine, C 1~4 Alkylthienyl, C 1~4 Alkylpyridinyl, C 1~4 Alkylisoxazolidinyl, C 1~4 Alkylthiazolyl, C 1~4Alkylmorpholinyl, and C 1~4 The alkylpyrazinyl is optionally C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, R and / or R are substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment, heteroarylalkyl is C 1~4 Alkylpyrrolyl, C 1~4 Alkylfuryl, C 1~4 Alkylpyridyl, C 1~4 Alkyl-1,2,4-thiadiazolyl, C 1~4 Alkylthienyl, C 1~4 Alkylisothiazolyl, C 1~4 Alkyl imidazolyl, C 1~4 Alkyltetrazolyl, C 1~4 Alkylpyrazinyl, C 1~4 Alkylpyrimidyl, C 1~4 Alkylquinolyl, C 1~4 Alkylpyrazolyl, C 1~4 Alkylisoquinolyl, C 1~4 Alkylthiophenyl, C 1~4 Alkylbenzothienyl, C 1~4 Alkylisobenzofuryl, C 1~4 Alkyl indolyl, C 1~4 Alkylpurinyl, C 1~4 Alkylcarbazolyl, C 1~4 Alkylbenzimidazolyl, and C 1~4 alkylisoxazolyl.

[0101] 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, -CXH2, -CX2H, C2-C4 alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X2p+1 , OR m , S.R. m , N.R. m R n , N.R. 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, 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.

[0102] In one embodiment, R1 is H, CH3, CH2Ph, CH2-(4-CF3-Ph), CH2-(4-F-Ph), CH2-(4-Cl-Ph), CH2-(OCH3-Ph), CH2-((2-Cl)-Ph), CH2-(2-thienyl), CH2-(3-thienyl), CH2-2-pyridinyl, CH2-4-methyl-2-thiazolyl, CH2-2-pyrazinyl, CH2CH2Ph, CH2CH 2(4-N-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-(3,4-diCl-Ph), CH2-(3,4-diF-Ph), CH2-(3,5-diF-Ph), CH2-((2-CH3)-Ph), CH2CH(OH)Ph, (4-F-Ph)-4-oxobutyl, CH2CH2NHCOOC(CH3)3, CH2CH2CH2NH2, and CD2C6D5. In one embodiment, R2 is H, CH3, CH2Ph, CH2-(4-CF3-Ph), CH2-((2-Cl)-Ph), CH2-((2-F)-Ph), CH2-(2-thienyl), CH2CH2Ph, CH2CH2(4-N-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-(2,4-diCl-Ph), CH2-(3,4-diCl-Ph), CH2-(3,4-diF-Ph), CH2-(3,5-diF-Ph), CH2-((2-CH 3)-Ph), CH2(2-CH3,4-F-Ph), CH2-((4-OCH3)-Ph), CH2-(3-pyridinyl), CH2-(3-isoxazolidinyl), CH2CH2-(4-morpholinyl), CH2-(2-F,4-CF3-Ph), CH2CH(OH)Ph, (CH2)3CO-4F-Ph, (4-F-Ph)-4-oxobutyl, CH2CH2NHCOOC(CH3)3, CH2CH2CH2NH2, and CD2C6D5.

[0103] In one embodiment, R is H. In one embodiment, R is an unsubstituted or substituted arylalkyl, such as a benzyl (CHPh) or phenylethyl (CHCHPh) group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0104] In one embodiment, R2 is substituted or unsubstituted arylalkyl, such as benzyl or phenylethyl. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from halo, CH3, CF3, or OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as piperazinylalkyl or morpholinoalkyl. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as pyridylmethyl or isoxazolidinylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is selected from the group consisting of C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with at least one substituent selected from halo, CH3, CF3, or OCH3.

[0105] In one embodiment, compound (10) has the structure of formula (80):

[0106] [ka]

[0107] In the formula, R a1 , R a2 , R a3 , R a4 , R a5 , R b1 , R b2 , R b3 , R b4 , and R b5are each independently H, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C2-C4 alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. m R n , N.R. m C(O)R n , SOR m , SO2R m , C(O)R m , and C(O)OR m and R m and R n is independently selected from H or C1-C4 alkyl, p is an integer from 2 to 20, and X is halogen.

[0108] In one embodiment, compound (10) has the structure of formula (90), represented by the following formula:

[0109] [ka]

[0110] where R2 is as defined above and R b1 , R b2 , R b3 , R b4 , and R b5 are each independently H, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C 2~4 Alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. mR n , N.R. m C(O)R n , SOR m , SO2R m , C(O)R m , and C(O)OR m and R m and R n is H or C 1~4 alkyl, p is an integer from 2 to 20, and X is halogen.

[0111] In one embodiment, compound (10) has the structure of formula (40):

[0112] [ka]

[0113] wherein R1 is as defined above and R a1 , R a2 , R a3 , R a4 , and R a5 are each independently H, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C 2~4 Alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. m R n , N.R. m C(O)R n , SOR m , SO2R m , C(O)R m , and C(O)OR m Selected from R m and R n is H or C 1~4In one embodiment, R1 is independently selected from alkyl, p is an integer from 2 to 20, and X is halogen. In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, such as benzyl or phenylethyl. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the benzyl is substituted with one or more halo. In one embodiment, the benzyl is substituted with one or more substituents selected from halo, CH, CF, and OCH. In one embodiment, the benzyl is substituted with one halo, e.g., F, at the ortho or para position. In one embodiment, the benzyl is substituted with two halogens, e.g., F, at both meta positions.

[0114] In one embodiment, compound 40 has the structure of compound 45, which is represented by the following formula:

[0115] [ka]

[0116] In the formula, R a1 , R a2 , R a3 , R a4 , and R a5 is as defined above. In one embodiment, benzyl is substituted with one or more halogens. In one embodiment, benzyl is substituted with one or more substituents selected from halo, CH, CF, and OCH. In one embodiment, R a1 or R a5 is halo, e.g., F. In one embodiment, R a2 and R a3 Both are halo, e.g., F.

[0117] In one embodiment, compound (10) has the structure of compound (50), which is represented by the following formula:

[0118] [ka]

[0119] wherein R1 is as defined above and R b is H, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C 2~4 Alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. m R n , N.R. m C(O)R n , SOR m , SO2R m , C(O)R m , and C(O)OR m Selected from R m and R n is H or C 1~4 alkyl, p is an integer from 2 to 20, X is halogen, and R a1 , R a2 , R a4 , and R a5 are each independently H, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C 2~4 Alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. m R n , N.R. m C(O)R n , SOR m , SO2R m , C(O)R m , and C(O)ORm Selected from R m and R n is H or C 1~4 In one embodiment, R1 is independently selected from alkyl, p is an integer from 2 to 20, and X is halogen. In one embodiment, R1 is H. In one embodiment, R1 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, R b is selected from halo, CH, CF, and OCH. In one embodiment, R a1 , R a2 , R a4 , and R a5 is selected from halo, CH, CF, and OCH. a1 , R a2 , R a4 , and R a5 is H and R b is selected from halo, CH, CF, and OCH. In one embodiment, R b is a halogen, e.g., F, and R a1 is CH3. In one embodiment, R b is F or Cl, and R a2 is F or Cl. In one embodiment, R b is CF. In one embodiment, R b is OCH. In one embodiment, R b and R a1 is Cl.

[0120] In one embodiment, compound (50) has the structure of compound (55), which is represented by the following formula:

[0121] [ka]

[0122] In the formula, R a1, R a2 , R a4 , R a5 , and R b is as defined above. In one embodiment, R b is selected from halo, CH, CF, and OCH. In one embodiment, R a1 , R a2 , R a4 , and R a5 is selected from halo, CH, CF, and OCH. a1 , R a2 , R a4 , and R a5 is H and R b is selected from halo, CH, CF, and OCH. In one embodiment, R b is a halo, e.g., F, and R a1 is CH3. In one embodiment, R b is F or Cl, and R a2 is F or Cl. In one embodiment, R b is CF. In one embodiment, R b is OCH. In one embodiment, R b and R a1 is Cl.

[0123] In one embodiment, compound (10) has the structure of compound (60), which is represented by the following formula:

[0124] [ka]

[0125] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, such as benzyl or phenylethyl. In one embodiment, 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-benzyl-piperazine), CH2-(3-isoxazolidinyl), CH2-2-pyridinyl, CH2-3-pyridinyl, and CH2CH2-(4-morpholinyl). In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, benzyl is substituted with one or more halogens. In one embodiment, benzyl is substituted with one or more substituents selected from halo (e.g., F), CH, CF, and OCH. In one embodiment, benzyl is substituted at the para position with a halo, CH, CF, or OCH substituent. In one embodiment, R is fluorophenyloxobutyl or hydroxyphenylethyl.

[0126] Scheme 3 illustrates the synthesis of compounds of formula (10).

[0127] [ka]

[0128] Compounds of formula (10) (imipyridones) are synthesized starting from substituted piperidones and converted by reaction with substituted aminoimidazolines to give the core compounds (10). Two routes exist: one where the R1 substituent is present in the piperidone (e.g., 68). In this route, 68 is acylated with dimethyl carbonate using sodium hydride in toluene at 80 °C to form the piperidone ester (69). Commercially available methylthioimidazoline HI salt (63) is reacted with an amine in dioxane at 70 °C to give the R2-substituted aminoimidazoline (64) as its HI salt. 64 can be directly reacted with piperidone ester (69) in 1-butanol and refluxed for 3–6 h with water removal via a Dean-Stark trap to give the tricyclic compounds (10). In a variation of this scheme, N-BOC-protected piperidone 61 is converted to BOC-protected compound 65 by the same method, treated with HCl in dioxane to remove the BOC group, and then extracted with methylene chloride and converted to the free base of 66 with 1 N NaOH. Subsequent treatment of 66 with halide 67 or epoxide 70 affords the desired compound 10.

[0129] The crude product can be purified by column chromatography eluting with methylene chloride:methanol or by HPLC using acetonitrile:TFA:HO to yield either the free base or the TFA salt as the final product. Treatment of the free base with HCl in dioxane or lyophilization of the TFA salt yields product (10) as the HCl or TFA salt. Alternatively, the free base can be treated with another inorganic or organic acid to form other salts selected from those generally known to be pharmaceutically acceptable. Salts of compound (10) are usually solids, and examples have been crystallized from ethanol or other solvents to obtain high-quality crystals. The tricyclic structure of compound (1) has been conclusively confirmed by X-ray crystallography and NMR.

[0130] The compounds described herein, with or without an aminoalkyl linker (e.g., compound (33)), can be used to identify molecules (e.g., proteins) that interact with them in a cellular context. Expression of these binding targets can be used to predict response to an imipyridone or its analog (i.e., serve as a biomarker). These compounds can also be used to screen structurally unrelated molecules using competition assays known in the art to identify agents that can outcompete the target interaction with higher affinity. Additionally, these molecules may have improved drug properties or enable additional applications by altering drug properties, including safety, efficacy, pharmacokinetics, biodistribution, or metabolism.

[0131] [Table 1-1]

[0132] [Table 1-2]

[0133] IV. Evaluation of the sensitivity and efficacy of treatment regimens

[0134] Measuring the expression, genetic mutations, or gene copy number of dopamine receptors or other G protein-coupled receptors (GPCRs) can be used to predict response or susceptibility to the therapeutic methods described herein, or to identify subjects likely to respond to the therapeutic methods described herein, such as treatment with a compound of Formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof. In one aspect, provided herein is a method for identifying whether a subject having a condition is likely to respond to a therapeutic regimen described herein. In one embodiment, the method includes: (i) obtaining a biological sample from the subject; (ii) measuring the expression level of at least one dopamine receptor or GPCR in the sample; (iii) comparing the level measured in the sample with a predetermined standard; and (iv) determining whether the subject is likely to respond to the therapeutic regimen based on the level measured in the sample relative to the level for the predetermined standard. In one embodiment, measuring the expression level in the sample comprises (i) contacting the sample with an antibody or antigen-binding fragment that specifically binds to the receptor to form a complex between the antibody or antigen-binding fragment and the receptor, and (ii) measuring the amount of the complex. In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the cancer is a neuro-oncology disease. In one embodiment, the cancer is a neuroendocrine tumor. In one embodiment, the cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, and diffuse intrinsic pontine glioma. In one embodiment, the subject has or is at risk of having a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, bipolar disorder, and major depressive disorder. In one embodiment, the subject has or is at risk of having an infection, such as a bacterial infection. In one embodiment, the infection is a gram-positive bacterial infection. In one embodiment, the infection is a gram-negative bacterial infection.In one embodiment, the infection is a bacterial infection selected from Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. In one embodiment, the Gram-positive bacterial infection is a Staphylococcus infection. For example, the Staphylococcus infection is a S. aureus infection (e.g., a methicillin-resistant S. aureus (MRSA) infection). In one embodiment, the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof. In one embodiment, the dopamine receptor is from the D2-like family. In one embodiment, the dopamine receptor is DRD2, DRD3, or both. In one embodiment, the dopamine receptor is DRD4. In one embodiment, the GPCR is a class A GPCR. In one embodiment, the GPCR is GPR132. In one embodiment, the GPCR is selected from GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, and combinations thereof. In one embodiment, the dopamine receptor is DRD5, the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and an increase in measured DRD5 expression in the sample compared to a predetermined standard indicates that the subject will respond, or is unlikely to respond, to the treatment regimen.

[0135] In another aspect, provided herein are methods for evaluating the effectiveness of a therapeutic regimen described herein, for monitoring, or for providing a prognosis for a subject with a condition. In one embodiment, the method includes: (i) obtaining a biological sample from the subject; (ii) measuring the expression level of at least one dopamine receptor or GPCR in the sample; (iii) comparing the level measured in the sample with a predetermined standard; and (iv) determining a prognosis or whether the subject will respond to the therapeutic regimen based on the level measured in the sample relative to the level for the predetermined standard. In one embodiment, the step of measuring the expression level of a dopamine receptor or GPCR in the sample includes: (i) contacting the sample with an antibody or antigen-binding fragment that specifically binds to the receptor to form a complex between the antibody or antigen-binding fragment and the receptor; and (ii) measuring the amount of the complex. In one embodiment, the method includes: (i) obtaining a biological sample from a subject; (ii) measuring gene copy numbers or mutations in at least one dopamine receptor in the sample; (iii) comparing the copy numbers measured in the sample or the mutations found in the sample with those for a predetermined standard; and (iv) determining whether the subject will respond to a treatment regimen based on the copy numbers measured in the sample or the mutations found in the sample relative to those for the predetermined standard. In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the cancer is a neuro-oncology disease. In one embodiment, the cancer is a neuroendocrine tumor. In one embodiment, the cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, and diffuse intrinsic pontine glioma. In one embodiment, the subject has or is at risk of having a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, bipolar disorder, and major depressive disorder. In one embodiment, the subject has or is at risk of having an infectious disease, such as a bacterial infection. In one embodiment, the infectious disease is a gram-negative bacterial infection. In one embodiment, the infection is a gram-positive bacterial infection.In one embodiment, the infection is a bacterial infection selected from Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. In one embodiment, the Gram-positive bacterial infection is a Staphylococcus infection. For example, the Staphylococcus infection is a S. aureus infection (e.g., a methicillin-resistant S. aureus (MRSA) infection). In one embodiment, the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof. In one embodiment, the dopamine receptor is selected from DRD2, DRD2S, DRD2L, and DRD3. In one embodiment, the dopamine receptor is from the D2-like family. In one embodiment, it is from the D1-like family. In one embodiment, the dopamine receptor is DRD1. In one embodiment, the dopamine receptor is DRD2. In one embodiment, it is DRD3. In one embodiment, it is DRD4. In one embodiment, it is DRD5. In one embodiment, the dopamine receptor is DRD2, DRD3, or both. In one embodiment, the GPCR is a class A GPCR. In one embodiment, it is GPR132. In one embodiment, the GPCR is selected from GPR132, GPR91, MTNR1A, GPR162, GPR137, BAI3, LGR4, PTGIR, CXCR7, and combinations thereof.

[0136] In one embodiment, the dopamine receptor is DRD5, the treatment regimen comprises administering an effective amount of a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and an increase in DRD5 expression level measured in the sample relative to a predetermined standard indicates that the treatment regimen is effective or ineffective. In one embodiment, the dopamine receptor is DRD5, the treatment regimen comprises administering an effective amount of a therapeutic agent, for example, a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and a mutation in the DRD5 gene measured in the sample indicates that the treatment regimen is effective or ineffective. In one embodiment, the dopamine receptor is DRD5, the treatment regimen comprises administering an effective amount of a therapeutic agent, for example, a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and a missense mutation Q366R in the DRD5 gene measured in the sample indicates that the treatment regimen is effective or ineffective.

[0137] In another aspect, provided herein is a method for identifying whether a subject with a pathological condition is likely to respond to a therapeutic regimen described herein. In one embodiment, the method includes: (i) obtaining a biological sample from the subject; (ii) measuring gene copy numbers or mutations in at least one dopamine receptor in the sample; (iii) comparing the measured copy numbers or mutations found in the sample with those for a predetermined standard; and (iv) determining whether the subject is likely to respond to the therapeutic regimen based on the measured copy numbers or mutations found in the sample relative to those for the predetermined standard. In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the cancer is a neuro-oncology disease. In one embodiment, the cancer is a neuroendocrine tumor. In one embodiment, the cancer is selected from the group consisting of meningioma, ependymoma, glioma, neuroblastoma, and diffuse intrinsic pontine glioma. In one embodiment, the subject has or is at risk of having a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, schizophrenia, bipolar disorder, and major depressive disorder. In one embodiment, the subject has or is at risk of having an infection, such as a bacterial infection. In one embodiment, the infection is a gram-negative bacterial infection. In one embodiment, the infection is a gram-positive bacterial infection. In one embodiment, the infection is a bacterial infection selected from Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. In one embodiment, the gram-positive bacterial infection is a Staphylococcus infection. For example, the Staphylococcus infection is a S. aureus infection (e.g., a methicillin-resistant S. aureus (MRSA) infection). In one embodiment, the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof. In one embodiment, the dopamine receptor is from the D2-like family of dopamine receptors.In one embodiment, the dopamine receptor is DRD1. In one embodiment, the dopamine receptor is DRD2. In one embodiment, the dopamine receptor is DRD3. In one embodiment, the dopamine receptor is DRD4. In one embodiment, the dopamine receptor is DRD5. In one embodiment, the dopamine receptor is DRD2, DRD3, or both. In one embodiment, the dopamine receptor is DRD5, and the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and a mutation in the DRD5 gene measured in the sample indicates that the subject will respond, or is unlikely to respond, to the treatment regimen. In one embodiment, the dopamine receptor is DRD5, and the treatment regimen comprises administering an effective amount of a therapeutic agent, e.g., a compound of Formula (10) or a pharmaceutically acceptable salt thereof, and a missense mutation Q366R in the DRD5 gene measured in the sample indicates that the subject will respond, or is unlikely to respond, to the treatment regimen.

