Pharmaceutical composition and use thereof for treating glioma

Abstract

Emipridone selectively modulates class A G protein coupled receptors (GPCRs), such as the D2-like subfamily of dopamine receptors, and can be used to treat conditions and disorders in need of such modulation, such as cancer. In particular, the cancers involve the midline structure of the brain, cancers with histone H3 mutations, or both. In addition, methods of identifying whether a subject having these conditions is likely to respond to a treatment regimen, such as emiridone administration, are provided. In addition, methods of assessing the effectiveness of a treatment regimen, such as administration of emiridone, monitoring subjects suffering from these conditions, or providing prognosis for subjects suffering from these conditions are also provided.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 370,520, filed on August 5, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] 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 a founding member of a class of anticancer compounds known as imipridones in Phase II clinical trials for a variety of 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 involved in the integrated stress response, leading to DR5 tumor upregulation and dual Akt / ERK inactivation, and subsequent Foxo3a activation leading to upregulation of the death ligand TRAIL. ONC201 is orally active in animal models with infrequent dosing, causes sustained pharmacodynamic effects, and is not genotoxic. The first-in-human clinical trial of ONC201 in advanced aggressive refractory solid tumors demonstrated that it was well tolerated.

[0004] H3 K27M (a specific mutation of the histone H3 protein) was found as an oncogenic mutation in the context of cancers that involve midline structures of the brain, i.e., the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. Due to the location of these tumors (a brain region involved in key physiological functions), these tumors have historically been inoperable (particularly in the brainstem where the pons are located). This means that until recently, midline gliomas such as diffuse intrinsic pontine glioma (DIPG) were diagnosed only on the basis of radiographic images. Advances in neurosurgical techniques and increased parental consent for postmortem tumor tissue extraction have resulted in the availability of sufficient biological specimens to allow systematic genomic evaluation of DIPG and other midline gliomas. Gliomas in the midline of the brain are among the most aggressive types of primary malignant brain cancers. The disease originates from glial cells, which form tissues that surround and protect other nerve cells found in the brain and spinal cord.

[0005] Standard treatment for midline gliomas involves neurosurgery followed, when feasible, by fractionated external beam radiotherapy.Gliomas in the midline of the brain are considered some of the most lethal forms of cancer due to their location in the brain, aggressiveness, and low survival times.

[0006] No medical therapy has been shown to prolong survival in adult and pediatric glioma patients with H3 K27M mutations. Standard of care DIPG treatment (55 Gy focal radiation fractionated over 6 weeks) is associated with an overall survival of 9 to 11 months. Adult H3 K27M gliomas are typically treated with the same regimen as glioblastoma involving concomitant radiation and maintenance temozolomide. Despite its use in treating this newly defined disease in adults, the efficacy of this regimen has not been specifically evaluated in adult patients with mutant H3 K27M gliomas.

[0007] The functions of histones are primarily protein-DNA and protein-protein interactions; they do not act as enzymes, which represents the majority of targeted cancer therapies (kinases, HDAC inhibitors, etc.). Therefore, no therapy directly targets the mutant H3 protein itself (such as the case for mutant BRAF); instead, therapeutic efforts have focused on targeting features of tumor cells with H3 K27M mutations, such as their epigenetic and transcriptional dependencies. Inhibition of proteins involved in epigenetics (such as histone deacetylases, histone demethylases, or bromodomains) has produced efficacy in preclinical models, however, its ability to improve clinical outcomes has not yet been shown.

[0008] The main challenge of effective H3 K27M glioma therapy is the need to penetrate the blood-brain barrier, which is a rare feature of current cancer therapies. The location of these tumors in midline brain structures (which have been shown to be more difficult to penetrate than other brain locations) further enhances this. H3 K27M mutations also tend to occur in midline gliomas with dopamine present in the tumor environment and ubiquitous DRD2 expression.

[0009] The lack of treatment for H3 K27M gliomas results in a large unmet medical need regarding disease control, symptom relief, and survival. Patients with recurrent disease have no treatment options after radiation, but have a demonstrated survival benefit. Summary of the invention

[0010] In one aspect, provided herein is a compound of formula (10): Wherein R1 and R2 are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio and acyl. 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)(ONC201). In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(2,4-diF-Ph)(ONC206). In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(4-CF3-Ph)(ONC212). 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).

[0011] In another aspect, provided herein are methods for treating or preventing cancer in a subject in need thereof, the methods comprising: administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of compound (1)

[0012]

[0013] or a pharmaceutically acceptable salt thereof, wherein the cancer involves midline structures of the brain and has a histone H3 K27M mutation.

[0014] In another aspect, provided herein are methods for treating or preventing cancer in a subject in need thereof, the methods 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 are methods for treating or preventing cancer in a subject in need thereof, the methods 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 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, the method comprising: administering to a subject in need of such treatment a pharmaceutical composition according to a weekly regimen cycle of oral administration, the pharmaceutical composition comprising an amount of compound (1)

[0017]

[0018] or a pharmaceutically acceptable salt thereof, the weekly regimen cycle comprising two doses per week on two consecutive days, Day 1 and Day 2, followed by a rest period, Day 3 to Day 7.

[0019] On the one hand, the weekly regimen cycle is repeated. On the one hand, the dosage is one or more of 125 mg, 250 mg, 375 mg, 500 mg or 625 mg. On the one hand, the dosage is 625 mg. On the one hand, the selected dosage is calculated based on the subject's body weight (kg) in mg / kg. On the one hand, one or more subsequent doses are reduced from the initial dose. On the one hand, each dose is administered to the subject on an empty stomach, without food, within two (2) hours before or after each dose. On the one hand, the weekly regimen is modified after one or more cycles to include a dose once a week, day 1, followed by a rest period, day 2 to day 7. On the one hand, the method further includes performing radiation therapy before starting the weekly regimen cycle. On the one hand, the radiation is 54 to 60 Gy at 1.8 to 2.2 Gy / fraction. On the one hand, the cancer involves the midline structure of the brain. On the one hand, the cancer has a histone H3 K27M mutation. On the one hand, the cancer is a central nervous system tumor, a brain tumor, a glioma, a peripheral nervous system tumor, a pheochromocytoma, a paraganglioma, an adrenocortical carcinoma, an adrenal tumor, and a neuroendocrine tumor. On the one hand, the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. On the one hand, the histone H3 K27M mutation is H3.3K27M or H3.1 K27M. On the one hand, DRD2 is overexpressed in the tissue, DRD5 is underexpressed in the tissue, or both. On the one hand, the subject is a human. On the one hand, the subject is a pediatric subject and the dosage is calculated based on the weight of the pediatric subject. On the one hand, the dosage is rounded to the nearest 125mg increment. On the one hand, administration begins 2, 3, 4, 5, or 6 weeks after radiation is completed. On the one hand, administration includes one or more doses of temozolomide. On the one hand, the method further includes administering one or more doses of bevacizumab. In one aspect, the method further comprises performing one or more radiation treatments after initiating the administration.

[0020] One embodiment of the present disclosure includes a use or compound for use in the preparation of a medicament for the treatments outlined herein.

[0021] In one embodiment, the present disclosure includes a compound (1)

[0022]

[0023] 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 for oral administration comprising two doses per week on two consecutive days, Day 1 and Day 2, followed by a rest period, Day 3 to Day 7.