[0138] Additionally, measuring the expression, post-translational modification, or activity level or mutation of eIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used to predict response or sensitivity to the therapeutic methods described herein and to identify subjects likely to respond to the therapeutic methods described herein, such as treatment with a compound of Formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof. Additionally, measuring the expression, post-translational modification, or activity level or mutation of eIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used to evaluate the efficacy of or monitor the therapeutic methods described herein. Furthermore, measuring the expression, post-translational modification, or activity level or mutation of eIF2-α, ATF4, CHOP, DR5, or cleaved or total cytokeratin 18 can be used to screen structurally unrelated anti-cancer molecules in vivo, in vitro, or in silico. For example, competition and other assays can be used to identify agents that can outcompete the target interaction with higher affinity, and compare changes in those levels to the respective changes produced by the compound of Formula (10) or its analogs. Assays can also be performed on live mammalian cells or microsomal extracts prepared from cultured cell lines, which more closely resemble the effects of specific serum drug levels in the body.

[0139] In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the treatment regimen comprises administering an effective amount of an imipyridone, e.g., ONC201, or an analog thereof. In one embodiment, the treatment regimen comprises administering an effective amount of ONC201. In one embodiment, the treatment regimen comprises administering an effective amount of a compound of Formula (10). In one embodiment, the compound of Formula (10) is a compound of Formula (40), e.g., a compound of Formula (45). In one embodiment, the compound of Formula (10) is a compound of Formula (50), e.g., a compound of Formula (55). In one embodiment, the compound of Formula (10) is a compound of Formula (80). In one embodiment, the compound of Formula (10) is a compound of Formula (90). In one embodiment, the compound of Formula (10) is a compound of Formula (60). In one embodiment, the analog of compound (1) has a structure selected from the structures of formula (25), formula (26), formula (27), formula (28), formula (29), formula (30), or formula (31):

[0140] The level of the predetermined standard can be, for example, the average level or median level measured in samples from a subject. The level of the predetermined standard can be measured under the same or substantially similar experimental conditions as when measuring the sample from the subject. The level of the predetermined standard can be obtained from a subject who is responsive to treatment with an imipyridone, e.g., ONC201, or an analog thereof. In one embodiment, the predetermined standard is obtained from a subject who is responsive to treatment with the compound, and if the level in the sample from the subject and the level in the standard are similar, the subject can be classified as likely to respond to treatment. The level of the predetermined standard can be obtained from a subject who is not responsive to treatment with the compound. In one embodiment, the predetermined standard is obtained from a subject who is not responsive to treatment with the compound, and if the level in the sample from the subject and the predetermined standard are different (e.g., up-regulated or down-regulated), the subject can be classified as likely to respond to treatment. The level of the predetermined standard can be obtained from a normal, healthy subject.

[0141] Immunoassays can be used to assay protein or methylation levels in a sample, including enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), flow cytometry, immunoblot, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescence immunoassay (LIA), fluorescence immunoassay (FIA), and radioimmunoassay. 6 A mRNA methylation level can be obtained by methylated RNA immunoprecipitation (Me-RIP) or other quantitative biochemical assays known in the art.

[0142] Nucleic acid mutations can be determined by any of several procedures. For example, a biological sample is obtained from a subject. Non-limiting examples of biological samples include bodily fluids (e.g., urine, saliva, plasma, or serum) or tissue samples (e.g., buccal tissue samples or buccal cells). The biological sample can then be sequenced or scanned using known methods. For example, a DNA array can be used to analyze at least a portion of the subject's genomic sequence. Furthermore, whole-genome or partial-genome sequence information can be used. Such sequences can be determined using standard sequencing methods, including chain termination (Sanger dideoxynucleotide), dye-terminator sequencing, and SOLID™ sequencing (Applied Biosystems). The whole genome can be cleaved by restriction enzymes or (mechanically) sheared into shorter fragments for sequencing. DNA sequences can also be amplified using methods such as PCR and vector-based cloning methods (e.g., Escherichia coli). In one embodiment, 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, for example, using conventional DNA sequencers or chip-based technologies, to identify the presence or absence of mutations or copy number variations.

[0143] In one aspect, provided herein are methods for identifying and treating a subject having a condition who is likely to respond to a therapeutic regimen described herein. In one embodiment, the method includes (i) identifying whether a subject having the condition is likely to respond to a therapeutic regimen described herein, and (ii) treating the subject determined to be likely to respond to the therapeutic regimen with the therapeutic regimen. In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the therapeutic regimen includes administering an effective amount of an imipyridone, e.g., ONC201, or an analog thereof. In one embodiment, the therapeutic regimen includes administering an effective amount of Compound (1). In one embodiment, the therapeutic regimen includes administering an effective amount of a compound of Formula (10). In one embodiment, the compound of Formula (10) is a compound of Formula (40), e.g., a compound of Formula (45). In one embodiment, the compound of Formula (10) is a compound of Formula (50), e.g., a compound of Formula (55). In one embodiment, the compound of Formula (10) is a compound of Formula (80). In one embodiment, the compound of Formula (10) is a compound of Formula (90). In one embodiment, the compound of Formula (10) is a compound of Formula (60). In one embodiment, the analog of Compound (1) has a structure selected from the structures of Formula (25), Formula (26), Formula (27), Formula (28), Formula (29), Formula (30), or Formula (31).

[0144] The level of the predetermined standard can be, for example, the average level or median level measured in samples from subjects. The level of the predetermined standard can be measured under the same or substantially similar experimental conditions as those used to measure samples from subjects. The level of the predetermined standard can be obtained from a subject responsive to treatment with an imipyridone, e.g., ONC201, or an analog thereof. In one embodiment, the predetermined standard is obtained from a subject responsive to treatment with the compound, and if the level in the sample from the subject is similar to the level in the standard, the subject can be classified as likely to respond to treatment. The level of the predetermined standard can be obtained from a subject not responsive to treatment with the compound. In one embodiment, the predetermined standard is obtained from a subject not responsive to treatment with the compound, and if the level in the sample from the subject is different (e.g., upregulated or downregulated) from the level in the predetermined standard, the subject can be classified as likely to respond to treatment. The level of the predetermined standard can be obtained from a normal, healthy subject. Immunoassays can be used to assay protein levels in a sample.

[0145] In one aspect, provided herein are methods of treating a subject having a condition and assessing the effectiveness of the treatment in the subject. In one embodiment, the method includes (i) treating the subject according to the treatment methods described herein and (ii) assessing the effectiveness of the treatment as described herein. In one embodiment, the subject has or is at risk of having cancer. In one embodiment, the treatment regimen includes administering an effective amount of an imipyridone, e.g., ONC201, or an analog thereof. In one embodiment, the treatment regimen includes administering an effective amount of Compound (1). In one embodiment, the treatment regimen includes administering an effective amount of a compound of Formula (10). In one embodiment, the compound of Formula (10) is a compound of Formula (40), e.g., a compound of Formula (45). In one embodiment, the compound of Formula (10) is a compound of Formula (50), e.g., a compound of Formula (55). In one embodiment, the compound of Formula (10) is a compound of Formula (80). In one embodiment, the compound of Formula (10) is a compound of Formula (90). In one embodiment, the compound of Formula (10) is a compound of Formula (60). In one embodiment, the compound of Formula (1) analog has a structure selected from Formula (25), Formula (26), Formula (27), Formula (28), Formula (29), Formula (30), or Formula (31).

[0146] Other conditions that may be suitable for the methods described herein include attention deficit disorder, addiction, epilepsy, viral infections, 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 cardiac hypertrophy, myocarditis, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, rheumatic heart disease, diabetes, and light chain amyloidosis.

[0147] V. Composition

[0148] In one aspect, there is provided a pharmaceutical composition comprising a compound of formula (10) or formula (1) represented by the following formula and a pharmaceutically acceptable salt thereof:

[0149] [ka]

[0150] [ka]

[0151] In one embodiment, the salt is a pharmaceutically acceptable monosalt of the compound. In one embodiment, the salt is a pharmaceutically acceptable di-salt of the compound. In one embodiment, the salt is a pharmaceutically acceptable mono- or poly-salt (e.g., di- or tri-salt) thereof, and is selected from hydrochloride, hydrobromide, bisulfate, sulfate, phosphate, fumarate, succinate, oxalate, lactate, bisulfate, hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate, maleate, fumarate, sulfonate, methylsulfonate, formate, acetate, and carboxylate. In one embodiment, the salt is selected from the group consisting of p-toluenesulfonate, benzenesulfonate, citrate, methanesulfonate, oxalate, succinate, tartrate, fumarate, and maleate. In one embodiment, the salt is a salt selected from the group consisting of ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and / or counterions (e.g., methylamino, dimethylamino, diethylamino, and triethylamino counterions). In one embodiment, the salt is a dihydrochloride or dihydrobromide salt.

[0152] Compound (1) (ONC201) has the same chemical structure as would be revealed by structural analysis (e.g., NMR, X-ray diffraction) of compound NSC 350625, available from the National Cancer Institute's Developmental Therapeutics Program Repository.

[0153] In one embodiment, the pharmaceutical composition comprises a di-salt (e.g., dihydrochloride salt) of ONC201 or an analog thereof (e.g., imipyridone). Salts (e.g., di- or tri-salts) of ONC201 analogs can be prepared from ONC201 analogs, which can be synthesized as described herein or using standard chemical synthesis methods known to those skilled in the art.

[0154] In one embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier. Non-limiting examples of suitable pharmaceutically acceptable carriers include those described in Handbook of Pharmaceutical Excipients, 7 th ed., Raymond C. Rowe et al., American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London and previous editions. Exemplary pharmaceutically acceptable carriers, methods for preparing pharmaceutical compositions and various dosage forms, and modes of administration are well known in the art and are described in detail, for example, in Pharmaceutical Dosage Forms: Tablets, Larry L. Augsburger & Stephen W. Hoag., eds., London: Informa Healthcare, 2008, and L.V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8 th ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004, ARGennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21 sted., 2005, especially chapter 89, and J. Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 10 th ed.,2001.

[0155] In one embodiment, the pharmaceutical composition is formulated for ocular administration. In one embodiment, the pharmaceutical composition is formulated for topical administration. In one embodiment, the pharmaceutical composition is formulated as drops, an ointment, or a liquid. In one embodiment, the pharmaceutical composition comprises a conventional pharmaceutical carrier, such as an aqueous, powder, or oily base, a thickener, or the like.

[0156] In one embodiment, the pharmaceutical composition is a formulation for intravenous administration. In one embodiment, the intravenous formulation comprises a compound of Formula (10) or a pharmaceutically acceptable salt thereof dissolved in a solvent. In one embodiment, the solvent comprises water. In one embodiment, the intravenous formulation comprises the 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, the intravenous formulation comprises the compound or a salt thereof at a concentration of about 0.05, 0.5, or 5 mg / mL to about 1, 10, or 100 mg / mL. In one embodiment, the intravenous formulation comprises about 0.005%, 0.05%, or 0.5% to about 0.1%, 1%, or 10% of the compound or a salt thereof. In one embodiment, the intravenous formulation comprises about 0.05%, 0.5%, or 5% of the compound or a salt thereof. In one embodiment, the intravenous formulation contains a higher or lower concentration of the compound or salt thereof.

[0157] In one embodiment, the intravenous formulation has a pH of about 3. In one embodiment, the formulation is adjusted to pH 3 with a phosphate buffer. In one embodiment, the intravenous formulation comprises dextrose or sodium chloride. In one embodiment, the intravenous formulation comprises the compound or a salt thereof at a concentration of about 5 mg / mL and a pH of 3, forming a stable solution. In one embodiment, the intravenous formulation comprises the compound or a salt thereof at a concentration of about 5 mg / mL and a pH of less than 5, forming a stable solution. In one embodiment, the intravenous formulation comprises the compound or a salt thereof and one or more antioxidants. In one embodiment, the intravenous formulation comprises a mixture of the monohydrochloride and dihydrochloride salts of the compound. In one embodiment, the intravenous formulation comprises the compound or a salt thereof as a 1% solution at a concentration of about 10 mg / mL. For example, the intravenous formulation is a solution having a pH of about 3.3. In one embodiment, the pH is less than 4.0.

[0158] In one embodiment, a suitable pharmaceutically acceptable carrier comprises an aqueous carrier. In one embodiment, the aqueous carrier comprises sterile water. In one embodiment, the formulation comprises dextrose, sodium chloride, or both. In one embodiment, the pharmaceutically acceptable carrier comprises an oil.

[0159] In one embodiment, the intravenous formulation comprises 25 mg / mL of ONC201 or an analog thereof, or its dihydrochloride salt, dissolved in water. In one embodiment, the formulation is adjusted to pH 3 with a phosphate buffer. In one embodiment, the formulation comprises dextrose, sodium chloride, or both. In one embodiment, the formulation comprises a higher or lower concentration of the dihydrochloride salt of ONC201 or an analog thereof. In one embodiment, the formulation comprises ONC201 or an analog thereof, or its dihydrochloride salt, at a concentration of about 5 mg / mL. In one embodiment, the about 5 mg / mL formulation forms a stable solution and a pH of 3. In one embodiment, the about 5 mg / mL formulation has a pH of less than 5 and forms a stable solution. In one embodiment, the intravenous formulation comprises ONC201 or an analog thereof, or its dihydrochloride salt, and one or more antioxidants. In one embodiment, the intravenous formulation comprises a mixture of the monohydrochloride and dihydrochloride salts of ONC201 or an analog thereof. In one embodiment, the intravenous formulation comprises ONC201 or an analog thereof, or its dihydrochloride salt, as a 1% solution at a concentration of about 10 mg / mL. For example, the intravenous formulation is a solution having a pH of about 3.3. In one embodiment, the pH is less than 4.0.

[0160] In one embodiment, the intravenous formulation contains about 0.5% to about 10% (or about 5 mg / mL to about 100 mg / mL) ONC201 or an analog thereof, or a di-salt thereof. In one embodiment, the formulation contains about 5% (or about 50 mg / mL) ONC201 or an analog thereof, or a di-salt thereof. In one embodiment, the intravenous infusion rate can be slowed to potentially reduce side effects of ONC201 or an analog thereof, or a di-salt thereof.

[0161] In one embodiment, the pharmaceutical composition comprises about 0.1-99% of an ONC201 salt or analog thereof and a pharmaceutically acceptable carrier, such as an oil or sterile water or other aqueous carrier. In one embodiment, for oral dosage forms, the composition comprises a mono- or di-salt of ONC201 or an analog thereof in the range of about 5% to about 50%.

[0162] In one embodiment, the pharmaceutical composition contains an antioxidant. Suitable antioxidants include ascorbic acid derivatives (e.g., ascorbic acid, erythorbic acid, sodium ascorbate), thiol derivatives (e.g., thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sulfites (e.g., sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium sulfite, and sodium thiosulfate), and nordihydroguaiaretic acid. Antioxidants used in aqueous formulations typically include sodium sulfite, sodium metabisulfite, sodium formaldehyde sulfoxylate, or ascorbic acid, and combinations thereof, while antioxidants used in oily solutions and organic solvents typically include BHT, BHA, or propyl gallate, and combinations thereof. In yet other embodiments, the antioxidant may be one or more of flavanoids, isoflavones, monothioglycerol, L-cysteine, thioglycolic acid, α-tocopherol, 6-palmitoyl ascorbic acid, dihydrolipoic acid, BHT, BHA, vitamin E, propyl gallate, β-carotene, and ascorbic acid. Antioxidants are typically used at about 0.1% to 1.0% by weight, more typically about 0.2% by weight.

[0163] In one embodiment, the pharmaceutical composition comprises an imipyridone, such as ONC201, or an analog thereof, or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent, such as hormone analogs and antihormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors, growth factor antibodies, 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, IGF-1R inhibitors, ErbB receptor inhibitors, rapamycin analogues, BTK inhibitors, CRM1 inhibitors (e.g., KPT185), P53 modulators (e.g., Natrin), antiangiogenic agents (e.g., axitinib, aflibercept, sorafenib, and regorafenib), amifostine, anagrelide, clodronate, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, 2-chlorodesoxyadenosine Syn, 2-fluorodesoxy-cytidine, 2-methoxyestradiol, 2C4,3-arretin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A105972, A204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, alanose, arglabin, arzoxifene, Atamestane, atrasentan, auristatin PE, ABT-199 (venetoclax), ABT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY162, ARRY438162, 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, ARQ680, AS-703026 (primasertib), Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodal dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7 787, BIBW2992 (afatinib, tomtobok), BIBF1120 (valgatef), BI836845, BI2536, BI6727, BI836845, BI847325, BI853520, 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, carboxyphthalatoplatin, CCl-77 9, CC-115, CC-223, CEP-701, CEP-751, CBT-1, cefixime, cefratonin, ceftriaxone, celecoxib, cermoleukin, cemadotin, CH4987655 / RO-4987655, chlorotrianisene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, combretastatin A4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3, cryptophyscin 52, CTP-37, CTLA- 4 monoclonal antibodies, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diflomotecan, didox, 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-18-0CH3, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, gallium maltolate, gefitinib, gemtuzumab, gimatecan, glufosfamide, G CS-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, Gra Nicetron, Herceptin, Hexamethylmelamine, Histamine, Homoharringtonine, Hyaluronic Acid, Hydroxyurea, Hydroxyprogesterone Caproate, Ibandronic Acid, Ibrutinib, Ibritumomab, Idatrexate, Idenestrol, IDN-5109, IGF-1R Inhibitors, IMC-1C11, IMC-A12 (Cixutumumab), Immunol, Indisulam, Interferon α-2a, Interferon α-2b, Pegylated Interferon α-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, Kahalid F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuporlin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU223651, lurtotecan, LY-S6AKT1,LY-2780301, mafosfamide, marimastat, mechloroethamine, 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, motexaf in gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, nexavar, neovastat, nilotinib, Nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, Oncophage, oncoVEX GM-CSF, olmiplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel Paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, 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 (vemurafenib), PLX-3603 / RO-5212054, PT-100, PWT-33597, PX-866, picoplatin , pivaloyloxymethyl butyrate, pixantrone, phenoxodiol O, PKI166, previtrexed, plicamycin, polyprenoic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY869766, RDEA-436, rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors, regorafenib, revimid, RG-7167, RG-7304, RG-7421, RG7440,Rhizoxin, rhu-MAb, linfavert, risedronate, rituximab, lobatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalubicin, SAHA, sargramostim, satraplatin, SB408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustine, seocalcitol, SM-113 55, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, Sutent, T900607, T138067, TAK-733, TAS-103, tacedinaline, talaporfin, tarceva, taliquitar, tasisulam, taxotere, taxoplexin, tazarotene, tegafur, temozolamide, tesmilifene, testosterone, testosterone propionate, tesmilifene tetraplatin, tetrodotoxin, tezacitabine, thalidomide, Theralux, telarubicin, thymalfasin, thymectacin, tiazofurin, tipifarnib, tomudex, tirapazamine, tocladesine, tremofin, trabectedin, TransMID-107, trans-retinoic acid, tretinoin, trastuzumab, tremelimumab, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286, TXD258, Tykerb / Tyverb, Urocidine, Valrubicin The agent is selected from the group consisting of cinnamyl, vatalanib, vincristine, vinflunine, virudin, 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 combinations thereof.

[0164] In one embodiment, the other therapeutic agent comprises a hormone analog, 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, the other therapeutic agent comprises one or more LHRH agonists selected from goserelin acetate, luprolide acetate, triptorelin pamoate, and combinations thereof, and the LHRH antagonist is selected from degarelix, cetrorelix, abarelix, ozarelix, degarelix, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more growth factor inhibitors selected from inhibitors of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER), and hepatocyte growth factor (HGF). In one embodiment, the other therapeutic agent comprises one or more inhibitors of human epidermal growth factor selected from HER2, HER3, and HER4. In one embodiment, the other therapeutic agent comprises one or more tyrosine kinase inhibitors selected from cetuximab, gefitinib, imatinib, lapatinib, and trastuzumab, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more aromatase inhibitors selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof. In one embodiment, the other therapeutic agents include one or more antimetabolites that are antifolates selected from methotrexate, raltitrexed, and pyrimidine analogs. In one embodiment, the other therapeutic agents include one or more antimetabolites that are pyrimidine analogs selected from 5-fluorouracil, capecitabine, and gemcitabine. In one embodiment, the other therapeutic agents include 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,The other therapeutic agent includes one or more antitumor antibiotics selected from anthracyclines, doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin, dactinomycin, plicamycin, streptozocin, and combinations thereof. In one embodiment, the other therapeutic agent includes one or more platinum derivatives selected from cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment, the other therapeutic agent includes one or more alkylating agents selected from estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, and combinations thereof. In one embodiment, the other therapeutic agent includes a nitrosourea selected from carmustine, lomustine, thiotepa, and combinations thereof. In one embodiment, the other therapeutic agent includes an antimitotic agent selected from vinca alkaloids and taxanes. In one embodiment, the other therapeutic agent comprises one or more taxanes selected from paclitaxel, docetaxel, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more vinca alkaloids selected from vinblastine, vindesine, vinorelbine, vincristine, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more topoisomerase inhibitors that are epipodophyllotoxins. In one embodiment, the other therapeutic agent comprises one or more epipodophyllotoxins selected from etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more serine / threonine kinase inhibitors selected from PDK1 inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors, inhibitors of CDKs, Aurora kinase inhibitors, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more tyrosine kinase inhibitors that are PTK2 / FAK inhibitors. In one embodiment, the other therapeutic agent comprises one or more tyrosine kinase inhibitors that are IAP, Mcl-1, MDM2 / MDMX,and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more rapamycin analogs selected from everolimus, temsirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment, the 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, the other therapeutic agent is 2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyestradiol, 2C4,3-arretin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A105972, A204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, alt. Retamin, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, alanose, arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199 (venetoclax), ABT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY16 2, ARRY438162, 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, ARQ680, AS-703026 (primasertib), Avastin, AZD-2014, and azacitidine , azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodal dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807,BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtobok), BIBF1120 (valgatef), BI836845, BI2536, BI6727, BI836845, BI847325, BI853520, 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, carboxybenzoate Phthalatoplatin, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1, cefixime, cefratonin, ceftriaxone, celecoxib, cermoleukin, cemadotin, CH4987655 / RO-4987655, chlorotrianisene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, combretastatin A4, COT inhibitors, CHS-828, CH-5132799, CLL-Thera, CMT-3, cryptophyscin 5 2, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone, dexrazoxane, diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin 10, doranidazole, DS-7423, E7010, E-6201, edatrexate, edotreotide, efaproxiral, eflornithine, EGF R inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulind, 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-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795 , GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronic acid, ibrutinib, ibritumomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (cixutumumab), Immunol, indisulam, interferon α-2a, interferon α-2b, pegylated interferon α-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, Kahalid F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuporlin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU223651, lurtotecan, LY- S6AKT1, LY-2780301, mafosfamide, marimastat, mechloroethamine, 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, motexaf in gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, nexavar, neovastatNilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, olmiplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, Estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, Pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitor, PI3K / mT OR inhibitors, PG-TXL, PG2, PLX-4032 / RO-5185426 (vemurafenib), PLX-3603 / RO-5212054, PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethylbutyrate, pixantrone, phenoxodiol O, PKI166, previtrexed, plicamycin, polyprenoic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY8697 66, RDEA-436, rebeccamycin analog, receptor tyrosine kinase (RTK) inhibitor, revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG7440, rhizoxin, rhu-MAb, rinfavert, risedronate, rituximab, lobatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalubicin, SAHA, sargramostim, satrapra Chin, SB408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustine, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, Sutent, T900607, T138067, TAK-733, TAS-103, tacedinaline, talaporfin, tarceva, taliquitar, tasisulam, taxotere, taxoplexin, tazarotene,Tegafur, temozolamide, tesmilifene, testosterone, testosterone propionate, tesmilifene, tetraplatin, tetrodotoxin, tezacitabine, thalidomide, cerarax, telarubicin, thymalfasin, thymectacin, tiazofurin, tipifarnib, tomudex, tirapazamine, tocladesine, tremofin, trabectedin, TransMID-107, trans-retinoic acid, tretinoin, trastuzumab, tremelimumab, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD258, ta and one or more therapeutic agents selected from Ikerub / Tyverb, Urocidin, Valrubicin, Vatalanib, Vincristine, Vinflunine, Virulizine, 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 combinations thereof.

[0165] In one embodiment, the other therapeutic agent comprises a steroid, including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazol. In one embodiment, the other therapeutic agent comprises an antiemetic, including 5-HT3 receptor agonists (e.g., dolasetron, granisetron, ondansetron, tropisetron, palonosetron, and mirtazapine), dopamine agonists (e.g., domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine, and metoclopramide), NK1 receptor antagonists (e.g., aprepitant and casopitan), Antihistamines (e.g., cyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine), cannabinoids (e.g., cannabis, dronabinol, nabilone, and sativex), benzodiazepines (e.g., midazolam and lorazepam), anticholinergics (e.g., hyoscine), trimethobenzamide, ginger, emetrol, propofol, peppermint, muscimol, and ajowan.

[0166] In one embodiment, the other therapeutic agent comprises an anti-cancer agent comprising an antimitotic agent. In one embodiment, the antimitotic agent comprises a taxane. In one embodiment, the antimitotic agent comprises a taxane selected from paclitaxel and docetaxel.

[0167] In one embodiment, the pharmaceutical composition comprises an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and at least one anticancer agent, including acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, amethanthrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacytidine, azetepa, or azepam. Zotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, visnafide dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calsterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin acetaminophen, decitabine, dexorumaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate, eflomitine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, elbrozole, esorubicin, estramustine, etanidazole, etoposide, etopurine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine, fosquidone, fosquidone Striesin, fulvestrant, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irmofosine, interleukin II (including IL-2, 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, megestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocalcine, mitochromine, mitogillin, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone, mycophenolate, nelarabine, nocodazole, nogalamycin, ormunaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, ribopurine, rogletimide, safingol, semustine, simtrazene, sparfosate , sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxylone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestron, triciribine, trimetrexate, triptorelin, tubrozole, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglisinate, vinleurosine, vinorelbine, vinrocidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, zorubicin, and combinations thereof.

[0168] In one embodiment, the pharmaceutical composition comprises an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and at least one anticancer agent, including lorlatinib (LORBRENA), pembrolizumab (KEYTRUDA), talazoparib (TALZENNA), emicizumab-kxwh injection (HEMLIBRA), cemiplimab-rwlc (LIBTAYO), dacomitinib tablets (VIZIMPRO), duvelisib (COPIKTRA), moxetumomab pasudotox-tdfk (LUMOXITI), nivolumab (OPDIVO), mogamulizumab-kpkc (Poteligeo), lusutrombopag (Mulpleta), iobenguane I-131 (AZEDRA), or ivosidenib. (Tibsovo), ribociclib (Kisqali), enzalutamide (XTANDI), ipilimumab (YERVOY), encorafenib and binimetinib (BRAFTOVI and MEKTOVI), bevacizumab (Avastin), venetoclax (VENCLEXTA), methoxypolyethylene glycol-epoetin beta (Mircera), Flufila (pegfilgrastim-jmdb), avatrombopag (Doptelet), Retacrit (epoetin alfa-epbx), tisagenlecleucel (KYMRIAH), dabrafenib (TAFINLAR), trametinib (MEKINIST), fostamatinib disodium hexahydrate tablets (TAVALISSE), everolimus tablets for oral suspension (Afinitor Disperz), rucaparib, nilotinib (TASIGNA), abemaciclib (VERZENIO), apalutamide, abiraterone acetate (Zytiga), lutetium Lu177 dotatate (LUTATHERA), afatinib (Gilotrif), olaparib tablets (Lynparza), pertuzumab (PERJETA), bosutinib (BOSULIF), cabozantinib (Cabometyx), ogibri (trastuzumab-dkst), sunitinib malate (Sutent), obinutuzumab (GAZYVA), emicizumab-kxwh (HEMLIBRA), dasatinib (SPRYCEL), brentuximab vedotin (ADCETRIS), alectinib (ALECENSA), vemurafenib (ZELBORAF), acalabrutinib (Calquence), axicabtagene Ciloleucel (YESCARTA), abemaciclib (VERZENIO), copanlisib (ALIQOPA), Muvasi (bevacizumab-awwb), gemtuzumab ozogamicin (Mylotarg), inotuzumab ozogamicin (BESPONSA), liposome-encapsulated daunorubicin and cytarabine combination (VYXEOS), ibrutinib (Imbruvica), enasidenib (IDHIFA), neratinib (NERLYNX), betrixaban (BEVYXXA), dabrafenib and trametinib (TAFINLAR and MEKINIST), rituximab and hyaluronidase human (RITUXAN HYCELA), aminolevulinic acid hydrochloride (ALA HCl; Gleolan), avelumab (BAVENCIO), durvalumab (IMFINZI), brigatinib (ALUNBRIG), midostaurin (RYDAPT), regorafenib (STIVARGA), palbociclib (IBRANCE), osimertinib (TAGRISSO), niraparib (ZEJULA), avelumab (BAVENCIO), ribociclib (KISQALI), lenalidomide (Revlimid), rucaparib (R These include one or more of: UBRACA, daratumumab (DARZALEX), olaratumumab (LARTRUVO), atezolizumab (TECENTRIQ), erlotinib (TARCEVA), cabozantinib (CABOMETYX), defibrotide sodium (Defitelio), crizotinib capsules (Xalkori), everolimus (Afinitor), eribulin (HALAVEN injection), and combinations thereof.

[0169] In one embodiment, the pharmaceutical composition comprises an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and at least one anticancer agent, including elotuzumab (EMPLICITI), necitumumab (PORTRAZZA), ixazomib (NINLARO), cobimetinib (COTELLIC tablets), talimogene laherparepvec (IMLYGIC), trabectedin (Yondelis injection), irinotecan liposome injection (O NIVYDE), idarucizumab (Praxbind injection), trifluridine / tipiracil (LONSURF), carfilzomib (Kyprolis), sonidegib (Odomzo capsules), gefitinib (IRESSA), ramucirumab (CYRAMZA), dinutuximab (Unituxin), filgrastim-sndz (ZARXIO injection), panobinostat (FARYDAK capsules), lenvatinib (Lenvima), lanreotide (Somatuline) Depot injection), ruxolitinib (Jakafi), blinatumomab (BLINCYTO), idelalisib (Zydelig tablets), belinstat (BELEODAQ), ceritinib (ZYKADIA), mercaptopurine (Purixan), siltuximab (Sylvant injection), ofatumumab (Arzerra injection), sorafenib (NEXAVAR tablets), crizotinib (Xalkori), ibrutinib (IMBRUVICA), paclitaxel protein-bound particles (albumin-bound) (Abraxane for injectable suspension), denosumab (Xgeva injection), radium Ra 223 dichloride (Xofigo injection), ado-trastuzumab emtansine (KADCYLA for injection), pomalidomide (POMALYST capsules), doxorubicin hydrochloride liposome injection, and combinations thereof.

[0170] Examples of suitable anti-cancer drugs include those described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12 thEd., Laurence Brunton, Bruce Chabner, Bjorn Knollman, McGraw Hill Professional, 2010.

[0171] In some exemplary embodiments, the pharmaceutical composition comprises an imipyridone, e.g., ONC201, or a salt (e.g., mono- or di-salt) of an analog thereof, and at least one other therapeutic agent, wherein the other therapeutic agent comprises an antiangiogenic agent, e.g., bevacizumab. In one embodiment, the antiangiogenic agent is aflibercept, axitinib, angiostatin, endostatin, 16 kDa prolactin fragment, laminin peptide, fibronectin peptide, tissue inhibitor of metalloproteinases (TIMP1, 2, 3, 4), plasminogen activator inhibitor (PAI-1, 2), tumor necrosis factor alpha (high dose, in vitro), TGF-β1, interferon (IFN-α, β, γ), ELR-CXC chemokine, IL-12; SDF -1; MIG; platelet factor 4 (PF-4); IP-10, thrombospondin (TSP), SPARC, 2-methoxyestradiol, proliferin-related protein, suramin, sorafenib, regorafenib, thalidomide, cortisone, linomide, fumagillin (AGM-1470; TNP-470), tamoxifen, retinoids, CM101, dexamethasone, leukemia inhibitory factor (LIF), hedgehog inhibitors, and combinations thereof.

[0172] A pharmaceutical combination can include the first and second therapeutic agents in any desired ratio, so long as a synergistic or cooperative effect still occurs. A synergistic pharmaceutical combination preferably has the first therapeutic agent and the second therapeutic agent in a ratio of about 1:9 to about 9:1. In one embodiment, the synergistic combination of the first therapeutic agent and the second therapeutic agent is 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, about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, or about 1:2 to about 2:1. In one embodiment, the synergistic combination of therapeutic agents is in a ratio of about 1:1.

[0173] In one embodiment, the 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, gemcitabine HCl, hydroxyurea, ifosfamide, 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 combinations thereof.

[0174] In one embodiment, the second therapeutic agent comprises a small molecule multikinase inhibitor, e.g., sorafenib or regorafenib. In one embodiment, the second therapeutic agent comprises a hedgehog pathway inhibitor, e.g., vismodegib. In one embodiment, the second therapeutic agent comprises an agent selected from Table 2 below.

[0175] [Table 2-1]

[0176] [Table 2-2]

[0177] In one embodiment, the second therapeutic agent comprises an agent that targets a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor. In one embodiment, the second therapeutic agent comprises recombinant TRAIL or an agonist antibody that activates one or more TRAIL receptors. In one embodiment, the second therapeutic agent comprises one or more antibodies or recombinant TRAIL that activate signaling through DR4, DR5, or both. In one embodiment, the second therapeutic agent comprises one or more of AMG-655, LBY-135, mapatumumab, lexatumumab, Apomab, and rhApo2L / TRAIL. In one embodiment, the 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 (a gossypol derivative), ABT-263, ABT-737, and GX-15-070 (obatoclax), vorinostat, cetuximab, panitumumab, bevacizumab, ganitumab, interferon gamma, sorafenib, XIAP antagonists, Bcl-2 antagonists, and Smac mimetics.

[0178] VI. Dose

[0179] In one embodiment, the pharmaceutical composition comprises an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, in a dose ranging 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 from about 0.4, 0.5, 0.6, or 1 mg to about 20 mg, wherein the weights may be based on the compound in its free base form. In one embodiment, the pharmaceutical composition comprises a dose of 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, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg. or 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 of an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises: from about 40 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 4 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, or 200 mg; or at dose levels ranging 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, comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof.In one embodiment, the pharmaceutical composition comprises a dose of 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, or 200 mg; or at dose levels ranging 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, comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises from about 100 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 mg, or 2000 mg; from about 10 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 mg, or 2000 mg; or 200 mg; or at dosage levels ranging 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, comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises 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, or 200 mg; or at a dose level ranging from about 2 mg to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg, comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof.In one embodiment, the pharmaceutical composition comprises from about 400 mg to about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg; from about 40 mg to about 50, 60, 70, 80, 90, 100, 110, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg, based on the compound in its free base form. or 200 mg; or at dosage levels ranging from about 4 mg to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg, comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises from about 50 mg to about 60, 70, 80, 90, or 100 mg; from about 60 mg to about 70, 80, 90, or 100 mg; from about 70 mg to about 80, 90, or 100 mg; from about 80 mg 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 mg to about 7, 8, 9, or 10 mg; from about 7 mg to about 8, 9, or 10 mg; from about 8 mg to about 9 or 10 mg; about 9 mg to about 10 mg; about 0.5 mg to about 0.6, 0.7, 0.8, 0.9, or 1 mg; about 0.6 mg to about 0.7, 0.8, 0.9, or 1 mg; about 0.7 mg to about 0.8, 0.9, or 1 mg; about 0.8 mg to about 0.9, or 1 mg; or about 0.9 mg to about 1 mg of an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof.