[0024] On the one hand, the weekly regimen cycle is repeated. On the one hand, the dosage is one or more of 125 mg, 250 mg, 375 mg, 500 mg or 625 mg. On the one hand, the dosage is 625 mg. On the one hand, the selected dosage is calculated based on the subject's body weight (kg) by mg / kg. On the one hand, one or more subsequent doses are reduced from the initial dose. On the one hand, each dose is administered to the subject on an empty stomach, without food, within two (2) hours before or after each dose. On the one hand, the weekly regimen is modified after one or more cycles to include a dose once a week, day 1, followed by a rest period, day 2 to day 7. On the one hand, the use or compound for use provides radiation therapy before starting the weekly regimen cycle. On the one hand, the radiation is 54 to 60 Gy at 1.8 to 2.2 Gy / fraction. On the one hand, the cancer involves the midline structure of the brain. On the one hand, the cancer has a histone H3 K27M mutation. In one aspect, wherein the cancer is a central nervous system tumor, a brain tumor, a glioma, a peripheral nervous system tumor, a pheochromocytoma, a paraganglioma, an adrenocortical carcinoma, an adrenal tumor, and a neuroendocrine tumor. In one aspect, the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord. In one aspect, the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M. In one aspect, DRD2 is overexpressed in tissue, DRD5 is underexpressed in tissue, or both. In one aspect, the subject is a human. In one aspect, the subject is a pediatric subject and the dosage is calculated based on the weight of the pediatric subject. In one aspect, the dosage is rounded to the nearest 125 mg increment. In one aspect, administration is started 2, 3, 4, 5, or 6 weeks after radiation is completed. In one aspect, the use or compound for use further includes administering one or more doses of temozolomide. In one aspect, the use or compound for use further comprises administering one or more doses of bevacizumab. In one aspect, the use or compound for use further comprises administering one or more radiation treatments after initiating administration.

[0025] One embodiment of the present disclosure includes a pharmaceutical composition comprising compound (1)

[0026]

[0027] Or a pharmaceutically acceptable salt thereof, at a dosage of twice a week for two consecutive days, day 1 and day 2, followed by a rest period, day 3 to day 7. On the one hand, the dosage is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg. On the one hand, the dosage is 625 mg. On the one hand, the selected dosage is calculated based on the subject's body weight (kg) by mg / kg. On the one hand, the subject is a pediatric subject and the dosage is calculated based on the body weight of the pediatric subject. On the one hand, wherein the dosage is rounded to the nearest 125 mg increment.

[0028] Although not specifically described, one or more embodiments or aspects may be incorporated into different embodiments or aspects. That is, all embodiments and aspects may be combined in any manner or combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above summary of the invention and the following detailed description of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the invention is not limited to the precise arrangements and tools shown. In the accompanying drawings:

[0030] Figure 1 ONC201 antagonizes dopamine receptors (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5).

[0031] Figure 2 Tumor type sensitivity of the Genomics of Drug Sensitivity in Cancer (GDSC) cell line collection. Mean estimated IC based on cell viability assays performed 72 hours after treatment 50 The values ​​determine the mean sensitivity. The numbers above the bars indicate the number of cell lines per tumor type.

[0032] Figure 3 GBM cell lines with higher DRD2 or lower DRD5 expression responded more strongly to ONC201. (A) Inhibitory effect of NCI60 GBM cell lines according to ONC201 concentration. (B) Log ONC201 GI of each GBM cell line 50 (M) Comparison of DRD2 expression. 2 = 0.8707. (C) Low DRD5 expression is significantly associated with improved ONC201 efficacy in the NCI60 panel of cancer cell lines.

[0033] Figure 4 ONC206 and ONC212 demonstrated anticancer efficacy in various tumor types in the NCI60 cancer cell line panel. ONC203 was an inactive negative control.

[0034] Figure 5Bone cancer responds more strongly to ONC206 than ONC201.

[0035] Figure 6 Ewing's sarcoma is the bone cancer subtype that responds most strongly to ONC206.

[0036] Figure 7 ONC206 had anticancer efficacy in the nanomolar range in 14 of 16 Ewing sarcoma cell lines; it showed superior efficacy compared to ONC201 in all cell lines.

[0037] Figure 8 .Leukemia responded more strongly to ONC212 than to ONC201.

[0038] Fig. 9 Regardless of the subtype, ONC212 showed anticancer efficacy in the nanomolar range in 55 leukemia cell lines (and had superior efficacy compared to ONC201).

[0039] Fig.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.

[0040] Fig.11 ONC212 was effective in ONC201-resistant AML xenograft models (MV411 AML cells (5×10 6 ) Efficacy of subcutaneous implantation in the flank of athymic nude mice. ONC212 and ONC201 were administered orally (PO) as indicated. Tumor volume (A and B) and body weight (C) were measured on the indicated days (n=10). * indicates p<0.05 relative to vehicle.

[0041] Fig.12 ONC206 was expressed in Ewing's sarcoma xenograft model (MHH-ES-1 Ewing's sarcoma cells (5×10 6 ) Efficacy of subcutaneous implantation into the flank 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) were measured on the indicated days (n=4).

[0042] Fig.13ONC213 has similar in vitro anticancer efficacy to ONC212 in HCT116 / RPMI8226 cancer cells, but has reduced in vitro toxicity to normal cells compared with ONC212.

[0043] Fig.14 .Immune induction is associated with tumor shrinkage in glioblastoma.

[0044] Fig.15 A 74-year-old woman with recurrent H3 K27M glioblastoma. MRI 8 weeks after first treatment showed complete disappearance of tumor lesions.

[0045] Fig.16 A 10-year-old girl with H3 K27M diffuse intrinsic pontine glioma had improvement in facial palsy and lesion shrinkage after 16 doses.

[0046] Fig.17 A 3-year-old girl with H3 K27M diffuse intrinsic pontine glioma. MRI 6 weeks after first treatment showed stable tumor lesions.

[0047] Fig.18 Progression-free survival of patients with recurrent high-grade glioma presenting at baseline was determined by MRI before starting ONC201 therapy. The cohort was divided into two groups: one with known H3 K27M mutation (red curve) and the other with wild-type or unknown H3 status (blue curve).

[0048] Fig.19 Waterfall plots for adult patients with recurrent H3 K27M mutant glioma treated with ONC201. Change in tumor size was calculated as the change from baseline at best treatment from the sum of the products of the perpendicular diameters of all measurable enhancing lesions. PD - progressive disease according to RANO; SD - stable disease according to RANO; PR - partial response according to RANO; NE - not evaluable for response (<1 cm multifocal lesions) according to RANO.

[0049] Fig. 20 (A) Gadolinium-enhanced MRI of 2 lesions at baseline and 72 weeks after starting ONC201 in a patient with recurrent H3 K27M mutant midline thalamic glioma receiving 625 mg ONC201 every three weeks. (B) Total tumor size relative to baseline over the duration of ONC201 administration is shown.

[0050] Fig.21 Gadolinium-enhanced MRI at baseline and 8 weeks after initiation of ONC201 (625 mg once weekly) in patients with recurrent H3 K27M mutant glioma.

[0051] Fig. 22 Gadolinium-enhanced MRI at baseline and 15 weeks after initiation of ONC201 (625 mg once weekly) in patients with recurrent H3 K27M mutant glioma.

[0052] Fig.23 is a graphical representation of the results of testing the K27M mutant compared to wild type using compounds of the present disclosure.

[0053] Fig.24 Shown are the results of ONC201 washout experiments in U110 glioblastoma cells. DETAILED DESCRIPTION

[0054] Scientific and technical terms used herein are intended to have the meanings that are commonly understood by one of ordinary skill in the art. Such terms may be found and used in the context of various standard references, illustratively including the following: Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition, 2001; FM Ausubel, ed., Short Protocols in Molecular Biology, Current Protocols; 5th edition, 2002; B. Alberts et al., Molecular Biology of the Cell, 4th edition, Garland, 2002; Nelson and Cox, Lehninger Principles of Biochemistry, 4th edition, WH Freeman & 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., Manipulating the Mouse Embryo: A Laboratory Manual, 3rd 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. Pat. No. 8,673,923. The contents of each of the above references are incorporated herein by reference in their entirety.

[0055] The term "substituted" means that one or more hydrogens on the designated atom are replaced by a selection from the indicated group, provided that the normal valence of the designated atom is not exceeded and that the substitution results in a stable compound. When the substituent is keto (i.e., =0), then 2 hydrogens on the atom are replaced. Keto substituents are not present on aromatic moieties. Ring double bonds are double bonds formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N).