[0180] In one embodiment, the pharmaceutical composition comprises an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, at a dose ranging from about 1 to about 40 mg / kg, about 0.1 to about 4 mg / kg, or about 0.01 to about 0.40 mg / kg. In one embodiment, the pharmaceutical composition comprises 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; or from about 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 mg / kg. g / kg to about 40 mg / kg; 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; 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; 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 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; 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; about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, or 0.19 mg / kg to about 0.20, 0.30, or 0.40 mg / kg g / kg; about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, or 0.29 mg / kg to about 0.30 or 0.400 mg / kg; or 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 of an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof.

[0181] In one embodiment, the pharmaceutical composition comprises about 37.5 mg / m 2 ~about 1500mg / m 2 , about 3.75mg / m 2 ~about 150mg / m 2 , or about 0.4 mg / m 2 ~about 15mg / m 2and an imipyridone, such as ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, at a dose ranging from 0.1 to 1.0. In one embodiment, the pharmaceutical composition comprises a medicament containing at least about 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, 5, 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, 62 0, 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, 91 5, 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, 1075, 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 35, 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, 1495mg / m, 2 ~about 1500mg / m 2 ; approx. 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, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 mg / m 2 ~about 150mg / m 2or 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 / m 2 ~about 15mg / m 2 and an imipyridone, such as ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, at a dose ranging from 0.1 to 1.0.

[0182] VII. Dosage Form

[0183] Pharmaceutical compositions for use in the methods described herein can be formulated into dosage forms that can be administered to a patient. In one embodiment, the composition is in the form of an oral dosage unit or a parenteral dosage unit. In one embodiment, it is in the form of an oral dosage unit. In one embodiment, the oral dosage unit is divided into several smaller doses, which are administered to a subject over a predetermined period of time to reduce the toxicity of the administered therapeutic agent. In one embodiment, the oral dosage unit is administered as a tablet or capsule containing a controlled-release formulation, which may include multiple particles, granules, pellets, mini-tablets, or tablets. In some cases, the composition is in the form of a parenteral dosage unit. For example, the parenteral dosage unit is selected from intravenous (IV), subcutaneous (SC), intramuscular (M), rectal (PR), or transdermal dosage units. In one embodiment, the dosage form is selected from a sterile solution, suspension, suppository, tablet, and capsule. In one embodiment, the oral dosage form is selected from a tablet, caplet, capsule, lozenge, syrup, liquid, suspension, and elixir. In one embodiment, the oral dosage form is selected from a tablet, a hard shell capsule, a soft gelatin capsule, a bead, a granule, an agglomerate, a powder, a gel, a solid, and a semi-solid.

[0184] In one embodiment, the pharmaceutical composition for use in the methods described herein comprises a dermatological composition suitable for topical administration to the skin. For example, the dermatological composition comprises a pharmaceutically or cosmetically acceptable medium. Dermatological compositions for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. In some cases, conventional pharmaceutical carriers, aqueous, powder, or oily bases, skin enhancers, thickeners may be necessary or desirable, and therefore may be used. Suitable enhancers include ethers such as diethylene glycol monoethyl ether (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; alcohols such as ethanol, propanol, octanol, and benzyl alcohol; polyethylene glycol and its esters, such as polyethylene glycol monolaurate; amides and other nitrogen compounds, such as urea, 2-pyrrolidone, 1-methyl-2-pyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF), ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, especially citric acid and succinic acid. AZONE® and sulfoxides such as DMSO and CI0MSO may also be used, but are less preferred.

[0185] In one embodiment, the dosage form is selected from sustained release forms, controlled release forms, delayed release forms, and responsive release forms.

[0186] VIII.How to use

[0187] The compositions and methods described herein are useful for treating many disease states, including cancer (e.g., colon cancer, brain cancer, and glioblastoma). In one embodiment, the compositions and methods described herein are used to treat diseases such as ocular melanoma, desmoplastic round cell tumor, chondrosarcoma, leptomeningeal disease, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal or rectal cancer, appendix cancer, astrocytoma, and atypical teratoid / rhabdoid tumor. In one embodiment, the compositions and methods described herein are used to treat diseases such as basal cell carcinoma, basal cell nevus syndrome, Gorlin nevus syndrome, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, and spinal tumor. In one embodiment, the compositions and methods described herein are used to treat diseases such as carcinoid tumors, cancer of unknown primary, central nervous system atypical teratoid / rhabdoid tumor, leptomeningeal disease, central nervous system embryonal tumors, central nervous system lymphoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, and cutaneous T-cell lymphoma (including Sézary syndrome and mycosis fungoides (MF)). In one embodiment, the compositions and methods described herein are used to treat diseases such as central nervous system embryonal tumors, endometrial cancer, epithelial blastoma, epithelioma, esophageal cancer, Ewing's sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, and eye cancers, including intraocular melanoma and retinoblastoma. In one embodiment, the compositions and methods described herein are used to treat diseases such as gallbladder cancer, gastric cancer (stomach cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, and glioma. In one embodiment, 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 carcinoma (liver cancer), histiocytosis, Hodgkin's lymphoma, and hypopharyngeal carcinoma. In one embodiment, 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, 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, non-small cell lung cancer and lung cancer, including small cell lung cancer, non-Hodgkin's lymphoma, and primary central nervous system lymphoma. In one embodiment, the compositions and methods described herein are used to treat diseases such as Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, multiple endocrine neoplasia syndrome, oral cancer, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, complex karyotype, blastic phase leukemia, myelodysplastic / myeloproliferative neoplasm, multiple myeloma, and myeloproliferative disorders. In one embodiment, the compositions and methods described herein are used to treat cancer. In one embodiment, the compositions and methods described herein are used to treat diseases such as nasal and paranasal sinus cancer, nasopharyngeal cancer, and neuroblastoma. In one embodiment, the compositions and methods described herein are used to treat diseases such as oral cavity cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer, and ovarian low malignant potential tumor. In one embodiment, the compositions and methods described herein are used to treat diseases such as pancreatic cancer, papilloma, paranasal and paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma, gestational breast cancer, primary central nervous system lymphoma, and prostate cancer. In one embodiment, the compositions and methods described herein are used to treat a cancer selected from the group consisting of rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter cancer, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, and rhabdomyosarcoma. In one embodiment, the compositions and methods described herein are used to treat aggressive prostate cancer. In one embodiment, the compositions and methods described herein are used to treat intermediate-grade prostate cancer. In one embodiment, the compositions and methods described herein are used to treat indolent prostate cancer.In one embodiment, the compositions and methods described herein are used to treat castration-resistant prostate cancer. In one embodiment, the compositions and methods described herein are used to treat nervous system tumors. In one embodiment, the compositions and methods described herein are used to treat central nervous system tumors. In one embodiment, the compositions and methods described herein are used to treat peripheral nervous system tumors. In one embodiment, the compositions and methods described herein are used to treat paraganglioma. In one embodiment, the compositions and methods described herein are used to treat pheochromocytoma.

[0188] In in vitro models, animal models, and human clinical trials, compound (1) (ONC201) has broad anticancer activity and exhibits low toxicity with few, if any, adverse effects, low genotoxicity, and high bioavailability, including oral bioavailability. These characteristics make ONC201 and various analogs particularly well suited for pediatric patients. These characteristics also make ONC201 and various analogs particularly well suited for chronic therapy, high-risk patients, and for ensuring long-term responses or stable disease or preventing disease recurrence.

[0189] In another aspect, provided herein is a method of treating or preventing cancer in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of compound (1) represented by the following formula:

[0190] [ka]

[0191] In one embodiment, the cancer involves the thalamus, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, spinal cord, or medulla. In one embodiment, the cancer is not a spinal cord tumor. In one embodiment, the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M. In one embodiment, the histone H3 K27M mutation is in one or more histone genes selected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J. In one embodiment, DRD2 is overexpressed in cancer tissue, DRD5 is underexpressed, or both. In one embodiment, the subject is a human. In one embodiment, the subject is a domestic pet. In one embodiment, the subject is a pediatric subject.

[0192] In another aspect, provided herein is a method for treating or preventing cancer in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (10) or an analog thereof, or a pharmaceutically acceptable salt thereof, wherein the cancer has a histone H3 mutation. In one embodiment, the cancer is selected from the group consisting of central nervous system tumors, brain tumors, peripheral nervous system tumors, pheochromocytoma, paraganglioma, adrenocortical carcinoma, adrenal tumors, and neuroendocrine tumors. In one embodiment, the cancer is of glial origin and is selected from the group consisting of meningioma, epithelioma, oligodendroglioma, astrocytoma, optic nerve glioma, pineal tumor, rhabdoid tumor, and diffuse intrinsic pontine glioma. In one embodiment, the cancer is of neural origin and is selected from the group consisting of medulloblastoma, neuroblastoma, ganglioneuroma, primary neuroectodermal tumor, and schwannoma. In one embodiment, the cancer involves the thalamus, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, spinal cord, or medulla. In one embodiment, the histone H3 mutation is H3.3 K27M or H3.1 K27M. In one embodiment, the cancer has a K27M mutation in one or more histone genes selected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J. In one embodiment, DRD2 is overexpressed in cancer tissue, DRD5 is underexpressed, or both. In one embodiment, the compound is ONC201. In one embodiment, the subject is a human. In one embodiment, the subject is a domestic pet. In one embodiment, the subject is a pediatric subject.

[0193] In another aspect, provided herein is a method of treating or preventing cancer in a subject in need thereof, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (10) or an analog thereof, or a pharmaceutically acceptable salt thereof, wherein the cancer involves midline structures of the brain. In one embodiment, the cancer involves the thalamus, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, spinal cord, or medulla. In one embodiment, the cancer is not a spinal cord tumor. In one embodiment, the cancer has a histone H3 mutation, and the histone H3 mutation is H3.3 K27M or H3.1 K27M. In one embodiment, the cancer has a histone H3 K27M mutation in one or more histone genes selected from H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, or HIST1H3J. In one embodiment, DRD2 is overexpressed in the cancer tissue, DRD5 is underexpressed, or both. In one embodiment, the compound is ONC201. In one embodiment, the subject is a human. In one embodiment, the subject is a domestic pet. In one embodiment, the subject is a pediatric subject.

[0194] In one embodiment, the compositions and methods described herein are used to treat pediatric cancer (e.g., pediatric solid tumors, pediatric sarcoma, pediatric Ewing's sarcoma, pediatric glioma, pediatric central nervous system cancer, pediatric neuroblastoma, pediatric leukemia, and pediatric lymphoma).

[0195] In one embodiment, the compositions and methods described herein are used to treat proliferative skin disorders such as psoriasis. In one embodiment, the compositions and methods described herein are used to treat a cancer selected from the group consisting of salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, eye cancer, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical carcinoma of unknown primary site, and supratentorial primitive neuroectodermal tumor. In one embodiment, the compositions and methods described herein are used to treat a cancer selected from the group consisting of T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, and gestational trophoblastic tumor. In one embodiment, 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, rare pediatric cancers, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, and uterine sarcoma. In one embodiment, the compositions and methods described herein are used to treat a cancer selected from the group consisting of vaginal cancer and vulvar cancer. In one embodiment, the compositions and methods described herein are used to treat a cancer selected from the group consisting of Wilms' tumor and women's cancer.

[0196] In one embodiment, the compositions and methods described herein are used as first-line therapy (sometimes referred to as primary therapy). In one embodiment, the compositions and methods described herein are used as second-line therapy. In one embodiment, the compositions and methods described herein are used as third-line therapy. In one embodiment, the compositions and methods described herein are used as salvage therapy. The term "salvage therapy" refers to a therapeutic agent that may be taken in any regimen after a subject's initial treatment regimen has failed or after the subject's condition has not responded to the initial treatment. In one embodiment, the compositions and methods described herein are used as salvage therapy. In one embodiment, the compositions are used as rescue agents to counter the effects of the initial treatment. In one embodiment, the compositions are used as rescue agents administered to a subject who has developed resistance to the standard or initial treatment. In one embodiment, the compositions and methods described herein are used as neoadjuvant therapy. In one embodiment, neoadjuvant therapy involves administering one or more of the therapeutic agents described herein to a subject prior to the main or first-line treatment. In one embodiment, neoadjuvant therapy reduces the size or extent of the cancer being treated before administering the main or first-line treatment to the subject being treated. In one embodiment, the compositions and methods described herein are used as adjuvant therapy. In one embodiment, adjuvant therapy comprises administering to a subject one or more therapeutic agents described herein, including one or more therapeutic agents that modify the effect of other therapeutic agents already administered to the subject, or administered simultaneously with the subject, or subsequently administered to the subject.

[0197] In one embodiment, the compositions and methods described herein exhibit a reduced potential for drug-drug interactions. In one embodiment, the imipyridone, e.g., ONC201, or an analog thereof, is cleared from the patient's body before it can interact with another pharmaceutically active agent.

[0198] In one embodiment, the compositions and methods described herein exhibit a level of toxicity that facilitates their use in combination with other pharmaceutical agents.

[0199] The methods and compositions described herein are not limited to a particular animal species. In one embodiment, the subject treated according to the methods and using the compositions described herein can be a mammal or a non-mammal. In one embodiment, mammalian subjects include, but are not limited to, humans, non-human primates, rodents such as mice, rats, or guinea pigs, domestic pets such as cats or dogs, horses, cows, pigs, sheep, goats, or rabbits. In one embodiment, non-mammalian subjects include, but are not limited to, birds such as ducks, geese, chickens, or turkeys. In one embodiment, the subject is a human. In one embodiment, the subject can be of either gender and of any 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.

[0200] The methods and compositions described herein are not limited to a particular age of the subject. In one embodiment, the subject treated according to the methods and using the compositions described herein is 50 years of age or older, 55 years of age or older, 60 years of age or older, or 65 years of age or older. In one embodiment, the subject treated according to the methods and using the compositions described herein is under 50 years of age, under 55 years of age, under 60 years of age, or under 65 years of age.

[0201] In one embodiment, the subject treated according to the methods and compositions described herein is a pediatric patient. In one embodiment, the pediatric patient is under 18 years old, under 17 years old, under 16 years old, under 15 years old, under 14 years old, under 13 years old, under 12 years old, under 11 years old, under 10 years old, under 9 years old, 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, or under 1 year old. In one embodiment, the pediatric patient is under 12 months old, under 11 months old, under 10 months old, under 9 months old, under 8 months old, under 7 months old, under 6 months old, under 5 months old, under 4 months old, under 3 months old, under 2 months old, or under 1 month old. In one embodiment, the pediatric patient is under 4 weeks old, under 3 weeks old, under 2 weeks old, or under 1 week old. In one embodiment, the 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, the pediatric patient is a newborn. In one embodiment, the pediatric patient is a premature infant.

[0202] In one embodiment, the 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, weighs less than 10 kg, weighs less than 5 kg, weighs less than 4 kg, weighs less than 3 kg, weighs less than 2 kg, or weighs less than 1 kg.

[0203] In one embodiment, the subject has received at least one prior therapeutic agent. In one embodiment, the subject has received at least two, at least three, or at least four prior therapeutic agents. In one embodiment, the prior therapeutic agent is ibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, or lenalidomide.

[0204] In one embodiment, the subject is being treated with radiation, in one embodiment, the subject is being treated with surgery, in one embodiment, the subject is being treated with adoptive T cell therapy.

[0205] In one embodiment, the cancer no longer responds to treatment with ibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, lenalidomide, radiation, surgery, or a combination thereof.

[0206] In one embodiment, the compositions and methods described herein have a dose-response relationship in cancer cells that differs from the dose-response relationship of the same compositions and methods in normal cells. The dose-response relationship of ONC201 on proliferation and cell death in normal and tumor cells was determined by measuring cell viability after 72 hours of treatment with various concentrations of ONC201. The tumors tested included a human colon cancer cell line (HCT116), a breast tumor cell line (MDA-MB-231), and a human primary glioblastoma cell line (U87). The normal cells tested also 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 in normal fibroblasts. Cell viability of the normal cells tested was at least approximately 75% at approximately 1-5 mg / mL of ONC201, whereas tumor cell viability was significantly lower (e.g., below 50%) at the same ONC201 concentrations. Furthermore, as the ONC201 concentration increased beyond approximately 5 mg / mL, tumor cell viability decreased to less than 25%, while normal cell viability remained at approximately 75%. Cell viability assays in human fetal lung fibroblast (MRC-5) cells were performed after 72 hours of treatment with compound (1) (5 μM) or DMSO and a complete post-treatment recovery period in drug-free medium. Cell recovery was observed with ONC201 but not with DMSO.

[0207] In one embodiment, the compositions and methods described herein are useful for treating cancer in a subject. In one embodiment, the compositions and methods described herein are useful for treating cancer in a human subject. In one embodiment, the method of treatment comprises administering to a subject in need of such treatment a pharmaceutically effective amount of an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0208] In one embodiment, a method of treatment comprises administering to a subject in need of such treatment (i) a first therapeutic agent comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially. The second therapeutic agent can be any suitable therapeutic agent, including a pharmaceutically active agent disclosed herein. Pharmaceutically acceptable ONC201 salts include the dihydrochloride salt:

[0209] [ka]

[0210] It is understood that the dihydrochloride salt of ONC201 or an analog thereof (including the compound of formula (10)), or an alternative disalt thereof, which are apparent from the teachings of the present disclosure, may be substituted for ONC201 or an analog thereof in the compositions or dosing regimens described herein.

[0211] In one embodiment, a method of treatment comprises simultaneously or sequentially administering to a subject in need of such treatment a synergistic pharmaceutical combination, the synergistic pharmaceutical combination comprising (i) a first therapeutic agent comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, and (ii) a second therapeutic agent. In one embodiment, a method of treatment comprises simultaneously or sequentially administering to a subject in need of such treatment a therapeutically synergistically effective amount of the first therapeutic agent in combination with the second therapeutic agent. In one embodiment, a method of treatment comprises administering to a subject in need of such treatment an effective amount of the first therapeutic agent in combination with an effective amount of the second therapeutic agent, wherein the combination provides a synergistic effect in the in vivo treatment of a cancer susceptible to the combination, and wherein the first and second therapeutic agents are administered simultaneously or sequentially. In one embodiment, a method of treatment comprises administering to a subject in need of such treatment an effective amount of a first therapeutic agent in combination with an effective amount of a second therapeutic agent, the combination providing a synergistic effect in the in vivo treatment of minimal residual disease susceptible to the combination, wherein the first and second therapeutic agents are administered simultaneously or sequentially. In one embodiment, the second agent is administered before or prior to the first agent.

[0212] In one embodiment, the treatment targets a cancer selected from the group consisting of a solid tumor, a liquid tumor, a lymphoma, a leukemia, or a myeloma.

[0213] In one embodiment, the treatment is targeted to a solid tumor, and the solid tumor is selected from the group consisting of cervical cancer, endometrial cancer, extracranial germ cell tumor, extragonadal germ cell tumor, germ cell tumor, gestational trophoblastic tumor, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer, and ovarian low malignant potential tumor, penile cancer, prostate cancer, gestational breast cancer, high-grade prostate cancer, intermediate-grade prostate cancer, low-grade prostate cancer, castration-resistant prostate cancer, breast cancer, cholangiocarcinoma, extrahepatic bile duct cancer, gallbladder cancer, hepatocellular carcinoma (liver cancer), kidney cancer (renal cell carcinoma), liver cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter, basal cell carcinoma, basal cell nevus syndrome, Gorlin nevus syndrome, melanoma, Merkel cell carcinoma, papilloma, multiple endocrine neoplasia syndrome, pancreatic cancer, parathyroid cancer, ocular melanoma, eye cancer, retinoblastoma, tumor, malignant fibrous histiocytoma, Ewing's sarcoma family of tumors, desmoplastic round cell tumor, chondrosarcoma, Kaposi's sarcoma, rhabdomyosarcoma, spinal cord tumor, leptomeningeal disease, central nervous system embryonal tumor, chordoma, central nervous system embryonal tumor, ependymoblastoma, ependymoma, neuroblastoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, adrenocortical carcinoma, bone cancer, osteosarcoma, malignant fibrous histiocytoma and osteosarcoma of bone, osteosarcoma and bone Malignant fibrous histiocytoma, carcinoid tumor, cancer of unknown primary origin, bronchial tumor, lung cancer, pleuropulmonary blastoma, respiratory tract cancer involving the NUT gene on chromosome 15, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system atypical teratoid / rhabdoid tumor, craniopharyngioma, glioma, brain cancer, medulloblastoma, medulloepithelioma, supratentorial primitive neuroectodermal tumor, pituitary tumor, gastric cancer cancer) (stomach cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), bladder cancer, anal or rectal cancer, appendix cancer, esophageal cancer, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous cell neck cancer of unknown primary, oral cavity cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity cancer, lip and oral cavity cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, head and neck cancer, and mesothelioma.

[0214] In one embodiment, the therapy targets a lymphoma selected from the group consisting of diffuse large B-cell lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, Sézary syndrome, mycosis fungoides (MF), histiocytosis, Burkitt lymphoma, and central nervous system lymphoma, non-Hodgkin's lymphoma, and primary central nervous system lymphoma, Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, mycosis fungoides, primary central nervous system lymphoma, lymphoplasmacytic lymphoma, and primary central nervous system lymphoma. In one embodiment, the therapy targets a non-Hodgkin's lymphoma (NHL) selected from the group consisting of mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, lymphoplasmacytic NHL, Waldenstrom's macroglobulinemia, and cutaneous lymphoma.

[0215] In one embodiment, the treatment method targets a leukemia selected from the group consisting of acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloproliferative disorder, hairy cell leukemia, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and Langerhans cell histiocytosis. In one embodiment, the treatment targets an acute leukemia selected from the group consisting of acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, and myeloproliferative disorder.

[0216] In one embodiment, the treatment targets a myeloma selected from the group consisting of: IgA myeloma, IgG myeloma, IgM myeloma, IgD myeloma, IgE myeloma, light chain myeloma, non-secretory myeloma, complex karyotype, blast phase leukemia, multiple myeloma / plasma cell neoplasm, multiple myeloma, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, and myeloproliferative disorder.

[0217] In some cases, the treatment targets a peripheral nervous system tumor. In some cases, the treatment targets a paraganglioma. In some cases, the treatment targets a pheochromocytoma.

[0218] In one embodiment, treating cancer comprises preventing tumor growth in a subject with cancer. In one embodiment, treating cancer comprises preventing the formation of cancer metastases in a subject with cancer. In one embodiment, treating cancer comprises targeted treatment of minimal residual disease in a subject with cancer known to have or at risk of having minimal residual disease in cancer.

[0219] This may occur after surgical treatment of the primary tumor and / or after chemotherapy (radiotherapy) has been initiated or determined to be effective. Disseminated tumor cells may be dormant and often cannot be attacked by chemotherapy (radiotherapy). Patients treated in this manner are seemingly cured and are referred to as having "minimal residual disease." Nevertheless, dormant tumor cells may form metastases if they become metastatic cells after a longer period of dormancy due to growth stimulation.

[0220] The term "minimal residual disease" refers to the small number of cancer cells remaining in a subject when the subject is in remission (exhibiting no symptoms or signs of the disease) during or after treatment. The methods described herein are preferably applied to the forms of the diseases listed herein, including adult and pediatric forms of these diseases.

[0221] In one embodiment, the method of treatment is useful for treating autoimmune diseases, including, but not limited to, alopecia areata, antiphospholipid, autoimmune hepatitis, celiac disease, type 1 diabetes, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, and vitiligo.

[0222] In one embodiment, the therapeutic methods are useful for treating metabolic disorders including diabetes, B12, and folate vitamin deficiencies, chemotherapy drugs and agents used to treat HIV, toxins that cause peripheral nerve damage, cancers that cause peripheral neuropathy, and autoimmune and inflammatory disorders of the peripheral nervous system such as amyotrophic lateral sclerosis (Lou Gehrig's disease), based on a variety of causes including paraneoplastic syndromes, alcohol abuse, chronic kidney disease, injuries that cause compression of nerves and other lesions, infections (e.g., Lyme disease), Guillain-Barré syndrome, connective tissue diseases, rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, certain inflammatory conditions (e.g., sarcoidosis), celiac disease, genetic disorders (e.g., Charcot-Marie-Tooth syndrome), Friedreich's ataxia, and / or idiopathic diseases.

[0223] In one embodiment, the method of treatment is useful for treating autoimmune and inflammatory disorders associated with ocular conditions, including, but not limited to, ocular cicatricial pemphigoid, Mooren's corneal ulcer, various forms of uveitis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis 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 ulcer, and less common entities such as choroiditis, retinal vasculitis, episcleral segments, retinal detachment, and / or macular edema.

[0224] In one embodiment, the method of treatment is useful for treating acute allograft rejection in transplant patients. In one embodiment, the method of treatment is useful for treating ischemic stroke. In one embodiment, the method of treatment is useful for treating inflammatory diseases including arthritis, psoriasis, asthma, and colitis.

[0225] In one embodiment, the therapeutic agent comprises a pharmaceutically acceptable mono-salt of ONC201 or an analog thereof (e.g., a compound of Formula (10)). In one embodiment, the therapeutic agent comprises a pharmaceutically acceptable di-salt of ONC201 or an analog thereof (e.g., a compound of Formula (10)). As described herein, some of the analogs may be tri-salts. In one embodiment, the therapeutic agent comprises ONC201 or an analog 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, hydrogensulfate, sulfate, phosphate, fumarate, succinate, oxalate, and lactate, bisulfate, hydroxyl, tartrate, nitrate, citrate, bitartrate, carbonate, malate, maleate, fumarate sulfonate, methylsulfonate, formate, acetate, and carboxylate. In one embodiment, the therapeutic agent comprises ONC201 or an analog 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, the therapeutic agent comprises ONC201 or an analog thereof in the form of a pharmaceutically acceptable mono- or di-salt having a counterion selected from the group consisting of ammonium, sodium, potassium, calcium, magnesium, zinc, and lithium, and / or a counterion such as a methylamino, dimethylamino, diethylamino, triethylamino counterion, and combinations thereof. In one embodiment, the therapeutic agent comprises a compound described herein in the form of a halide di-salt, such as a dihydrochloride or dihydrobromide salt.

[0226] In one embodiment, the second therapeutic agent comprises an anticancer agent. In one embodiment, the second therapeutic agent comprises acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, amethanthrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleoma Isin, brequinar, bropirimine, busulfan, cactinomycin, calsterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesilate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, desaguanine, desaguanine mesilate, diaziconazole, docetaxel, doxorubicin, Droloxifene, dromostanolone, duazomycin, edatrexate, eflomitine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, elbrozole, esorubicin, estramustine, etanidazole, etoposide, etopurine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine, fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irmofo sine, interleukin II (including IL-2, 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, megestrol acetate, melphalan,Menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone, mycophenolate, nelarabine, nocodazole, nogalamycin, ormunaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, ribopurine, rogletimide, safingol, semustine, simtrazene, sparfosate, sparsomycin, spirogermanium , spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxylone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestron, triciribine, trimetrexate, triptorelin, tubrozole, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglisinate, vinleurosine, vinorelbine, vinrocidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, zorubicin, and combinations thereof.

[0227] In one embodiment, the second therapeutic agent is selected from the group consisting of hormone analogs and antihormones, aromatase inhibitors, LHRH agonists and antagonists, inhibitors of growth factors, growth factor antibodies, 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 alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, 2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyestradiol, 2C4,3-arretin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A105972, A204197, abiraterone, aldesleukin, Alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, alanose, arglabin, arzoxifene, atamestane, atrasentan, auristatin PE, ABT-199 (venetoclax), ABT-263 (nabitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY162, ARRY438162, ARRY-300, ARRY-142886 / AZD-6244 (selmethicone) Nib), ARRY-704 / AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ680, AS-703026 (primasertib), Avastin, AZD-2014, azacitidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodal dicitrate, BCX-1777, BKM-120, bleocin, BLP-25,BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtobok), BIBF1120 (valgatef), BI836845, BI2536, BI6727, BI836845, BI847325, BI853520, 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, carboxyphthalatoplatin, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1, cefixime, cefratonin, ceftriaxone, celecoxib, celmoleukin, cemadotin, CH4987655 / RO-4987655, chlorotrianisene, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine cytidine, combretastatin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3, cryptophycin 52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin 1 0, 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-18-0CH3, ethinylcytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, exisulind, 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-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 ( Dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronic acid, ibritumomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), Immunol, indisulam, interferon interferon alfa-2a, interferon alfa-2b, pegylated interferon alfa-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, Kahalid F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuporlin, lexidronam, LGD -1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechloroethamine, MEK inhibitor, 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,Gadolinium-containing motexaf, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 6-benzylguanine, oblimersen, omeprazole, Oncophage, oncoVEX GM-CSF, olmiplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, estrogen, panitumumab, patupilone, pegfil rastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, Pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitor, PI3K / mTOR inhibitor, PG-TXL, PG2, PLX-4032 / RO-5185426 (vemurafenib), PLX-3603 / RO-5212054, PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethyl butyrate, pixantrone, phenoxodiol O, PKI166, previtrexed, plicamycin, polyprenoic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY869766, RDEA-436, rebeccamycin analogs, receptor RTK inhibitors, Revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG7440, rhizoxin, rhu-MAb, rinfavert, risedronate, rituximab, lobatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalubicin, SAHA, sargramostim, satraplatin, SB408075, Se-015 / Ve-015,SU5416, SU6668, SDX-101, semustine, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, Sutent, T900607, T138067, TAK-733, TAS-103, tacedinaline, talaporfin , Tarceva, Taliquitar, Tasisulam, Taxotere, Taxoplexin, Tazarotene, Tegafur, Temozolamide, Tesmilifene, Testosterone, Testosterone propionate, Tesmilifene, Tetraplatin, Tetrodotoxin, Tezacitabine, Thalidomide, Theralux, Terarubicin, Thymalfasin, Thimectacin, Tiazofurin, Tipifarnib, Tomudex, Tirapazamine , tocladesine, tremofine, trabectedin, TransMID-107, trans retinoic acid, tretinoin, trastuzumab, tremelimumab, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD258, Tykerb / Tyverb, urocidin, valrubicin, vatalanib, vincristine, vinflunine, virudin, WX-UK1, WX-554, Selected from 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 combinations thereof.

[0228] In one embodiment, the second therapeutic agent is 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, the second therapeutic agent is selected from an LHRH agonist and an LHRH antagonist. In one embodiment, the LHRH agonist is selected from goserelin acetate, luprolide acetate, triptorelin pamoate, and combinations thereof. In one embodiment, the second therapeutic agent comprises an LHRH antagonist selected from degarelix, cetrorelix, abarelix, ozarelix, and combinations thereof of degarelix. In one embodiment, the second therapeutic agent comprises a growth factor inhibitor. In one embodiment, the growth factor inhibitor is selected from inhibitors of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER), hepatocyte growth factor (HGF), and combinations thereof. In one embodiment, the human epidermal growth factor (HER) is selected from HER2, HER3, and HER4.

[0229] In one embodiment, the second therapeutic agent comprises a tyrosine kinase inhibitor. In one embodiment, the tyrosine kinase inhibitor is selected from cetuximab, gefitinib, imatinib, lapatinib, and trastuzumab, and combinations thereof. In one embodiment, the second therapeutic agent comprises an aromatase inhibitor. In one embodiment, the aromatase inhibitor is selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof.

[0230] In one embodiment, the second therapeutic agent comprises an antimetabolite. In one embodiment, the antimetabolite comprises an antifolate. In one embodiment, the antifolate is selected from methotrexate, raltitrexed, a pyrimidine analog, and combinations thereof. In one embodiment, the antimetabolite is a pyrimidine analog. In one embodiment, the pyrimidine analog is selected from 5-fluorouracil, capecitabine, gemcitabine, and combinations thereof. In one embodiment, the antimetabolite is a purine analog or an adenosine analog. In one embodiment, the purine analog or adenosine analog is selected from mercaptopurine, thioguanine, cladribine, pentostatin, cytarabine, fludarabine, and combinations thereof. In one embodiment, the second therapeutic agent comprises an antitumor antibiotic. In one embodiment, the antitumor antibiotic is selected from anthracyclines, doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C, bleomycin, dactinomycin, plicamycin, streptozocin, and combinations thereof. In one embodiment, the second therapeutic agent comprises a platinum derivative. In one embodiment, the platinum derivative is selected from cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment, the second therapeutic agent comprises an alkylating agent. In one embodiment, the alkylating agent is selected from estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, and combinations thereof. In one embodiment, the second therapeutic agent comprises a nitrosourea. In one embodiment, the nitrosourea is selected from carmustine, lomustine, thiotepa, and combinations thereof. In one embodiment, the second therapeutic agent comprises an antimitotic agent. In one embodiment, the antimitotic agent is selected from a vinca alkaloid and a taxane. In one embodiment, the taxane is selected from paclitaxel, docetaxel, and combinations thereof. In one embodiment, the vinca alkaloid is selected from vinblastine, vindesine, vinorelbine, vincristine, and combinations thereof. In one embodiment, the second therapeutic agent comprises a topoisomerase inhibitor. In one embodiment,The topoisomerase inhibitor is an epipodophyllotoxin. In one embodiment, the topoisomerase epipodophyllotoxin is selected from etoposide, etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone, and combinations thereof. In one embodiment, the second therapeutic agent comprises a serine / threonine kinase inhibitor. In one embodiment, the serine / threonine kinase inhibitor is selected from a PDK1 inhibitor, a B-Raf inhibitor, an mTOR inhibitor, an mTORC1 inhibitor, a PI3K inhibitor, a dual mTOR / PI3K inhibitor, an STK33 inhibitor, an AKT inhibitor, a PLK1 inhibitor, an inhibitor of CDK, an Aurora kinase inhibitor, and combinations thereof. In one embodiment, the second therapeutic agent comprises a tyrosine kinase inhibitor. In one embodiment, the second therapeutic agent comprises a PTK2 / FAK inhibitor. In one embodiment, the second therapeutic agent comprises a protein-protein interaction inhibitor. In one embodiment, the protein-protein interaction inhibitor is selected from IAP, Mcl-1, MDM2 / MDMX, and combinations thereof. In one embodiment, the second therapeutic agent comprises a rapamycin analog. In one embodiment, the rapamycin analog is selected from everolimus, temsirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment, the 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, the second therapeutic agent is 2-chlorodesoxyadenosine, 2-fluorodesoxy-cytidine, 2-methoxyestradiol, 2C4,3-arretin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A105972, A204197, abiraterone, aldesleukin, alitretinoin, allovectin-7, altretamine, alvocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, alanose, arglabin, arzoxifene, atamestane, atrasentan, auristatin PE,ABT-199 (venetoclax), ABT-263 (navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY162, ARRY438162, 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, ARQ680, AS-703026 (primasertib), Avastin, AZD-2 014, azacitidine, azaepothilone B, azonafide, BAY-43-9006, BAY80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodal dicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtobok), BIBF1120 (valgatef), BI8 36845, BI2536, BI6727, BI836845, BI847325, BI853520, 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, carboxyphthalatoplatin, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1, cefixime, Furatonin, ceftriaxone, celecoxib, cermoleukin, cemadotin, CH4987655 / RO-4987655, chlorotrianisene, 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, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine,D24851, decitabine, deoxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, desoxyepothilone B, dexamethasone, dexrazoxane, diethylstilbestrol, diflomotecan, didox, 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-18-0CH3, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulind, 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-0980, GDC-0032 , GDC-0068, GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexamethylmelamine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea , hydroxyprogesterone caproate, ibandronic acid, ibritumomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), Immunol, indisulam, interferon α-2a, interferon α-2b, pegylated interferon α-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, Kahalid F, ketoconazole, KW-2170, KW-2450, lobaplatin, leflunomide, lenograstim, leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, mechloroethamine, 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, motexaf in gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 6-benzylguanine, oblimersen, omega-3 fatty acids ... Prazol, Oncophage, oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-046915 02, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, Pentrix, perifosine, perillyl alcohol, pertuzumab, PI3K inhibitor, PI3K / mTOR inhibitor, PG-TXL, PG2, PLX-4032 / RO-5185426 (vemurafenib), PLX-3603 / RO-5212054, PT-100, PWT-33597, PX-866, picoplatin, pivaloyloxymethyl butyrate, pixantrone, phenoxodiol O, PKI166, previtrexed, plicamycin,Polyprene, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY869766, RDEA-436, rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors, revimid, RG-716, 7, RG-7304, RG-7421, RG-7321, RG7440, rhizoxin, rhu-MAb, linfavert, risedronate, rituximab, lobatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalubicin, SAHA, sargramostim, satraplatin, SB408075, Se-015 / Ve-015, SU5416, SU6668, SDX -101, semustine, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, Sutent, T900607, T138067, TAK-733, TAS-103, tacedinaline, talaporfin, tarceva, taliquitar, tasisulam, taxotere, taxoplexin, tazarotene, tegafur, temozolamide, tesmilifen, testosterone, tes tosterone propionate, tesmilifen, tetraplatin, tetrodotoxin, tezacitabine, thalidomide, Theralux, telarubicin, thymalfasin, thymectacin, tiazofurin, tipifarnib, tirapazamine, tocladesine, tomudex, tremofine, trabectedin, TransMID-107, trans-retinoic acid, trastuzumab, tremelimumab, tretinoin, triacetyluridine, triapine, triciribine, trimetrexate, TLK-286TXD258, Tykerb / Tyverb , urocidin, valrubicin, vatalanib, vincristine, vinflunine, virudin, 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 combinations thereof.