[0056] In variables (e.g., R 4 ) occurs more than once in a constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown as being composed of 0 to 3 R 4 Partially substituted, the group may optionally be substituted with up to three R 4 Partially substituted and R 4 In each occurrence, independently selected from R 4 Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0057] When an atom or chemical moiety is followed by a numerical range in a subscript (e.g., C 1-6 ), it will be understood that this encompasses every number within that range and all intermediate ranges. For example, “C 1-6 The term “alkyl” is meant to include alkyl groups having 1, 2, 3, 4, 5, 6, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, and 5 to 6 carbons.

[0058] "Alkyl" includes both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. 1-6 Alkyl includes C1, C2, C3, C4, C5 and C6 alkyl. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, neopentyl and n-hexyl. In some cases, straight or branched alkyl has six or less carbon atoms in its main chain (e.g., C1-C6 for straight chain and C3-C6 for branched chain); in other cases, straight or branched alkyl has four or less carbon atoms. Similarly, cycloalkyl can have three to eight carbon atoms in its ring structure; in some cases, cycloalkyl has five or six carbons in the ring structure. Most preferably, C 1-6 Alkyl, in particular ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

[0059] The term "substituted alkyl" means an alkyl substituted by one, two or three substituents selected from halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C1-C4)alkyl, -C(=O)NH2, -SO2NH2, -C(=NH)NH2 and -NO2; preferably one or two substituents selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2 and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyl include 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

[0060] Unless the number of carbons is otherwise specified, "lower alkyl" is an alkyl group having one to six carbon atoms, preferably one to four carbon atoms in its backbone chain. "Lower alkenyl" and "lower alkynyl" have a chain length of 2 to 6 carbon atoms, and preferably 2 to 4 carbon atoms.

[0061] "Alkenyl" includes unsaturated aliphatic groups that are similar in length to the above-mentioned alkyl and may be substituted but contain at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl (e.g., vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched alkenyl, cycloalkenyl (e.g., alicyclic) (e.g., cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl and cycloalkyl or cycloalkenyl substituted alkenyl. In some cases, straight or branched alkenyl has six or less carbon atoms in its main chain (e.g., C2-C6 for straight chain, C3-C6 for side chain). Similarly, cycloalkenyl can have three to eight carbon atoms in its ring structure; in some cases, cycloalkenyl has five or six carbons in the ring structure. The terms "C2-C6" and "C3-C6" include alkenyl groups containing two to six carbon atoms and three to six carbon atoms, respectively.

[0062] "Alkynyl" includes unsaturated aliphatic groups that are similar in length to the above-mentioned alkyl and may be substituted but contain at least one triple bond. For example, "alkynyl" includes straight chain alkynyl (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl), branched chain alkynyl and cycloalkyl or cycloalkenyl substituted alkynyl. In some cases, straight chain or branched chain alkynyl has six or less carbon atoms in its main chain (e.g., C2-C6 for straight chain and C3-C6 for side chain). The terms "C2-C6" and "C3-C6" include alkynyl containing two to six carbon atoms and three to six carbon atoms, respectively.

[0063] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group in which each of the atoms forming the ring (i.e., the backbone atoms) is a carbon atom. In some cases, the cycloalkyl is saturated or partially unsaturated. In other cases, the cycloalkyl is fused to an aromatic ring. Cycloalkyl includes groups having 3 to 10 ring atoms. Examples of cycloalkyl include, but are not limited to, the following:

[0064]

[0065] 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 adamantane and norbornane. The term cycloalkyl includes "unsaturated non-aromatic carbocyclic radicals" or "non-aromatic unsaturated carbocyclic radicals", both of which refer to non-aromatic carbocyclic rings as defined herein, which contain at least one carbon-carbon double bond or one carbon-carbon triple bond.

[0066] The term "cycloalkylalkyl" refers to an alkyl group substituted by a cycloalkyl group. Example cycloalkylalkyl groups include cyclopropylalkyl, cyclohexylalkyl.

[0067] The term "heterocycloalkyl" refers to a non-aromatic heterocycle in which one or more ring-forming atoms in the ring-forming atoms are heteroatoms such as O, N or S atoms. Heterocycloalkyl includes monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) ring systems and spirocycles. Examples of heterocycloalkyl include morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxol, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl and imidazolidinyl. The definition of heterocycloalkyl may also include a portion having one or more aromatic rings fused to a non-aromatic heterocycle (i.e., having a common bond), for example, quinolyl, isoquinolyl and a benzo derivative of a heterocycle. Heterocycloalkyl with one or more fused aromatic rings is connected by an aromatic or non-aromatic moiety. The definition of heterocycloalkyl also includes a part in which one or more ring-forming atoms can be replaced by 1 or 2 oxo groups or sulfide groups. In some cases, heterocycloalkyl has 1 to about 20 carbon atoms, and in further cases has about 3 to about 20 carbon atoms. In some cases, heterocycloalkyl contains 3 to about 20, 3 to about 14, 3 to about 7 or 5 to 6 ring-forming atoms. In some cases, heterocycloalkyl contains 1 to about 4, 1 to about 3 or 1 to 2 heteroatoms. In some cases, heterocycloalkyl contains 0 to 3 double bonds. In some cases, heterocycloalkyl contains 0 to 2 triple bonds.

[0068] The term "heterocycloalkylalkyl" refers to an alkyl group substituted with a heterocycloalkyl group. Example heterocycloalkylalkyl groups include morpholinylalkyl and piperazinylalkyl.

[0069] The term "aryl" refers to a monocyclic or polycyclic (eg, having 2, 3 or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl. In some cases, the aryl group has 6 to 20 carbon atoms.

[0070] The term "arylalkyl" refers to an alkyl group substituted with an aryl group. Example arylalkyl groups include benzyl and phenethyl.

[0071] The term "heteroaryl" refers to an aromatic heterocycle having at least one heteroatom ring member (such as O, S or N atom). Heteroaryl includes monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. The ring-forming N atoms in the heteroaryl can also be oxidized to form N-oxo moieties. Examples of heteroaryl include pyridyl, N-oxopyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl. In some cases, the heteroaryl group has 1 to about 20 carbon atoms, and in some cases has about 3 to 20 carbon atoms. In some cases, the heteroaryl group contains 3 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some cases, the heteroaryl group contains 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms.

[0072] "Heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. An example of a heteroarylalkyl group is pyridinylmethyl.

[0073] 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 a halogen replacing a hydrogen on one or more carbons of a carbon-hydrogen backbone. C1-C6 haloalkyl includes straight or branched chain alkyl groups having six or fewer backbone carbon atoms and a halogen replacing a hydrogen on one or more backbone carbons.

[0074] The term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl and alkynyl radicals covalently linked 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 alkoxyl radicals include methoxy, ethoxy, isopropoxy, propoxy, butoxy and pentoxy. Preferred are (C1-C3) alkoxy, particularly ethoxy and methoxy. Examples of substituted alkoxy groups include halogenated alkoxy.

[0075] The term "hydroxy" or "hydroxyl" includes radicals having -OH or -O – The group.

[0076] 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; alkali metal 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 parent compounds containing basic or acidic moieties 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 an appropriate base or acid in water or an organic solvent, or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418; Journal of Pharmaceutical Science, 66, 2 (1977); and P. Stahl and C. Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd ed. Rev., Weinheim / Zürich: Wiley-VCH / VHCA (2011), each of which is incorporated herein by reference in its entirety.

[0077] Examples of suitable inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid or hydrobromic acid, while examples of suitable organic acids include carboxylic acids, sulpho acids 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, pamoic acid, nicotinic acid, isonicotinic acid, oxalic acid, gluconic acid, amino acids, methylsulfonic acid, ethylsulfonic acid, 2-hydroxyethylsulfonic 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.