[0231] In one embodiment, the other therapeutic agent comprises a steroid, including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazol. In one embodiment, the other therapeutic agent comprises an antiemetic. Antiemetics include 5-HT3 receptor agonists (e.g., dolasetron, granisetron, ondansetron, tropisetron, palonosetron, and mirtazapine), dopamine agonists (e.g., domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine, and metoclopramide), NK1 receptor antagonists (e.g., aprepitant and casopitant), antihistamines (e.g., fluticasone, fluoxetine ... Examples include, but are not limited to, cyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine), cannabinoids (e.g., cannabis, dronabinol, nabilone, and Sativex), benzodiazepines (e.g., midazolam and lorazepam), anticholinergics (e.g., hyoscine), trimethobenzamide, ginger, emetrol, propofol, peppermint, muscimol, and ajowan.

[0232] The pharmaceutical composition can be administered to a subject via any suitable route of administration. In one embodiment, the pharmaceutical composition is administered to a subject orally, parenterally, transdermally, or transmucosally. In one embodiment, the pharmaceutical composition is administered to a subject parenterally. In one embodiment, the pharmaceutical composition is administered to a subject via a parenteral route selected from intravenous (IV), subcutaneous (SC), and intramuscular (IM). In one embodiment, the pharmaceutical composition is administered to a subject via a route selected from rectal and transdermal. In one embodiment, the pharmaceutical composition is administered to a subject in a dosage form selected from the group consisting of a sterile solution, suspension, suppository, tablet, and capsule. In one embodiment, the pharmaceutical composition is administered to a subject in an oral dosage form selected from the group consisting of a tablet, caplet, capsule, lozenge, syrup, liquid, suspension, and elixir. In one embodiment, the pharmaceutical composition is administered to a subject in an oral dosage form selected from the group consisting of a tablet, hard shell capsule, soft gelatin capsule, bead, granule, aggregate, powder, gel, solid, and semisolid.

[0233] In one embodiment, the pharmaceutical composition is administered to the subject in a dosage form selected from a sustained release form, a controlled release form, a delayed release form, and a responsive release form.

[0234] In one embodiment, the pharmaceutical composition is administered to the subject once daily. In one embodiment, the pharmaceutical composition is administered to the subject according to an infrequent dosing regimen (e.g., administered once a week or less frequently). In one embodiment, the pharmaceutical composition is administered to the subject according to a frequent dosing regimen (e.g., administered more than once a week). In one embodiment, the pharmaceutical composition is administered to the subject once a week. In one embodiment, the pharmaceutical composition is administered to the subject once every four weeks. In one embodiment, the pharmaceutical composition is administered to the subject twice a week. In one embodiment, the pharmaceutical composition is administered to the subject once every two weeks. In one embodiment, the pharmaceutical composition is administered to the subject once every three weeks. In one embodiment, the pharmaceutical composition is administered to the subject in a repeating cycle of once a week, once every two weeks, once every three weeks, once every four weeks, or a combination thereof.

[0235] In one embodiment, a treatment method comprises administering to a subject in need of such treatment (i) a first therapeutic agent comprising a compound comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially, and further comprising assaying expression of an endoplasmic reticulum (ER) stress response gene in a biological sample. In one embodiment, the ER stress response gene is selected from the group including, but not limited to, C / EBP homologous protein (CHOP), activating transcription factor 3 (ATF3), and both CHOP and ATF3. In one embodiment, the ER stress response gene is selected from the group including, but not limited to, ATF3, activating transcription factor 4 (ATF4), CHOP, IRE1, binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and X-box binding protein 1 (XBP1). The biological sample may be a tumor, peripheral blood mononuclear cells, or a skin biopsy. Biological samples may be obtained before, during, or after administration of the agents. In one embodiment, the method of treatment further comprises adjusting the dose of the first therapeutic agent to achieve 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 greater than 600% induction of one or more ER stress genes. In one embodiment, the method of treatment further comprises adjusting the dose of the first therapeutic agent to achieve about 50% to about 100%, about 100% to about 150%, about 150% to about 200%, about 200% to about 250%, about 250% to about 300%, about 300% to about 350%, about 350% to about 400%, about 400% to about 450%, about 450% to about 500%, about 500% to about 550%, about 550% to about 600%, or greater than 600% induction of an ER stress gene. In one embodiment, the method of treatment further comprises adjusting the dose of the first therapeutic agent to achieve about 50% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300% to about 400%, about 400% to about 500%, about 500% to about 600%, or greater than 600% induction of the ER stress gene.

[0236] In one embodiment, a method of treatment comprises administering to a subject in need of such treatment (i) a first therapeutic agent comprising a compound comprising an imipyridone, e.g., ONC201 or an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially, and further comprising assaying the expression of proteasome activity in a biological sample. In one embodiment, the proteasome activity can be chymothricin-like, trypsin-like, and / or caspase-like activity. In one embodiment, the biological sample can be a tumor, peripheral blood mononuclear cells, or skin cells. The biological sample can be obtained before, during, or after drug administration. In one embodiment, the method of treatment further comprises adjusting the dose to achieve 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% inhibition of proteasome activity. In one embodiment, the method of treatment further comprises adjusting the dose to achieve at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% inhibition of proteasome activity. In one embodiment, the method of treatment further includes adjusting the dose to achieve 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 greater than 90% inhibition of proteasome activity.

[0237] In one aspect, provided herein are methods of treatment comprising administering to a subject in need of such treatment a first therapeutic agent comprising an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof (e.g., a di- or tri-salt), in combination with a second therapeutic agent, the method comprising: (i) administering a first therapeutic agent to a subject; (ii) waiting until a predetermined waiting time has elapsed and / or until the adverse event has resolved or is resolved after administering the first therapeutic agent to the subject; (iii) administering a second therapeutic agent to the subject, wherein the predetermined waiting time is selected to achieve 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, the predetermined waiting time is determined based on the clearance rate of the first therapeutic agent compound or a metabolite thereof. In one embodiment, the predetermined waiting time is determined by a quantitative assessment of renal function and renal parameters. In one embodiment, the predetermined waiting time is determined by an assay for determining renal function, wherein the assay is selected from the group consisting of serum levels of the first therapeutic agent compound or a metabolite thereof, clearance rate of the first therapeutic agent compound or a metabolite thereof, and 24-hour urinary clearance of the first therapeutic agent compound or a metabolite thereof.

[0238] In one embodiment, the predetermined waiting time is substantially equal to the time required for systemic clearance of the first therapeutic agent compound or its metabolites from the subject's body. In one embodiment, the predetermined waiting time is substantially equal to the time required for renal clearance of the first therapeutic agent compound or its metabolites from the subject's body. In one embodiment, the predetermined waiting time is substantially equal to the time required for hepatic clearance of the first therapeutic agent compound or its metabolites from the subject's body. In one embodiment, the predetermined waiting time is substantially equal to the time required for total clearance of the first therapeutic agent compound or its metabolites from the subject's body. In one embodiment, the predetermined waiting time is about 4 hours. In another embodiment, the waiting time is 1 day. In one embodiment, the waiting time is equal to the time required for total clearance of the first therapeutic agent compound or its metabolites from the subject's body. maxIn other embodiments, the waiting period is until the majority of adverse events have resolved or after the resolution period. In one embodiment, the predetermined waiting period is about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In one embodiment, the predetermined waiting period is in the range of about 1-7 days, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, or about 1-2 days. In one embodiment, the waiting period is up to 3 weeks. The foregoing are considered "treatment periods."

[0239] If the order of administration is reversed, the timing of administration of the first therapeutic agent or agents should be adjusted to coincide with the C of the second therapeutic agent (i.e., the first administered agent). max In one embodiment, administration of the first therapeutic agent can be after most or substantially all of the first agent has been eliminated from the body, or after or during the resolution of the toxic effects of the first agent.

[0240] In one embodiment, the treatment method further includes monitoring the level of the first therapeutic compound or its metabolite in the subject using pharmacokinetic profiling. In some such embodiments, monitoring the level of the first therapeutic compound or its metabolite in the subject using pharmacokinetic profiling includes constructing a pharmacokinetic profile of the first therapeutic compound or its metabolite in the subject using the concentrations of the first therapeutic compound or its metabolite in at least two samples obtained from the subject at time points suitable for constructing the pharmacokinetic profile. In one embodiment involving monitoring the level of the first therapeutic compound or its metabolite in the subject using pharmacokinetic profiling, the sample is collected from the subject at the point of care or point of use by sampling or self-sampling on a point-of-care or point-of-use device or on a matrix suitable for sample storage prior to quantification in a laboratory. In one embodiment, the point-of-care or point-of-use device is each capable of quantifying the first therapeutic compound or its metabolite. In one embodiment involving monitoring the level of a first therapeutic agent compound, or a metabolite thereof, in a subject, one or more samples are obtained from the subject at the point of care or point of use by a biopsy device for analysis at the point of care or point of use device, or for storage prior to laboratory analysis. In one embodiment, the biopsy is taken 3 to 8 hours after administering the first therapeutic agent to the subject. In one embodiment, the biopsy is taken 3 to 24 hours after administering the first therapeutic agent to the subject. In one embodiment, the biopsy is taken 8 to 24 hours after administering the first therapeutic agent to the subject. In one embodiment, the biopsy is taken 2 days after administering the first therapeutic agent to the subject. In one embodiment, the biopsy is taken 3 days after administering the first therapeutic agent to the subject. In one embodiment, the biopsy is taken 4 days after administering the first therapeutic agent to the subject.In one embodiment, the biopsy is performed after a period of 1 to 7 days following administration of the first therapeutic agent.

[0241] In one embodiment, the pharmacokinetic profile comprises pharmacokinetic parameters suitable for guiding the dosing of the first therapeutic agent to the subject being treated. In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent to the subject max is in the range of about 1000 ng / dL to 1500 ng / dL over the treatment period. max is less than 1500 ng / dL and greater than 85 ng / dL over the treatment period. In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent to the subject max is in the range of about 1000 ng / mL to 1500 ng / mL over the course of treatment. max is less than 1500 ng / mL and greater than 85 ng / mL throughout the treatment period.

[0242] In one embodiment, the maximum concentration ("C") of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) after administration is measured. max") are approximately 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 about 1500 ng / dL; 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 a C of about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng / dL to about 15 ng / dL; or a C of about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng / dL to about 15 ng / dL max is.

[0243] In one embodiment, the maximum concentration ("C") of the first therapeutic agent in the blood (whole blood, plasma, or serum) after administration of the first therapeutic agent is measured. max") are approximately 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 about 1500 ng / mL; 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 a C of 5, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng / mL to about 150 ng / mL; or a C of about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng / mL to about 15 ng / mL max is.

[0244] In one embodiment, the maximum concentration ("C") of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) after administration of the first therapeutic agent is measured. max ") is approximately 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 of the first therapeutic agent in the blood (whole blood, plasma, or serum) after administration of the first therapeutic agent is selected from the group consisting of:max ("C max ") 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 C of the first therapeutic agent after administration of the first therapeutic agent is max is selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng / dL.

[0245] In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent max are approximately 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, In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent is selected from the group consisting of 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. max 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 / mL. maxis selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14, or 14.5 ng / mL.

[0246] In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent max are approximately 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, 47 5, 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, 87 5, 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, 1495, or 1500 ng / dL. maxare approximately 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 8, 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 , 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng / dL. In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent max 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.

[0247] In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent maxare approximately 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, 47 5, 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, 87 5, 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, 1495, or 1500 ng / mL. maxare approximately 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 8, 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 , 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng / mL. In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent max 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.

[0248] In one embodiment, the C of the first therapeutic agent after administering the first therapeutic agent to the subject is max is in the range of about 85 ng / dL to 1500 ng / dL; about 8.5 ng / dL to 150 ng / dL; or about 0.85 ng / dL to 15 ng / dL. In one embodiment, the C of the first therapeutic agent in blood (whole blood, plasma, or serum) after administration of the first therapeutic agent is maxare approximately 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, 47 5, 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, 87 5, 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 about 1500 ng / dL;Approximately 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, 1 08, 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 to about 150 ng / dL; or 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 to about 15 ng / dL;

[0249] In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent max is in the range of about 85 ng / mL to 1500 ng / mL, about 8.5 ng / mL to 150 ng / mL, or about 0.85 ng / mL to 15 ng / mL. In one embodiment, the C of the first therapeutic agent after administration of the first therapeutic agent is maxare approximately 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, 47 5, 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, 87 5, 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 / mL to about 1500 ng / mL;Approximately 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, 1 08, 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 / mL to about 150 ng / mL; or 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 about 15 ng / mL;

[0250] In one embodiment, total drug exposure over time, measured as the area under the curve ("AUC") of a plot of the concentration of drug in a subject's blood (whole blood, plasma, or serum) after administration of the drug versus time after administration of the drug, ranges from about 150 ng-hr / mL to about 8000 ng-hr / mL, about 15 ng-hr / mL to about 800 ng-hr / mL, or about 1.5 ng-hr / mL to about 80 ng-hr / mL. In one embodiment, the AUC is less than 8000 ng-hr / mL and greater than or equal to 150 ng-hr / mL. In one embodiment, the AUC is less than 800 ng-hr / mL and greater than or equal to 15 ng-hr / mL. In one embodiment, the AUC is less than 800 ng-hr / mL and greater than or equal to 1.5 ng-hr / mL.

[0251] In one embodiment, the total drug exposure over time is an AUC of about 100 ng-hr / mL to about 8000 ng-hr / mL, about 10 ng-hr / mL to about 800 ng-hr / mL, or about 1 ng-hr / mL to about 80 ng-hr / mL. In one embodiment, the total drug exposure over time is an AUC of 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, 7600, or 7800 ng-hr / mL to about 8000 ng-hr / mL. In one embodiment, the total drug exposure over time is an AUC of 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-hr / mL to about 800 ng-hr / mL. In one embodiment, the total drug exposure over time is an AUC of 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 nghr / mL to about 80 nghr / mL.

[0252] In one embodiment, the total drug exposure over time is an AUC of about 100 ng-hr / mL to about 8000 ng-hr / mL, about 10 ng-hr / mL to about 800 ng-hr / mL, or about 1 ng-hr / mL to about 80 ng-hr / mL. In one embodiment, the total drug exposure over time is an AUC of about 150 ng-hr / 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, 1000, 800, 600, 400, or 200 ng-hr / mL. In one embodiment, the total drug exposure over time is an AUC of about 15 ng-hr / 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-hr / mL. In one embodiment, the total drug exposure over time is an AUC of about 1.5 ng-hr / 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-hr / mL. In one embodiment, the total drug exposure over time is an AUC of about 100 ng-hr / mL to about 200 ng-hr / mL, about 10 ng-hr / mL to about 20 ng-hr / mL, or about 1 ng-hr / mL to about 2 ng-hr / mL.

[0253] In one embodiment, the total drug exposure over time is an AUC selected from about 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 nghr / mL. In one embodiment, the total drug exposure over time is an AUC 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 800 nghr / mL. In one embodiment, the total drug exposure over time is an AUC 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 80 nghr / mL.