[0078] The term "antibody" encompasses the structure of the natural biological form of an antibody. In most mammals, including humans and mice, this form is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising the immunoglobulin domain V L and C L , and each heavy chain contains the immunoglobulin domain V H , Cγ1, Cγ2 and Cγ3. In each pair, the light chain and heavy chain variable regions (V L and V H ) are jointly responsible for binding to the antigen, 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 of which contains the immunoglobulin domain V H , Cγ2 and Cγ3. "Immunoglobulin (Ig)" herein refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have a variety of structural forms, including full-length antibodies; antibody fragments; and V H 、Cγ1、Cγ2、Cγ3、V L and C L A single immunoglobulin domain.

[0079] 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 several of these classes can be further divided into "subclasses", for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

[0080] The term "antibody" or "antigen-binding fragment" refers to an intact molecule and its functional fragments, such as Fab, scFv-Fc bivalent molecules, F(ab')2 and Fv, respectively, that can specifically interact with the desired target. In some cases, the antigen-binding fragment comprises:

[0081] (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule that can be produced by digesting a whole antibody with the enzyme papain to produce an intact light chain and a portion of one heavy chain;

[0082] (2) Fab', a fragment of an antibody molecule that can be obtained by treating a whole antibody with pepsin followed by reduction to produce an intact light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule;

[0083] (3) (Fab')2, a fragment of an antibody that can be obtained by treating a whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

[0084] (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain represented as two chains;

[0085] (5) single-chain antibodies ("SCAs"), genetically engineered molecules comprising a light chain variable region and a heavy chain variable region connected as a genetically fused single chain molecule by a suitable polypeptide linker; and

[0086] (6) scFv-Fc, which is produced by fusing a single-chain Fv (scFv) with a hinge region (such as IgG) and an Fc region from an immunoglobulin (Ig).

[0087] In one embodiment, the antibody provided herein is a monoclonal antibody. In one embodiment, the antigen-binding fragment provided herein is a single-chain Fv (scFv), a diabody, a tandem scFv, a scFv-Fc bivalent molecule, a Fab, a Fab', a Fv, a F(ab')2, or an antigen-binding scaffold (e.g., an affibody, a monoclonal antibody, an anticalin, a DARPin, a knottin).

[0088] The term "binds" or grammatical equivalents refers to compositions that have an affinity for each other, either directly or indirectly. "Specific binding" refers to the selective binding between two molecules. For example, specific binding occurs between an antibody and an antigen. Generally, when the dissociation constant (K D ) is less than about 1×10 -5 M or less than about 1×10 -6 M or 1×10 -7 When the binding site is determined by the method of the present invention, specific binding can be distinguished from nonspecific binding. For example, specific binding can be detected by ELISA, immunoprecipitation, coprecipitation, with or without chemical cross-linking, and two-hybrid assays. The use of appropriate controls can distinguish "specific" binding from "nonspecific" binding. "Affinity" is the strength of the binding interaction of two molecules (such as an antigen and its antibody), which is defined as the binding strength of the ligand at one specified binding site for antibodies and other molecules with more than one binding site. Although non-covalent attachment of a ligand to an antibody or other molecule is generally not as strong as covalent attachment, "high affinity" ligands are generally greater than 10 4 M -1 (usually 10 5 -10 11 M -1 ) affinity constant (K a ) is bound to an antibody or other molecule; as determined by inhibition ELISA or by equivalent techniques (such as Scatchard plots or using a K a The reciprocal K d / dissociation constant) determined by the equivalent affinity.

[0089] With respect to binding, inhibition, stimulation or modulation, the term "selectivity" means preferential binding, inhibition, stimulation or modulation of a first activity relative to a second activity (e.g., preferential binding of one receptor to another receptor; preferential inhibition relative to other receptors; or preferential inhibition of a mutant relative to a wild type or vice versa). In some cases, the binding has greater selectivity for a desired molecular target or pathway relative to an undesired molecular target or pathway than two-fold, greater than five-fold, greater than ten-fold, greater than fifty-fold, greater than 100-fold, or greater than 1000-fold. In some cases, under the same conditions, the compound will bind to the first molecular target or affect the pathway at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the second target or pathway. It will be understood that in preferred embodiments, with respect to the D1-like family of dopamine receptors or members thereof, the binding to the D2-like family of dopamine receptors or members thereof will have one of the aforementioned amounts of selectivity. The in vitro or in vivo activity of a molecular target or pathway can be measured by any suitable reproducible method.

[0090] The term "modulate" refers to "stimulate" or "inhibit" the activity of a molecular target or pathway. For example, if the composition stimulates or inhibits the activity 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%, at least about 99% or more relative to the activity of the molecular target or pathway under the same conditions but without the composition, then the composition modulates the activity of the molecular target or pathway. In another example, if the composition stimulates or inhibits the activity by at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times relative to the activity of the target or pathway under the same conditions but without the composition, then the composition modulates the activity of the molecular target or pathway. The activity of a molecular target or pathway can be measured by any reproducible method. For example, the activity of a molecular target or pathway can be measured in vitro or in vivo by any suitable assay known in the art for measuring activity. A control sample (not treated with the composition) can be assigned a relative activity value of 100%.

[0091] In one embodiment, the antibody, antigen-binding fragment, or affinity tag has 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 to 2 nM, 0.1 to 1 nM, 0.05 to 1 nM, 0.1 to 0.5 nM, or 0.1 to 0.2 nM. DBinds to its target. In one embodiment, it binds to its target directly. In one embodiment, it binds to its target indirectly, for example, a second antibody binds to an antibody that binds to the target.

[0092] The term "label" refers to a compound or composition that is conjugated or fused directly or indirectly to an agent such as a nucleic acid probe or antibody and that facilitates detection of the agent to which it is conjugated or fused. The label can be detectable by itself (e.g., a radioisotope or a fluorescent label) or, in the case of an enzyme label, can catalyze chemical alteration of a detectable substrate compound or composition.

[0093] The term "probe" refers to a synthetic or biologically produced nucleic acid containing a specific nucleotide sequence that hybridizes with a target nucleic acid sequence under stringent conditions. The term "labeled probe", "operably connected to a detectably labeled nucleic acid probe" or "operably connected to a detectably labeled nucleic acid chain" refers to a probe for detection prepared with a marker group or "detectable label". The marker group is internally connected at the 5' end, 3' end or a combination thereof. In other words, a probe can be connected to multiple markers. A preferred group is a distinguishing marker, such as a fluorophore. The labeled probe can also include a plurality of different nucleic acid sequences each labeled with one or more markers. Each marker can be the same or different. It may be beneficial to mark different probes (e.g., nucleic acid sequences) with different markers. This can be achieved by having a single distinguishable group on each probe. For example, probe A is connected to group X and probe B is connected to group Y. Alternatively, probe A is connected to groups X and Y, and probe B is connected to groups Z and W. Alternatively, probe A is connected to groups X and Y, and probe B is connected to groups Y and Z. All of the above probes "A" and "B" will be distinguishable and uniquely labeled.

[0094] "Tissue sample" means a collection of similar cells obtained from a tissue of a subject or patient, 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) neural tissue. Tissue sample sources can be solid tissue such as from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; blood or blood components; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from the subject's gestational or developmental period. Tissue samples can also be primary or cultured cells or cell lines. Tissue samples can contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients or antibiotics. Tissue sample "section" means a single portion or piece of a tissue sample, for example, a thin slice of tissue or cells cut from a tissue sample. Multiple sections of a tissue sample can be collected and analyzed. "Cell line" refers to a permanently established cell culture that will proliferate if given appropriate fresh media and space.

[0095] Detection Methods

[0096] In various aspects, the present invention provides a method for detecting or measuring a target receptor (e.g., dopamine receptor or GPCR) in a biological sample. The target is detected by contacting the sample with a target detection reagent (e.g., an antibody or a fragment thereof) and a labeling agent. The presence or absence of the target is detected by the presence or absence of a labeling agent. In some cases, the sample is contacted with a target detection object and a labeling agent simultaneously, for example, the detection reagent is a first antibody and the labeling agent is a fluorescent dye conjugated thereto. Alternatively, the biological sample is contacted with a target detection object and a labeling agent in sequence, for example, the detection reagent is a first antibody and the labeling agent comprises a second antibody. For example, under conditions allowing the formation of a complex between the detection reagent (and the labeling agent) and the target, the sample is incubated with the detection reagent (in some cases, with the labeling agent). After the complex is formed, the sample is optionally cleaned one or more times to remove unbound detection reagents (and labeling agents). When the sample is further contacted with a labeling agent of a specific binding detection reagent (the detection reagent is combined with the target), the sample can be optionally washed one or more times to remove unbound labeling agents. The presence or absence of the target in the sample is then determined by detecting the labeled agent.

[0097] The methods described herein provide for detecting multiple targets in a sample. Multiple targets are identified using the described methods by contacting a biological sample with an additional detection reagent and subsequently with an additional labeling reagent that is specific for the additional detection reagent.

[0098] The detection moiety (i.e., detectable label) is a substance used to facilitate target identification and / or quantification. The detection moiety is directly observed or measured or indirectly observed or measured. Non-limiting examples of detection moieties include radioactive labels that can be measured with a radio counting device; 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, wherein the output signal is generated by exciting a suitable molecular adduct and can be visualized by light excitation absorbed by the dye or can be measured with a standard fluorometer or imaging system. The detection moiety can be a luminescent substance, such as a phosphor or a fluorophore; a bioluminescent substance; a chemiluminescent substance, wherein the output signal is generated by chemical modification of a signal compound; a metal-containing substance; or an enzyme, wherein an enzyme-dependent secondary generation of a signal occurs, such as forming a colored product from a colorless substrate. The detection moiety can also be in the form of a chemical or biochemical or inert particle, including colloidal gold, microspheres, quantum dots, or inorganic crystals such as nanocrystals or phosphors. The term "detection moiety" or "detectable label" can also refer to a "tag" or hapten that can be selectively bound to a marker molecule so that when the marker molecule is subsequently added, a detectable signal is generated using the marker molecule. For example, biotin, iminobiotin or desthiobiotin can be used as a tag and then avidin or streptavidin conjugate of horseradish peroxidase (HRP) is used to bind to the tag, and then a chromogenic substrate (e.g., tetramethylbenzidine) or a fluorescent substrate (such as Amplex Red or Amplex Gold (Molecular Probes, Inc.)) is used to detect the presence of HRP. Similarly, the tag can be a hapten or antigen (e.g., digoxin), and an enzymatically, fluorescently or radioactively labeled antibody can be used to bind to the tag. Many labels are known to those skilled in the art and non-limiting examples include particles, fluorescent dyes, haptens, enzymes and chromogenic substrates thereof, fluorescent substrates, and chemiluminescent substrates.

[0099] Fluorophores are chemical groups that exhibit a maximum absorption above 280 nm and retain their spectral properties when covalently linked in a labeling agent. 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, boropolyazaindacene, xanthene, oxazine or benzoxazine, carbazine, phenalenone, coumarin, benzofuran and benzphenalenone and derivatives thereof. Oxazines include resorufin, aminooxazinones, diaminooxazines and benzo-substituted analogs thereof.

[0100] For xanthene fluorophores, the fluorophore can be fluorescein, rhodol or rhodamine. Fluorescents include benzo- or dibenzofluoresceins, seminaphthofluoresceins or naphthofluoresceins. Similarly, rhodol includes seminaphthorhodafluors. Alternatively, the fluorophore is a xanthene bound by a single covalent bond at position 9 of the xanthene. Preferred xanthenes include derivatives of 3H-xanthene-6-ol-3-one, 6-amino-3H-xanthene-3-one or 6-amino-3H-xanthene-3-imine. Fluorophores include xanthenes (rhodol, rhodamine, fluorescein and derivatives thereof), coumarins, cyanines, pyrenes, oxazines and boron polybenzazaindoles. Additionally, fluorophores can be sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins, and sulfonated cyanines. The choice of fluorophore in the labeling reagent will determine the absorption and fluorescence emission properties of the labeling reagent. The physical properties of fluorophore labels include spectral characteristics (absorption, emission, and Stokes shift), fluorescence intensity, lifetime, polarization, and photobleaching rate, all of which can be used to distinguish one fluorophore from another.

[0101] Typically, fluorophores contain one or more aromatic or heteroaromatic rings optionally substituted with one or more substituents comprising halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzo or other substituents commonly found on fluorophores known in the art.

[0102] Preferably, the detection moiety is a fluorescent dye. Fluorescent dyes include, for example, fluorescein, rhodamine, Texas Red, Cy2, Cy3, Cy5, Cy0, Cy0.5, Cy1, Cy1.5, Cy3.5, Cy7, VECTOR Red, ELF TM (enzyme-labeled fluorescence), FluorX, Calcein, Calcein-AM, CRYPTOFLUOR TM '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-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) (NBD), BODIPY TM , boron dipyrromethene difluoride, Oregon Green, MITOTRACKER TM Red, DiOC7 (3), DiIC18, phycoerythrin, phycobiliprotein 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 act as chromophores and therefore these fluorophores are also preferred chromophores.

[0103] In addition to fluorophores, enzymes are also found to be used as detectable moieties. Because the amplification of detectable signals can be achieved, resulting in enhanced assay sensitivity, enzymes are desirable detectable moieties. Enzymes themselves do not produce detectable responses, but when enzymes contact appropriate substrates, they decompose substrates so that converted substrates produce fluorescent signals, colorimetric signals, or luminescent signals. Because a kind of enzyme on a labeling agent can cause a variety of substrates to be converted into detectable signals, enzymes can amplify detectable signals. If there is a low amount of target in a sample or there is no fluorophore that produces a signal equivalent to or stronger than that of an enzyme, this is advantageous. However, fluorophores are preferred because they do not require additional assay steps, and therefore reduce the total time to complete the assay. Enzyme substrates are selected to produce preferred measurable products, for example, colorimetric, fluorescence, or chemiluminescence. Such substrates are widely used in the art.

[0104] Preferred colorimetric substrates or fluorogenic substrates and enzyme combinations use oxidoreductases such as horseradish peroxidase and substrates such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole-e (AEC), which produce distinctive colors (brown and red, respectively). Other non-limiting examples of colorimetric oxidoreductase substrates that produce detectable products include: 2,2-azino-bis(3-ethylbenzothiazole-line-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 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 variants thereof) and reduced dihydroxanthenes (including dihydrofluorescein and dihydrorhodamines including dihydrorhodamine 123). Peroxidase substrates that are tyramides represent a class of peroxidase substrates that may be inherently detectable prior to enzyme action but are "fixed in place" by the action of peroxidase in a process described as tyramide signal amplification (TSA). These substrates are widely used to label targets in cell, tissue or array samples for subsequent detection by microscopy, flow cytometry, optical scanning and fluorimetry.

[0105] Additional colorimetric substrates (in some cases fluorogenic substrates) and enzyme combinations use a phosphatase (such as acid phosphatase, alkaline phosphatase, or a recombinant version of such a phosphatase) in combination with a colorimetric substrate (such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate) or in combination with a fluorogenic substrate (such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy 4-methylcoumarinyl phosphate (DiFMUP) fluorescein diphosphate, 3-0-methylfluorescein phosphate, resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39, or related phosphates).

[0106] Glycosidases, in particular β-galactosidase, β-glucuronidase and β-glucosidase are other suitable enzymes. Suitable colorimetric substrates include: 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides and glucuronides, 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 structural variants thereof, 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.

[0107] Enzymes and their substrates that produce chemiluminescence are preferred for some assays. These include, for example, natural and recombinant luciferases and aequorin. For luciferase or aequorin, exemplary substrates are luciferin, ATP, Ca for phosphatases, glycosidases, and oxidases. ++ and coelenterazine chemiluminescent substrates, such as those containing stable dioxetanes, luminol, isoluminol, and acridinium esters are useful.