[0254] In another aspect, provided herein is a therapeutic method or use of 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, the method comprising: (i) administering to the subject a first therapeutic agent comprising an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof; (ii) monitoring the level of the first therapeutic compound or a metabolite thereof in the subject using pharmacokinetic profiling; and (iii) administering a second therapeutic agent conditional on the level of the first therapeutic agent in the subject. In one embodiment, the monitoring step includes constructing a pharmacokinetic profile of the first therapeutic agent compound or its metabolite in the subject using the concentrations of the first therapeutic agent compound or its metabolite in multiple samples obtained from the subject at time points suitable for constructing the pharmacokinetic profile. In one embodiment, at least two samples are collected at the point of care or point of use by sampling or self-sampling on a point-of-care or point-of-use device or on a matrix suitable for sample storage prior to quantification of the compound or its metabolite by a laboratory. In one embodiment, each point-of-care or point-of-use device is capable of quantifying the compound or its metabolite. In one embodiment, the pharmacokinetic profile includes pharmacokinetic parameters suitable for guiding dosing of the compound or its salt in the subject. In one embodiment, the samples include 2 to 12 samples. In one embodiment, the 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, the pharmacokinetic parameters include AUC, AUC inf , T max , C max The one or more pharmacokinetic parameters include at least one parameter selected from the group consisting of time above a threshold, steady-state concentration, absorption rate, clearance rate, distribution rate, terminal phase T1 / 2, or a parameter obtained from a non-compartmental pharmacokinetic (PK) analysis or a compartmental PK analysis, including a physiologically model-based compartmental PK analysis. In one embodiment, the treatment method further includes generating a report including the subject's pharmacokinetic profile. In one embodiment, the report includes dosing recommendations based on the subject's pharmacokinetic profile. In one embodiment, a reduction in the dosage of ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof is indicated to reduce the risk of toxicity based on one or more pharmacokinetic parameters. In one embodiment, a reduction in the dosage of the compound or salt thereof is indicated based on the time above a threshold, the threshold being the drug concentration above which toxicity occurs, or AUC, AUC inf, mean residence time (MRT), an exponential function defining the pharmacokinetic profile, volume of distribution at steady state (Vss), volume of distribution at terminal phase (Vz), or a combination of groups of pharmacokinetic variables to adequately describe the pharmacokinetic profile. In one embodiment, dose adjustment of the compound or salt thereof is shown to increase efficacy based on one or more pharmacokinetic parameters. In one embodiment, increasing the dosage of the compound or salt thereof increases AUC, AUC inf The concentration of the compound or its salt may be determined based on one or more of the following: MRT, an exponential function defining the pharmacokinetic profile, a steady-state volume of distribution (Vss), a terminal volume of distribution (Vz), or a combination of a group of pharmacokinetic variables to adequately describe the pharmacokinetic profile. In one embodiment, the dose of the compound or its salt is adjusted to within 5% to 25% of a desired target value. In one embodiment, each of the samples is applied to a point-of-care or point-of-use device for determining the concentration of the compound or its metabolite, the point-of-care or point-of-use device including a lateral flow strip having a structure and composition such that application of one or more of the samples to the lateral flow strip causes a portion of the drug in the sample to bind to components of the lateral flow strip, thereby generating a detectable signal proportional to the concentration of the drug in the applied sample. In one embodiment, the sample is applied to a matrix suitable for sample storage prior to quantification by a laboratory. In one embodiment, the sample is stored as a dried blood spot. In one embodiment, the drug concentration is measured by ELISA, LC-MS-MS, LC-UV, or LC-MS. In one embodiment, the pharmacokinetic parameters include steady-state concentration, absorption rate, and terminal T 1 / 2 In one embodiment, at least one of the samples is whole blood.

[0255] IX. Multimodal Treatment Methods

[0256] In one aspect, provided herein is a multimodal treatment method in which administration of an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof, to a subject in need thereof is supplemented with other treatment modalities. In one embodiment, the multimodal treatment comprises administering to the subject a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof, in conjunction with radiation therapy or after radiation therapy has been determined to be ineffective. In one embodiment, the multimodal treatment comprises administering to the subject a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof, in conjunction with radiation therapy, wherein the pharmaceutical composition comprising the imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof, and the radiation therapy are administered simultaneously or sequentially in any order. In one embodiment, the multimodal treatment involves administering a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog, or a pharmaceutically acceptable salt thereof, in a sequential arrangement in conjunction with radiation therapy. In one embodiment, the multimodal treatment involves administering a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog, or a pharmaceutically acceptable salt thereof, simultaneously with radiation therapy. In one embodiment, the multimodal treatment is used to treat cancer. In one embodiment, the multimodal treatment involves administering to a subject with cancer a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog, or a pharmaceutically acceptable salt thereof, and irradiating the cancer cells with a radiation beam. In one embodiment, the multimodal treatment uses conformal radiation therapy (CRT) techniques to deliver a prescribed dose-volume histogram (DVH) to the subject. In one embodiment, the multimodal treatment method uses intensity-modulated radiation therapy (IMRT) to deliver radiation to the cancer cells. In one embodiment, the multimodal treatment method uses techniques to compensate for tumor motion in a subject during treatment (e.g., a dose of radiation must be administered to a chest tumor that moves as the patient breathes).For example, multimodal treatments use four-dimensional computed tomography (4DCT) scanning technology to adjust the delivered radiation field and compensate for tumor motion over the respiratory cycle.

[0257] Any suitable type of radiation can be used with the multimodal treatment method, including gamma radiation with a given fraction, IMRT (intensity-modulated radiation therapy), gamma knife, proton therapy, and brachytherapy. Radiation therapy and administration of an imipyridone, e.g., ONC201, its analogs, or a pharmaceutically acceptable salt thereof, can be used to treat brain tumors, e.g., glioblastoma, or brain metastases from lung cancer. Multimodal treatment can be used to treat lung cancer, pancreatic cancer, rectal cancer, breast cancer, sarcoma, prostate cancer, gynecological malignancies, and lymphoma. Gamma knife is frequently used to treat brain metastases. In one embodiment, multimodal treatment involves the use of proton therapy to treat cancer, including brain tumors, prostate cancer, and any tumors in close proximity to vital organs, where minimizing toxicity to nearby normal tissues is critical.

[0258] In one embodiment, the multimodal treatment comprises administering to a cancer subject a pharmaceutical composition comprising an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof, in combination with an adoptive cell therapy (e.g., CAR-T (JCAR 14, 15, 16, 17, KTE-C19, or CTL019), other T cells (AFM13), or NK (CDNO-109 or NK-92)), either simultaneously or in combination.

[0259] In one embodiment, the multimodal treatment eliminates minimal residual disease without adding to the toxicities caused by treatment with an imipyridone, e.g., ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the multimodal treatment improves the prognosis in the subject receiving the treatment and / or reduces adverse side effects associated with the disease state or condition.

[0260] X. Additional Imipyridone Derivatives, Analogs, and Salts In one aspect, provided herein are analogs of the compound of Formula 10 and methods for making them. Those skilled in the art will understand that the general principles and concepts described above in conjunction with ONC201 and the compound of Formula 10 and their salts, including principles and concepts related to methods and pharmaceutical compositions, apply equally to the following analogs and salts thereof.

[0261] In one embodiment, the analog has the structure of compound (25), which is represented by the formula:

[0262] [ka]

[0263] wherein Y is NR or O, and R, R, R, and R 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, arylsulfonyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic radicals. In one embodiment, R, R, R, and R are optionally substituted. 。 In one embodiment, some or all of the hydrogens in R, R, R, and R are replaced by deuterium. 。 In other embodiments, R1, R2, R3, and R4 are independently H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C1~4 The benzylpiperazine is optionally C 1~4 In still other embodiments, R, R, R, and R are independently selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)CO-4F-Ph.

[0264] In one embodiment, the analog has the structure of compound (26), which is represented by the formula:

[0265] [ka]

[0266] wherein 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 heterocyclic radicals. In one embodiment, R1 and R2 are selected from the group consisting of H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4 Alkoxy, hydroxyl, perhalogenated C1~4 In one embodiment, R is independently selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)CO-4F-Ph. In one embodiment, R2 is independently selected from the group consisting of H, CH3, CH2Ph, CH2-((2-Cl)-Ph), CH2-(2-thienyl), CH2CH2Ph, CH2CH2(4-N-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, and (CH2)3CO-4F-Ph.

[0267] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 wherein p is an integer from 2 to 20, and X is a halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and 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, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 In the formula, p is an integer of 2 to 20, and X is a halogen.

[0268] In one embodiment, R is H. In one embodiment, R is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0269] In one embodiment, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3, and -OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, —CH 3 , —CF 3 , and —OCH 3 .

[0270] In one embodiment, the analog has the structure of compound (27), represented by the following formula:

[0271] [ka]

[0272] wherein R1 is 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 heterocyclic radicals. In one embodiment, R1 is H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4 Alkoxy, hydroxyl, perhalogenated C 1~4 In one embodiment, R is selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)CO-4F-Ph.

[0273] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1wherein p is an integer from 2 to 20, and X is a halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and more preferably F or Cl. In one embodiment, R1 is H. In one embodiment, R1 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is a C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0274] In one embodiment, the analog has the structure of compound (28), which is represented by the formula:

[0275] [ka]

[0276] wherein 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 heterocyclic radicals. In one embodiment, R1 and R2 are selected from the group consisting of H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4Alkoxy, hydroxyl, perhalogenated C 1~4 In one embodiment, R is selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, CHCH(4-N-benzyl-piperazine), and (CH)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-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, and (CH2)3CO-4F-Ph. In one embodiment, when R1 is CH2Ph, R2 is not CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2,4-diF-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(4-CF3-Ph).

[0277] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1wherein p is an integer from 2 to 20, and X is a halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and 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, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 In the formula, p is an integer of 2 to 20, and X is a halogen.

[0278] In one embodiment, R is H. In one embodiment, R is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0279] In one embodiment, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3, and -OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is C 1~4 Alkyl, C 1~4Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, —CH 3 , —CF 3 , and —OCH 3 .

[0280] In one embodiment, the analog has the structure of compound (29), represented by the formula:

[0281] [ka]

[0282] wherein 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 heterocyclic radicals. In one embodiment, R1 and R2 are selected from the group consisting of H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4 Alkoxy, hydroxyl, perhalogenated C 1~4In one embodiment, R is selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)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-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, and (CH2)3CO-4F-Ph. In one embodiment, when R1 is CH2Ph, R2 is not CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2,4-diF-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(4-CF3-Ph).

[0283] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 wherein p is an integer from 2 to 20, and X is halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and 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, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C pX 2p+1 , -OCX3, or -OC p X 2p+1 In the formula, p is an integer of 2 to 20, and X is a halogen.

[0284] In one embodiment, R is H. In one embodiment, R is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0285] In one embodiment, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3, and -OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, —CH 3 , —CF 3 , and —OCH 3 .

[0286] In one embodiment, the analog has the structure of compound (30), represented by the formula:

[0287] [ka]

[0288] wherein 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 heterocyclic radicals. In one embodiment, R1 and R2 are selected from the group consisting of H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4 Alkoxy, hydroxyl, perhalogenated C 1~4In one embodiment, R is selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)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-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, and (CH2)3CO-4F-Ph. In one embodiment, when R1 is CH2Ph, R2 is not CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2,4-diF-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(4-CF3-Ph).

[0289] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 wherein p is an integer from 2 to 20, and X is halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and 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, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C pX 2p+1 , -OCX3, or -OC p X 2p+1 In the formula, p is an integer of 2 to 20, and X is a halogen.

[0290] In one embodiment, R is H. In one embodiment, R is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0291] In one embodiment, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is selected from the group consisting of halo, hydroxyl, C 1~4 Alkyl, C 1~4 Alkoxy or perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3, and -OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, —CH 3 , —CF 3 , and —OCH 3 .

[0292] In one embodiment, the analog has the structure of compound (31), represented by the formula:

[0293] [ka]

[0294] wherein 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 heterocyclic radicals. In one embodiment, R1 and R2 are selected from the group consisting of H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C 1~4 Benzylpiperazine, and C 1~4 alkylthienyl, wherein C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketones, and C 1~4 The benzylpiperazine is optionally C 1~4 Alkyl, C 1~4 Alkoxy, hydroxyl, perhalogenated C 1~4In one embodiment, R is selected from the group consisting of H, CH, CHPh, CH-((2-Cl)-Ph), CH-(2-thienyl), CHCHPh, CHCH(4-N-benzyl-piperazine), CH-(2,4-diF-Ph), CH-((2-CH)-Ph), CHCHOHPh, and (CH)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-benzyl-piperazine), CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, and (CH2)3CO-4F-Ph. In one embodiment, when R1 is CH2Ph, R2 is not CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2-CH3-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(2,4-diF-Ph). In one embodiment, R1 is CH2Ph and R2 is CH2-(4-CF3-Ph).

[0295] 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: -CH3, -NO2, -OCH3, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, or -OC p X 2p+1 wherein p is an integer from 2 to 20, and X is a halogen, including F, Cl, Br, or I, preferably F, Cl, or Br, and 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, -CXH2, -CX2H, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X2p+1 , -OCX3, or -OC p X 2p+1 In the formula, p is an integer of 2 to 20, and X is a halogen.

[0296] In one embodiment, R is H. In one embodiment, R is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 It is substituted with alkyl or halo.

[0297] In one embodiment, R2 is a substituted or unsubstituted arylalkyl, such as a benzyl or phenylethyl group. In one embodiment, arylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the arylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3, and -OCH3. In one embodiment, R2 is a substituted or unsubstituted heterocycloalkylalkyl, such as a morpholinoalkyl or piperazinylalkyl group. In one embodiment, R2 is a substituted or unsubstituted heteroarylalkyl, such as an isoxazolidinylmethyl or pyridylmethyl group. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, —CH 3 , —CF 3 , and —OCH 3 .

[0298] In one embodiment, provided herein is a compound of formula (100), represented by the formula:

[0299] [ka]

[0300] wherein R and R are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, and acyl radicals. In one embodiment, R is CHPh and R is CH-(2-CH-Ph), which is a linear isomer of ONC201 having the formula:

[0301] [ka]

[0302] It lacks anti-cancer activity (Jacob et al., Angew. Chem. Int. Ed., (2014) 53:6628; Wagner et al., Oncotarget (2015) 5(24):12728). TIC-10 is a CXCR7 agonist. CXCR7 agonists can be used for liver regeneration and the prevention or treatment of liver fibrosis.

[0303] In one embodiment, R1 and R2 are H, C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl 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, independently selected from the group consisting of C 1~4 Alkyl, C 1~4 Alkylphenyl, C 1~4 Alkyl phenyl ketone, C1~4 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 The alkylpyrazinyl is optionally C 1~4 Alkyl, C 1~4 Alkoxyl, hydroxyl, perhalogenated C 1~4 In one embodiment, R and / or R are substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment, heteroarylalkyl is C 1~4 Alkylpyrrolyl, C 1~4 Alkylfuryl, C 1~4 Alkylpyridyl, C 1~4 Alkyl-1,2,4-thiadiazolyl, C 1~4 Alkylpyrimidyl, C 1~4 Alkylthienyl, C 1~4 Alkylisothiazolyl, C 1~4 Alkyl imidazolyl, C 1~4 Alkyltetrazolyl, C 1~4 Alkylpyrazinyl, C 1~4 Alkylpyrimidyl, C 1~4 Alkylquinolyl, C 1~4 Alkylisoquinolyl, C 1~4 Alkylthiophenyl, C 1~4 Alkylbenzothienyl, C 1~4 Alkylisobenzofuryl, C 1~4 Alkylpyrazolyl, C 1~4 Alkyl indolyl, C 1~4 Alkylpurinyl, C 1~4 Alkylcarbazolyl, C 1~4 Alkylbenzimidazolyl, and C 1~4 alkylisoxazolyl.

[0304] 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, -CXH2, -CX2H, C2-C4 alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1 , -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , S.R. m , N.R. m R n , N.R. 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, 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.

[0305] XI. Reference examples

[0306] It should be understood that the following description and examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The following examples are intended to illustrate the disclosed embodiments and should not be construed as limitations thereon. Additional compounds other than those described below can be made by the following reaction schemes or suitable variations or modifications thereof.

[0307] Reference 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). The reaction mixture was stirred at 70 °C under argon for 90 min. The solution was cooled to room temperature, filtered through a sintered funnel, washed with cold dioxane (2 mL), and dried under vacuum. Compound 4·HI (R2 = 2-chlorobenzyl) was obtained as a white solid (242 mg, 72%) and used without further purification.

[0308] Reference 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. The reaction mixture was stirred under argon at 7 °C for 30 minutes. Methylene chloride (5 mL) was then added and the mixture was stirred for an additional 5 minutes. The reaction mixture was extracted with methylene chloride (2 × 2.5 mL). The organic layer was dried over anhydrous NaSO, filtered, and evaporated. The resulting free base (150 mg, 100%) was obtained as a viscous liquid and was used in the next reaction without further purification. MS (ESI) 210 (M+H).

[0309] Reference Example 3. Synthesis of methyl-1-benzyl 4-oxo-3-piperidinecarboxylate (compound (6)). To stirred methyl-1-benzyl 4-oxo-3-piperidinecarboxylate hydrochloride (5.7 g, 20 mMol) in ethyl acetate (50 mL) was added triethylamine (6 mL) at 7 °C. The reaction mixture was stirred at 7 °C for 30 minutes under an argon atmosphere. The reaction mixture was extracted with ethyl acetate (2 × 50 mL) and washed with water (50 mL). The organic layer was dried over anhydrous NaSO, filtered, and evaporated. The free base residue (5, R = benzyl) obtained as a viscous oil was used in the next reaction without further purification. MS (ESI) 248 (M + H).

[0310] Reference Example 4. Synthesis of ONC202 (compound (14)) To a solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mMol) and methyl 1-benzyl 4-oxo-3-piperidinecarboxylate (5, R = benzyl) (195 mg, 0.79 mMol) in 1-butanol (2 mL) was added PPTS (10 mg), and the mixture was stirred at room temperature for 48 h. The reaction mixture was then refluxed at 125-130 °C for 2 h. The solvent was removed in vacuo, extracted with ethyl acetate (10 mL), and washed with saturated sodium bicarbonate solution (2 × 10 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 the TFA salt of ONC202 as a white solid (228 mg, 50% yield), MS (ESI) 407 (M+H).

[0311] Starting with different benzylamines, the same process was used to prepare various analogs, e.g., ONC203, 204, 205, 206, 912, 210, 211, 212, 213, 214, 217, 218, 219, 220, 221, 222, 223, 224, 225, and 226.

[0312] Reference Example 5. Synthesis of ONC207 (compound (19)) Dimethyl carbonate (4.32 g, 48.0 mMol) was added dropwise to a suspension of 60% sodium hydride (3.5 g, 88 mMol) in dry toluene (50 mL) under nitrogen at room temperature for 0.5 h. 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 over 1 h. The reaction mixture was stirred at the same temperature for 3 h, 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 adjusted to pH 8 with 5% sodium hydroxide solution. The organic layer was separated, and the aqueous layer was extracted with toluene (20 mL). The combined organic layers were 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%), which was used in the next reaction without further purification.

[0313] 2-Methylbenzylamino-2-imidazoline (190 mg, 1 mMol), methyl 1-tert-butoxycarbonyl-4-oxo-3-piperazinecarboxylate (315 mg, 1.1 mMol) in 1-butanol (2 mL) were added to PPTS (10.0 mg), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed at 125-130 °C for 2 hours. The solvent was removed under vacuum, extracted with ethyl acetate (10 mL), and washed with saturated sodium bicarbonate solution (2 × 10 mL) and water (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated. The crude free base was cleaved with 10% trifluoroacetic acid in dichloromethane and purified by RP HPLC (10%-40% acetonitrile / water) to afford the TFA salt of ONC207 (262 mg, 50%) as a white solid MS (ESI) 297 (M+H).