[0108] In addition to enzymes, haptens such as biotin are also useful detectable moieties. Biotin is in an enzyme system that can further amplify the detectable signal and can serve as a label in affinity chromatography for separation purposes. For detection, an enzyme conjugate with affinity for biotin is used, such as avidin-HRP. Then, a peroxidase substrate is added to produce a detectable signal. Haptens also include hormones, naturally occurring drugs and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates or nucleotides.

[0109] In some cases, the detectable portion is a fluorescent protein. For example, it includes green fluorescent protein (GFP), phycobiliprotein and its derivatives, luciferase or aequorin. Fluorescent proteins, particularly phycobiliproteins, are particularly useful for creating labeling agents labeled with tandem dyes. Tandem dyes include fluorescent proteins and fluorophores to obtain a larger Stokes shift that is far away from the absorption spectrum of the fluorescent protein. This is particularly advantageous for detecting low-amount targets in samples that optimize the emitted fluorescence to the greatest extent, in other words, the fluorescent protein will hardly reabsorb or even completely no longer absorb the emitted light. The fluorescent protein and the fluorophore act as an energy transfer pair, wherein the fluorescent protein emits a wavelength absorbed by the fluorophore, and then the wavelength emitted by the fluorophore is farther from the fluorescent protein than the wavelength that can be obtained with the fluorescent protein alone. Particularly useful combinations are phycobiliproteins and sulforhodamine fluorophores, sulfonated cyanine fluorophores; or sulfonated xanthene derivatives. Alternatively, the fluorophore is an energy donor and the fluorescent protein is an energy acceptor.

[0110] The method of visualizing the detected part depends on the marker.

[0111] In some cases, a wavelength of light is selected to produce a detectable optical response to illuminate the sample, and the sample is observed with a device that detects the response. Equipment for illuminating fluorescent compounds includes 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 standard or microfluorometers. The degree or position of the signal, compared to the standard or expected response, indicates whether and to what extent the sample has a given characteristic or desired target.

[0112] The optical response is detected by visual inspection or using one of the following devices: CCD camera, video camera, photographic film, laser scanning device, fluorometer, photodiode, quantum counter, epifluorescence microscope, scanning microscope, flow cytometer, fluorescence microplate reader or a device for amplifying the signal such as a photomultiplier tube. When the sample is examined using a flow cytometer, the examination of the sample optionally includes sorting a portion of the sample according to its fluorescence response.

[0113] When an indirect detectable label is used, then illumination generally includes the addition of a reagent to produce a detectable signal such as a colorimetric enzyme substrate. Radioisotopes are also considered to be indirectly detectable when no additional reagents are required but the radioisotope is exposed to X-ray film or other mechanisms to record and measure the signal. This is true for some chemiluminescent signals observed after exposure to film.

[0114] I. ONC201 (Compound (1)), Its Salts and Synthesis

[0115] Provided herein is ONC201 (compound (1)) Its analogs and pharmaceutically acceptable salts thereof and their synthesis. In in vitro models, animal models and human clinical trials, ONC201 has broad anticancer activity, low toxicity (including few side effects, if any), low genotoxicity and high bioavailability including oral administration. These characteristics make ONC 201 and various analogs very suitable for various applications. ONC201 can be synthesized as shown in Scheme 1.

[0116]

[0117] 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) (step 1) is neutralized with a base to produce compound (4), a free base. For example, compound (3) is neutralized with an inorganic base to produce compound (4). Alternatively, it is neutralized with an organic base to produce 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., NaHCO3 and n-butanol). In one embodiment, compound (3) is neutralized in the presence of n-butanol and triethylamine (Et3N).

[0118] In one embodiment, the synthesis comprises reacting compound (4) with (5) (step 2) to produce intermediate compound (1). In one embodiment, step 2 comprises heating compound (4) with (5). In one embodiment, step 2 comprises reflux the heated compound (4) with (5) in the presence of a solvent. In one embodiment, step 2 comprises removing water and / or methanol (MeOH) formed in the reaction using a Dean-Stark trap.

[0119] In one embodiment, ONC201 dihydrochloride is synthesized (step 3). In one embodiment, step 3 comprises treating ONC201 with dioxane containing HCl. In one embodiment, step 3 comprises treating ONC201 with dioxane containing 4N HCl. In one embodiment, the synthesis optionally comprises recrystallizing ONC201 disalt. Preferably, ONC201 dihydrochloride is synthesized as shown in Scheme 2.

[0120]

[0121] II. TNF-related apoptosis-inducing ligand ("TRAIL")

[0122] TRAIL protein can be measured in a sample obtained from a subject to detect TRAIL expression induced by compounds described herein and salts thereof. Immunoassays can be used, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, enzyme-linked immunofiltration assay (ELIFA), immunoblotting, 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 determinations are described in standard reference texts, including: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Breitling and 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-immunophenotypiccorrelation, Taylor&Francis, 2006; Crowther, JR, The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, 3rd Edition, Elsevier Science, 2005; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001. .

[0123] An assay for analyzing a sample for TRAIL to detect the effect of a pharmaceutical composition is described in US 8,673,923, which is incorporated herein by reference in its entirety.

[0124] In one embodiment, a TRAIL assay is used to monitor a subject. For example, samples are obtained from a subject one or more times before treatment with a pharmaceutical formulation and during and / or after treatment to assess the efficacy of the treatment. In another example, samples are obtained from a subject at various times to assess the course or progression of a disease or cure. In one embodiment, death receptors from circulating tumor cells are assayed to see if the treatment described herein increases the number or type of death receptors.

[0125] Cancers 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 to prevent as well as to ameliorate signs or symptoms of cancer. "Treatment" of cancer in a subject includes: preventing, inhibiting, or ameliorating the subject's cancer, such as slowing the progression of the cancer or alleviating or ameliorating signs or symptoms of the cancer. Examples of cancers treated using the methods and compositions described herein include breast cancer, CNS cancer, colon cancer, ovarian cancer, prostate cancer, leukemia, lung cancer, and lymphoma. III. Compounds of formula (10) and salts thereof

[0126] In one aspect, provided herein are compounds and salts of formula (10) and methods for preparing the same. Those skilled in the art will appreciate 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) are equally applicable to compounds of formula (10) and its salts.

[0127] In one embodiment, provided herein is a compound of formula (10): Wherein R1 and R2 are independently selected from H, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio and acyl. In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(2-CH3-Ph)(ONC201). In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(2,4-diF-Ph)(ONC206). In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(4-CF3-Ph)(ONC212). 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).

[0128] In one embodiment, R1 and R2 are independently selected from 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 Alkylpyridyl, C 1-4 Alkylisoxazolidinyl, C 1-4 Alkylmorpholinyl, C 1-4 Alkylthiazolyl and C 1-4 Alkylpyrazinyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine, C 1-4 Alkylthienyl, C 1-4 Alkylpyridyl, C 1-4 Alkylisoxazolidinyl, C 1-4 Alkylthiazolyl, C 1-4 Alkylmorpholinyl and C 1-4 The alkylpyrazinyl group is optionally substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, R1 and / or R2 are substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment, heteroarylalkyl is selected from C 1-4 Alkyl pyrrolyl, C1-4 Alkyl furanyl, 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 Alkyl tetrazolyl, C 1-4 Alkylpyrazinyl, C 1-4 Alkyl pyrimidinyl, C 1-4 Alkylquinolyl, C 1-4 Alkylpyrazolyl, C 1-4 Alkyl isoquinolyl, C 1-4 Alkylphenylthio, C 1-4 Alkylbenzothiophene, C 1-4 Alkyl isobenzofuranyl, C 1-4 Alkyl indolyl, C 1-4 Alkyl purine, C 1-4 Alkylcarbazolyl, C 1-4 Alkylbenzimidazole and C 1-4 Alkylisoxazolyl.

[0129] In one embodiment, R1 and / or R2 are 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 , SR m NR m R n NR m C(O)R n , SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C1-C4 alkyl; and 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; more preferably, F or Cl.

[0130] In one embodiment, R1 is selected from 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-pyridyl, CH2-4-methyl-2-thiazolyl, CH2-2-pyrazinyl, CH2CH2Ph, CH 2CH2(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 selected from 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), CH 2-((2-CH3)-Ph), CH2(2-CH3,4-F-Ph), CH2-((4-OCH3)-Ph), CH2-(3-pyridyl), 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.

[0131] In one embodiment, R1 is H. In one embodiment, R1 is unsubstituted or substituted arylalkyl, for example, benzyl (CH2Ph) or phenethyl (CH2CH2Ph). In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0132] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, for example, benzyl or phenethyl. In one embodiment, the arylalkyl group is C 1-4 Alkyl, C 1-4Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, arylalkyl is substituted with one or more substituents selected from halo, CH3, CF3 or OCH3. In one embodiment, R2 is substituted or unsubstituted heterocycloalkylalkyl, for example, piperazinylalkyl or morpholinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, for example, pyridylmethyl or isoxazolidinylmethyl. In one embodiment, heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with at least one substituent selected from halo, CH3, CF3 or OCH3.

[0133] In one embodiment, compound (10) has the structure of formula (80): Where R a1 , R a2 , R a3 , R a4 , R a5 , R b1 , R b2 , R b3 , R b4 and R b5 Each independently selected from the group consisting of: 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 ,SR m NR m R n NR m C(O)R n , SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n is independently selected from H or C1-C4 alkyl; and wherein p is an integer from 2 to 20 and X is halogen.

[0134] In one embodiment, compound (10) has the structure of formula (90): wherein R2 is as defined above, and wherein R b1 , R b2 , R b3 , R b4 and R b5 Each independently selected from the group consisting of: 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 ,SR m NR m R n NR m C(O)R n , SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C 1-4 alkyl; and wherein p is an integer from 2 to 20 and X is halogen.

[0135] In one embodiment, compound (10) has a structure of formula (40): wherein R1 is as defined above, and wherein R a1 , R a2 , R a3 , R a4 and R a5 Each independently selected from 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 ,SR m NR m R n NR m C(O)R n , SORm 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C 1-4 alkyl; p is an integer from 2 to 20; and X is a halogen. In one embodiment, R1 is H. In one embodiment, R1 is a substituted or unsubstituted arylalkyl, such as benzyl or phenethyl. In one embodiment, the arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated 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, CH3, CF3 and OCH3. In one embodiment, benzyl is substituted with one halo (e.g., F) at the ortho or para position. In one embodiment, benzyl is substituted with two halogens (e.g., F) at two meta positions.

[0136] In one embodiment, compound (40) has the structure of compound (45):

[0137] Where R a1 , R a2 , R a3 , R a4 and R a5 As defined above. In one embodiment, the benzyl group is substituted with one or more halogens. In one embodiment, the benzyl group is substituted with one or more substituents selected from halo, CH3, CF3 and OCH3. In one embodiment, R a1 or R a5 is halogenated, for example, F. In one embodiment, R a2 and R a3 are all halogenated, for example, F.

[0138] In one embodiment, compound (10) has the structure of compound (50): wherein R1 is as defined above, and wherein R b Selected from 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 , ORm , SR m NR m R n NR m C(O)R n , SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C 1-4 alkyl; and wherein p is an integer from 2 to 20 and X is a halogen, and wherein R a1 , R a2 , R a4 and R a5 Each independently selected from 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 , SR m NR m R n NR m C(O)R n , SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C 1-4 alkyl; and wherein p is an integer from 2 to 20 and X is a halogen. In one embodiment, R1 is H. In one embodiment, R1 is a substituted or unsubstituted arylalkyl, such as benzyl or phenethyl. In one embodiment, the arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, R b is selected from halo, CH3, CF3 and OCH3. In one embodiment, R a1 , R a2 , R a4 and R a5One or more of is selected from halo, CH3, CF3 and OCH3. In one embodiment, R a1 , R a2 , R a4 and R a5 is H, and R b is selected from halo, CH3, CF3 and OCH3. In one embodiment, R b is a halogen, for example, 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 CF3. In one embodiment, R b is OCH3. In one embodiment, R b and R a1 It is Cl.

[0139] In one embodiment, compound (50) has the structure of compound (55): Where R a1 , R a2 , R a4 , R a5 and R b As defined above. In one embodiment, R b is selected from halo, CH3, CF3 and OCH3. In one embodiment, R a1 , R a2 , R a4 and R a5 One or more of is selected from halo, CH3, CF3 and OCH3. In one embodiment, R a1 , R a2 , R a4 and R a5 is H, and R b is selected from halo, CH3, CF3 and OCH3. In one embodiment, R b is halogenated, for example, 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 CF3. In one embodiment, R b is OCH3. In one embodiment, R b and R a1 It is Cl.

[0140] In one embodiment, compound (10) has the structure of compound (60): In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, such as benzyl or phenethyl. 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 replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, benzyl is substituted with alkyl or halo. In one embodiment, benzyl is substituted with one or more halogen. In one embodiment, benzyl is substituted with one or more substituents selected from halo (e.g., F), CH3, CF3 and OCH3. In one embodiment, benzyl is substituted with halo, CH3, CF3 or OCH3 substituents at the para position. In one embodiment, R1 is fluorophenyloxobutyl or hydroxyphenylethyl.

[0141] Scheme 3 shows the synthesis of compounds of formula (10):

[0142]

[0143] The compound of formula (10) (imidone) is synthesized starting from a substituted piperidone, which is converted by reaction with a substituted aminoimidazoline to give the core compound (10). There are two approaches, one of which is where the R1 substituent is present in the piperidone (e.g., 68). In this approach, (68) is acylated with dimethyl carbonate using sodium hydride in toluene at 80°C to form the piperidone ester (69). The commercially available methylthioimidazoline HI salt (63) is reacted with an amine-containing dioxane at 70°C to give the R2-substituted aminoimidazoline (64) as its HI salt. (64) is reacted directly with 1-butanol containing the piperidone ester (69) under reflux, wherein water is removed by a Dean-Stark trap over 3 to 6 hours to give the tricyclic compound (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 converted to the free base of (66) with 1N NaOH, extracted with dichloromethane. (66) is subsequently treated with halide (67) or epoxide (70) to give the desired compound (10).

[0144] The crude product can be purified by column chromatography using dichloromethane: methanol or by HPLC using acetonitrile: TFA: H2O to produce free base or TFA salt as the final product. The free base or lyophilized TFA salt is treated with dioxane containing HCl to produce product (10) as HCl or TFA salt. Alternatively, the free base can be treated with another inorganic acid or organic acid to form other salts generally selected from known pharmaceutically acceptable salts. The salts of compound (10) are generally solid and examples have been crystallized from ethanol or other solvents to obtain high quality crystals. The tricyclic structure of compound (1) has been clearly confirmed by X-ray crystal structure and NMR.

[0145] The compounds described herein can be used with or without an aminoalkyl linker (e.g., compound (33)) to identify molecules (e.g., proteins) that interact with it in the cellular environment. The expression of these binding targets can be used to predict the response to imiprazole or its analogs (i.e., act as biomarkers). Using competition assays known in the art, these compounds can also be used to screen structurally unrelated molecules to identify drugs that can outcompete the target interaction with higher affinity. In addition, these molecules can have improved drug properties or allow additional applications by altering drug properties (including safety, efficacy, pharmacokinetics, biodistribution, or metabolism).