[0314] Reference Example 6. Synthesis of ONC209 (compound (21)) A mixture of ONC207 (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 h. The solvent was removed in vacuo, extracted with ethyl acetate (10 mL), and washed with water (5 mL). The organic layer was dried over anhydrous NaSO, filtered, and evaporated. The crude free base was purified by RP HPLC (10%-40% acetonitrile / water) to give the TFA salt of ONC209 (62 mg, 50%) as a white solid MS (ESI) 401 (M+H).

[0315] Starting with different halides, the same process was used to obtain ONCs 215 and 214. Starting with different benzylamines, compounds 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236 were prepared using similar processes from Reference Examples 1 and 5. The intermediate compounds where R1 is H are then treated with different halides as described above.

[0316] Compound ONC216 was prepared from ONC215 by treatment with TFA.

[0317] Compound (72) was prepared by reacting the precursor NH compound, prepared similarly to Reference Example 5, and treating it with styrene oxide.

[0318] Reference Example 7. Synthesis of ONC208 (compound (20)) To a solution of 2-methylbenzylamino-2-imidazoline (190.0 mg, 1.0 mmol) and methyl 1-methyl 4-oxo-3-piperidinecarboxylate (185.0 mg, 1.0 mmol) in 1-butanol (2.0 mL) was added PPTS (10.0 mg), and the mixture was stirred at room temperature for 48 h. The reaction mixture was then refluxed at 125-130 °C for 2 h. The solvent was removed in vacuo, extracted with ethyl acetate (10 mL), and washed with saturated sodium bicarbonate solution (2 × 10 mL) and water (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated. The crude free base was purified by HPLC 10%-40% acetonitrile and water to give the TFA salt of ONC208 (270.0 mg, 50%) as a white solid with MS (ESI) 311 (M+H).

[0319] Reference Example 8. Synthesis of ONC201 (compound (1)) Compound (3) (239.7 g, 0.845 mol, 1.6 equiv.) was added portionwise to 800 mL of stirred saturated NaHCO3 in a 2 L round-bottom flask. n-Butanol (500 mL) was added to the resulting mixture, and the mixture was stirred for 30 min 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, N2 inlet, thermocouple, condenser, and Dean-Stark trap. Compound (5) (100 g, 0.528 mol, 1 equiv.) 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 heated to reflux for 6 h. Water in the reaction mixture was separated into the Dean-Stark trap as needed. The reflux temperature was increased from 93 °C to 118 °C. The progress of the reaction was monitored by HPLC. The reaction was stopped when the peak area of compound (1) on HPLC with reaction time remained constant.

[0320] Reference Example 9. Synthesis of the disalt of ONC201 (compound (1)·2HCl) Without isolating compound (1), the reaction mixture from Reference 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 (500 mL × 2) and transferred to a 3 L three-neck round-bottom flask equipped with a mechanical stirrer, N2 inlet, thermocouple, condenser, and Dean-Stark trap. While stirring the reaction mixture, 1 N HCl in dioxane-MTBE solution was added dropwise until no solid precipitated from the reaction mixture upon addition of HCl (4 N HCl in dioxane: 300 mL, 1.2 mol, 2.27 equiv.; MTBE: 1200 mL). The reaction mixture was heated to reflux at 60-65 °C for 2 h. Water was separated into a Dean-Stark trap as needed. After cooling to room temperature, the solid was filtered through a sintered glass 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 give 200 g of a yellow solid.

[0321] The above solid (200 g) was added to a 2 L three-necked round-bottom flask equipped with a mechanical stirrer, N2 inlet, thermocouple, and condenser, followed by the addition of ethanol (1000 mL). The mixture was heated to reflux at 78 °C for 2 hours. After cooling to room temperature, the solid was filtered through a sintered glass funnel and washed with ethanol (200 mL x 3). The wet solid was dried in a vacuum oven at 85 °C for 3 days until the residual solvent met specifications. 120 g of compound (2) was obtained as a white solid in 49% yield with an HPLC purity of 99.7%.

[0322] Reference Example 10. Activity of imipyridones Based on the above synthesis, several imipyridones were prepared. Using each compound, the viability of human cancer cells was measured 72 hours after treatment. The change in efficacy (compared to ONC201) was determined and is shown in Table 3.

[0323] [Table 3]

[0324] [ka]

[0325] IC of ONC201 and ONC212 (5 nM to 5 μM, 72 h) during treatment of several acute myeloid leukemia (AML) cell lines (n = 3) 50 The results are shown in Table 4.

[0326] [Table 4]

[0327] Cell viability was measured for MV411 AML cells (n = 3) treated with ONC212 and cytarabine (5 nM to 5 μM, 24 h) (Figure 10A). Additionally, cell viability was measured for MOLM14, MV411 AML cells, MRC5 lung fibroblasts, and Hs27a bone marrow cells (n = 3) treated with ONC212 (5 nM to 5 μM, 72 h) (Figure 10B). Cell viability was measured for MOLM14 and MV411 AML cells treated with ONC212 (250 nM) for 4, 8, 24, 48, 72, and 96 h. The ONC212 medium was replaced with fresh medium at these time points, and cell viability was determined at 96 h for all samples (n = 2) (Figure 10C).

[0328] Additionally, a single oral or intraperitoneal dose of ONC212 administered to mice bearing human colon cancer xenografts resulted in significant tumor volume reduction compared with vehicle-treated control cohorts. ONC212 is well tolerated in mice at doses up to 225 mg / kg, providing a wide therapeutic window.

[0329] Furthermore, ONC212 demonstrated efficacy in an ONC201-resistant AML xenograft model (Figure 11). MV411 AML cells (5x10 6 ) were implanted subcutaneously into the flanks of athymic nude mice. ONC212 and ONC201 were administered orally (PO) as indicated. Tumor volumes (A and B) and body weights (C) (n=10) were measured on the indicated days. *indicates p<0.05 vs. vehicle.

[0330] The efficacy of ONC212 in AML was evaluated in vitro and was up to 400-fold more potent than ONC201 (Table 4). ONC212 was also effective in AML cells resistant to standard-of-care cytarabine (Figure 10A). Despite this robust improvement in efficacy, ONC212 maintains a wide therapeutic window in vitro and is nontoxic to normal cells at effective concentrations (Figure 10B). An 8-hour exposure to ONC212 at 250 nM was sufficient to cause a robust decrease in cell viability in MOLM14 and MV411 AML cells (Figure 10C). At least 24-48 hours of exposure with ONC201 was required for efficacy.

[0331] The efficacy of ONC212 was determined in a leukemia xenograft model with MV411 AML cells, which are resistant to standard-of-care cytarabine (Figure 11). ONC212 at 50 mg / kg orally administered once weekly significantly reduced leukemia xenograft tumor growth, whereas ONC201 was not effective in this model at similar doses (Figure 11A). Interestingly, ONC212 administered every other week at 25 mg / kg and every / every other week at 5 mg / kg were not effective (Figure 11B). None of these ONC212 administration regimens was associated with weight loss (Figure 11C) or macroscopic observations.

[0332] ONC212 25 mg / kg represents the NOAEL in non-GLP oral single-dose studies in mice and rats, and is also the effective dose in mouse xenograft studies. ONC212 is approximately 10 times more toxic than ONC201 (NOAEL 225 mg / kg in non-GLP oral single-dose studies in rats).

[0333] [ka]

[0334] ONC206 demonstrated efficacy in a Ewing sarcoma xenograft model. MHH-ES-1 Ewing sarcoma cells (5 × 10 6 ) were implanted subcutaneously into the flanks of athymic nude mice. ONC206 (PO) and methotrexate (IV) were administered on days 1 and 13, as indicated. Tumor volume (FIG. 12A) and body weight (FIG. 12B) (n=4) were measured on the indicated days.

[0335] In addition, the IC values of ONC201 and ONC206 upon treatment of several cell lines (n=3) were 50 (5 nM to 5 μM, 72 hours) were determined and are shown in Table 5 below.

[0336] [Table 5]

[0337] ONC206 demonstrated up to a 20-fold improvement in in vitro efficacy compared to ONC201, with no in vitro toxicity to normal cells at therapeutic doses (Table 5). ONC206 demonstrated only a 2-fold increase in overall toxicity (NOAEL 125 mg / kg) compared to ONC201 (NOAEL 225 mg / kg) in a non-GLP oral single-dose rat study. In vivo efficacy in a nontoxic Ewing's sarcoma model was demonstrated (Figure 12). The efficacy of ONC206 was comparable to the chemotherapy methotrexate, although the chemotherapy was associated with weight loss.

[0338] [ka]

[0339] In vitro profiling of GPCR activity using a heterologous reporter assay for arrestin recruitment, a hallmark of GPCR activation, showed that ONC213 selectively targets DRD2 / 3 and GPR132 / 91. Dual targeting of DRD2 / 3 and GPR132 / 91 represents a novel strategy for anticancer efficacy without toxicity. ONC213 is a DRD2 / 3 inhibitor and a GPR132 / 91 agonist. The DRD2 / 3 potency of ONC213 is greater than that of ONC201 but less than that of ONC206. The GPR132 potency of ONC213 is less than that of ONC212. Specifically, ONC213 exhibited similar in vitro anticancer efficacy in HCT116 / RPMI8226 cancer cells as ONC212, but its in vitro toxicity against normal cells was reduced compared to ONC212 (Figure 13). The safety profile of ONC213, confirmed in a mouse MTD study using a NOAEL of 75 mg / kg, is three times that of ONC212 (25 mg / kg). The GPR91 agonist activity of ONC213 offers opportunities for immunology, immuno-oncology, and hematopoietic applications (Nature Immunology 9:1261(2008), J Leukoc Biol. 85(5):837(2009)).

[0340] [ka]

[0341] In vitro profiling of GPCR activity using a heterologous reporter assay for arrestin recruitment, a hallmark of GPCR activation, showed that ONC237 selectively targets GPR132 and DRD5. ONC237 is a GPR132 agonist and DRD5 antagonist, and exhibits increased anti-cancer efficacy (IC) compared to ONC201. 50 31.2 μM). This data indicates that combining GPR132 agonism with DRD5 (D1-like dopamine receptor) antagonism results in poorer anticancer efficacy compared to ONC213, which combines GPR132 agonism and DRD2 / 3 antagonism.

[0342] [ka]

[0343] In vitro profiling of GPCR activity using a heterologous reporter assay for arrestin recruitment, a hallmark of GPCR activation, demonstrated that ONC236 is a highly selective GPR132 agonist. ONC236 exhibited comparable anticancer efficacy (IC) to ONC212 (10 nM), better than ONC206 / ONC201. 50 88 nM) and the completeness of the response in HCT116 cells is superior to ONC201 but not to ONC212.

[0344] [ka]

[0345] In vitro profiling of GPCR activity using a heterologous reporter assay for arrestin recruitment, a hallmark of GPCR activation, demonstrated that ONC234 is a broad-spectrum and potent GPCR targeting small molecule. ONC234 hits several GPCRs, including adrenergic, histamine, serotonin, CHRM, CCR, and DRD2 / 5 receptors, as well as antagonist activity against CXCR7 agonist activity. ONC234 exhibited similar anti-cancer efficacy (IC) to ONC206. 50 234 nM), with the same completeness of response as ONC212 and superior to ONC201 in HCT116 cells.

[0346] Reference Example 11. GPCR Antagonism of ONC201 ONC201 was evaluated in a whole-cell functional assay of β-arrestin G protein-coupled receptor (GPCR) activity, which directly measures dopamine receptor activity by detecting the interaction of β-arrestin with activated GPCR, which serves as a reporter. Cell lines overexpressing reporter constructs for each dopamine receptor (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5) were grown from freezer stocks. Cells were seeded in a total volume of 20 μL into white-walled 384-well microplates and incubated with antagonists at 37°C prior to testing, followed by EC204. 80 Agonist challenges were performed at concentrations of 5x EC 2.0 and 5x EC 2.0. Intermediate dilutions of sample stocks were made to generate 5x samples in assay buffer. 3.5 μL of 5x sample was added to cells and incubated at 37°C or room temperature for 30 minutes. The vehicle concentration was 1%. 5 μL of 6x EC 2.0 in assay buffer was added to cells and incubated at 37°C or room temperature for 30 minutes. 80 Agonists were added to the cells and incubated at 37°C or room temperature for 90 or 180 minutes before assay readout. % antagonism was calculated using the following formula: Antagonism = 100% x (1 - (mean RLU of test sample - mean RLU of vehicle control)) / (EC 80 Mean RLU of control - Mean RLU of vehicle control).

[0347] Reference Example 12: Selective antagonism of DRD2 by ONC201. ONC201 is a first-in-class small molecule discovered in a phenotypic screen for p53-independent inducers of tumor-selective pro-apoptotic pathways. Based on its remarkable efficacy and favorable safety profile in aggressive and refractory tumors, oral ONC201 is being evaluated as a novel therapeutic agent in five early-phase clinical trials for selected advanced cancers.

[0348] This reference example reports the prediction and validation of selective direct molecular interactions between ONC201 and specific dopamine receptor family members. Experimental GPCR profiling demonstrated that ONC201 selectively antagonizes the D2-like dopamine receptor subfamily but not the D1-like dopamine receptor subfamily. Reporter assays in heterologous expression systems revealed that ONC201 selectively antagonizes both the short and long isoforms of DRD2 and DRD3, with weaker potency against DRD4 and no antagonism of DRD1 or DRD5. Increased prolactin secretion is a clinical hallmark of DRD2 antagonism by several psychiatric drugs that potently target this receptor. In a first-in-human trial with advanced solid tumors, ELISA measurements in peripheral blood from patients treated with ONC201 determined that 10 / 11 patients evaluated showed induction of prolactin (mean 2-fold increase).

[0349] Using the TCGA database, the D2-like dopamine receptor subfamily, particularly DRD2, was found to be widespread and selectively overexpressed in several malignancies. Preclinical reports indicate that DRD2 inhibition confers antitumor efficacy without killing normal cells through ATF4 / CHOP induction and inhibition of Akt and ERK signaling, all attributes of ONC201.

[0350] method ONC201 dihydrochloride was obtained from Oncoceutics. Kinase inhibition assays against the kinome were performed as described (see Anastassiadis et al., Nat Biotech 29:1039 (2011)). GPCR arrestaristin recruitment and cAMP-regulated reporter assays were performed as described (see McGuinness et al., Journal of Biomolecular Screening 14:49 (2009)). PathHunter™ (DiscoveRx) β-arrestin cells expressing one of several GPCR targets were seeded in 384-well white solid-bottom assay plates (Corning 3570) at 5,000 cells per well in a volume of 20 μL in the appropriate cell seeding reagent. Cells were incubated at 37°C and 5% CO2 for 18–24 hours. Samples were prepared in buffer containing 0.05% fatty acid-free BSA (Sigma). For agonist mode testing, samples (5 μL) were added to pre-seeded cells and incubated at 37°C, 5% CO for 90 minutes. For antagonist mode testing, samples (5 μL) were added to pre-seeded cells and incubated at 37°C, 5% CO for 30 minutes, followed by EC 80 Agonist (5 μL) was added for 90 minutes at 37°C, 5% CO2. For Schild analysis, sample (5 μL) was added to pre-seeded cells and incubated for 30 minutes at 37°C, 5% CO2, followed by the addition of serially diluted agonist (5 μL) for 90 minutes at 37°C, 5% CO2. Control wells defining the maximum and minimum responses for each assay mode were run in parallel. Arrestin recruitment was measured by adding 15 μL of PathHunter detection reagent, incubated for 1-2 hours at room temperature, and read on a Perkin Elmer Envision Plate Reader. For agonist and antagonist testing, data were normalized for percent efficacy using appropriate controls and expressed as a sigmoidal dose-response (variable slope), Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC 50 -X) *The data were fitted to a logarithmic HillSlope (X) where X is the log compound concentration. For Schild analysis, data were normalized for percent efficacy using appropriate controls, Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC-X) * HillSlope)), Antag=1+(B / (10^(-1 * pA2)))^SchildSlope and LogEC = Log(EC 50 * Antag) using global fitting to Gaddum / Schild EC 50 Shift adapted. EC 50 / I C 50 Analyses were performed with the CBIS data analysis suite (Cheminnovation), and Schild analyses were performed with GraphPad Prism 6.0.5.

[0351] result ONC201 is a small molecule in Phase II clinical trials for selected advanced cancers. It was discovered in a phenotypic screen for p53-independent inducers of the pro-apoptotic TRAIL pathway. Although the contribution of ONC201-induced ATF4 / CHOP upregulation and inactivation of Akt / ERK signaling to its anticancer activity has been characterized (Allen et al., Science Translational Medicine 5, 171ra117-171ra117 (2013)), its molecular binding target has remained elusive.

[0352] In vitro profiling of GPCR activity using a heterologous reporter assay for arrestin recruitment, a hallmark of GPCR activation, showed that ONC201 selectively antagonized the D2-like (DRD2 / 3 / 4), but not the D1-like (DRD1 / 5), dopamine receptor subfamily (Figure 1). Antagonism of alpha-adrenergic receptors or other GPCRs was not observed under the conditions evaluated. Within the DRD2 family, ONC201 antagonized both the short and long isoforms of DRD2 and DRD3, with weaker potency against DRD4. Further characterization of ONC201-mediated antagonism of arrestin recruitment to DRD2L determined a dissociation constant of 2.9 μM for ONC201, equivalent to its effective dose in many human cancer cells (Gaddum / Schild EC). 50 Confirmatory results were obtained for cAMP modulation in response to ONC201, another measure of DRD2L activation, as assayed by shift analysis. The ability of dopamine to reverse the dose-dependent antagonism of ONC201 up to 100 μM suggests direct competitive antagonism of DRD2L. Consistent with ONC201 specificity predicted by BANDIT, no significant interactions were identified between ONC201 and nuclear hormone receptors, the kinome, or other drug targets for FDA-approved cancer therapies. Interestingly, a biologically inactive constitutive isomer of ONC201 (Wagner et al., Oncotarget 5:12728 (2014)) does not inhibit DRD2L, suggesting that antagonism of this receptor may be related to its biological activity. In summary, these studies confirm that ONC201 selectively antagonizes the D2-like dopamine receptor subfamily, which appears to be a promis...

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