[0146] Table 1: Examples of compounds having formula (10)

[0147]

[0148]

[0149] IV. Evaluating the sensitivity and efficacy of treatment options

[0150] Measuring the expression, gene mutation or gene copy number of dopamine receptors or other G protein coupled receptors (GPCRs) can be used to predict the response or sensitivity to the treatment methods described herein or to identify subjects who may respond to the treatment 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 are methods for identifying whether a subject with a condition is likely to respond to the treatment regimen described herein. In one embodiment, these methods include (i) obtaining a biological sample from a 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 the level of a predetermined standard; and (iv) determining whether the subject is likely to respond to the treatment regimen based on the comparison of the level measured in the sample with the level of the predetermined standard. In one embodiment, the step of measuring the expression level in the sample includes the following steps: (i) contacting the sample with an antibody or antigen binding fragment that specifically binds to the receptor to form a complex of the antibody or antigen binding fragment and the receptor; and (ii) measuring the amount of the complex. In one embodiment, the subject has cancer or is at risk of cancer. In one embodiment, the cancer is a neuro-oncology disease. In one embodiment, cancer is a neuroendocrine tumor. In one embodiment, cancer is selected from the group consisting of: meningioma, ependymoma, glioma, neuroblastoma and diffuse intrinsic pontine glioma. In one embodiment, the subject suffers from a psychiatric disorder or is at risk of suffering from a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, bipolar disorder and major depressive disorder. In one embodiment, the subject suffers from an infection such as a bacterial infection or is at risk of suffering from an 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 an infection of a bacterial species selected from: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter bacteria. In one embodiment, the gram-positive bacterial infection is a Staphylococcus aureus infection. For example, the staphylococcal infection is a Staphylococcus aureus infection (e.g., a methicillin-resistant Staphylococcus aureus (MRSA) infection). In one embodiment, the treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10), a pharmaceutically acceptable salt thereof, or an 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 DRD5 expression measured in the sample relative to a predetermined standard indicates that the subject is likely or unlikely to respond to the treatment regimen.

[0151] On the other hand, methods for assessing the effectiveness of the treatment regimen described herein, monitoring or providing a prognosis for a subject with a condition are provided herein. In one embodiment, these methods include (i) obtaining a biological sample from a 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 the level of a predetermined standard; and (iv) determining the prognosis or whether the subject responds to the treatment regimen based on the comparison of the level measured in the sample with the level of a predetermined standard. In one embodiment, the step of measuring the expression level of a dopamine receptor or GPCR in a sample includes the following steps: (i) contacting the sample with an antibody or antigen binding fragment that specifically binds to a receptor to form a complex of the antibody or antigen binding fragment and the receptor; and (ii) measuring the amount of the complex. In one embodiment, the methods include (i) obtaining a biological sample from a subject; (ii) measuring the gene copy number or mutation of at least one dopamine receptor in the sample; (iii) comparing the gene copy number measured in the sample or the mutation found with the gene copy number or mutation of a predetermined standard; and (iv) determining whether the subject responds to the treatment regimen based on the comparison of the gene copy number measured in the sample or the mutation found with the gene copy number or mutation of a predetermined standard. In one embodiment, the subject suffers from cancer or is at risk of 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 suffers from a psychiatric disorder or is at risk of a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, bipolar disorder and major depressive disorder. In one embodiment, the subject suffers from an infection such as a bacterial infection or is at risk of an 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, infection is selected from the infection of following bacterial species: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter bacteria. In one embodiment, gram-positive bacteria infection is Staphylococcus aureus infection. For example, Staphylococcus infection is Staphylococcus aureus infection (for example, methicillin-resistant Staphylococcus aureus (MRSA) infection).In one embodiment, the treatment regimen includes administering an effective amount of a therapeutic agent, for example, 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.

[0152] 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 the level of DRD5 expression 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, such as 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, such as 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.

[0153] On the other hand, methods are provided herein for identifying whether a subject with a condition is likely to respond to a treatment regimen described herein. In one embodiment, these methods include (i) obtaining a biological sample from a subject; (ii) measuring the gene copy number or mutation of at least one dopamine receptor in the sample; (iii) comparing the gene copy number measured in the sample or the mutation found with the gene copy number or mutation of a predetermined standard; and (iv) determining whether the subject is likely to respond to the treatment regimen based on the comparison of the gene copy number measured in the sample or the mutation found with the gene copy number or mutation of a predetermined standard. In one embodiment, the subject suffers from cancer or is at risk of 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 suffers from a psychiatric disorder or is at risk of a psychiatric disorder. For example, the psychiatric disorder is selected from psychosis, schizophrenia, bipolar disorder, and major depressive disorder. In one embodiment, the subject suffers from an infection such as a bacterial infection or is at risk of an 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 an infection selected from the following bacterial species: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter bacteria. In one embodiment, the Gram-positive bacterial infection is a Staphylococcus aureus infection. For example, Staphylococcus infection is a Staphylococcus aureus infection (e.g., methicillin-resistant Staphylococcus aureus (MRSA) infection). In one embodiment, the treatment regimen includes administering an effective amount of a therapeutic agent, such as 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, the treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, and the mutation in the DRD5 gene measured in the sample indicates that the subject is likely or unlikely to respond to the treatment regimen. In one embodiment, the dopamine receptor is DRD5, the treatment regimen comprises administering an effective amount of a therapeutic agent, such as a compound of formula (10) or a pharmaceutically acceptable salt thereof, and the missense mutation Q366R in the DRD5 gene measured in the sample indicates that the subject is likely or unlikely to respond to the treatment regimen.

[0154] In addition, 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 the response or sensitivity to the treatment methods described herein and to identify subjects who may respond to the treatment methods described herein (such as treatment with a compound of formula (10), a pharmaceutically acceptable salt thereof, or an analog thereof). In addition, 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 assess the effectiveness of the treatment methods described herein or to monitor the treatment methods described herein. In addition, 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 for structurally unrelated anti-cancer molecules in vivo, in vitro, or in silico. For example, competition and other assays can be used to identify drugs that can outperform target interactions with higher affinity to compare changes in those levels with corresponding changes produced by compounds of formula (10) or their analogs. Assays can also be performed on living mammalian cells, which more closely approximate the effects of specific serum drug levels in vivo, or on microsomal extracts prepared from cultured cell lines.

[0155] In one embodiment, the subject has cancer or is at risk of developing cancer. In one embodiment, the treatment regimen comprises administering an effective amount of emilione (such as 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), for example, a compound of formula (45). In one embodiment, the compound of formula (10) is a compound of formula (50), for example, 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 structure of the analog of compound (1) is selected from the structure of formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31).

[0156] The level of the predetermined standard can be, for example, the average or median level measured in a sample from a subject. The level of the predetermined standard can be measured under the same or substantially similar experimental conditions as measuring a sample from a subject. The level of the predetermined standard can be obtained from a subject who responds to treatment with imiridone (such as ONC201) or an analog thereof. In one embodiment, the predetermined standard is obtained from a subject who responds to treatment with a 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 possibly responding to treatment. The level of the predetermined standard can be obtained from a subject who does not respond to treatment with a compound. In one embodiment, the predetermined standard is obtained from a subject who does not respond to treatment with a compound, and if the level in the sample from the subject is different from the level in the predetermined standard (e.g., upregulated or downregulated), the subject can be classified as possibly responding to treatment. The level of the predetermined standard can be obtained from a normal healthy subject.

[0157] Immunoassays can be used to measure protein or methylation levels in a sample and include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), flow cytometry, immunoblotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescence immunoassay (LIA), fluorescence immunoassay (FIA), and radioimmunoassay. 6 AmRNA methylation levels can be obtained by methylated RNA immunoprecipitation (Me-RIP) or other quantitative biochemical assays known in the art.

[0158] Nucleic acid mutations can be determined by any of a number of procedures. For example, a biological sample from a subject is obtained. Non-limiting examples of biological samples include body fluids (e.g., urine, saliva, plasma, or serum) or tissue samples (e.g., cheek tissue samples or cheek cells). The biological sample can then be sequenced or scanned using known methods. For example, at least a portion of the subject's genomic sequence can be analyzed using a DNA array. In addition, whole or partial genomic sequence information can be used. Such sequences can be determined using standard sequencing methods, which include chain termination methods (Sanger dideoxynucleotides), dye terminator sequencing, and SOLID TM Sequencing (Applied Biosystems, Inc., USA). The entire genome can be cut by restriction enzymes or (mechanically) sheared into shorter fragments for sequencing. Methods such as PCR and vector-based cloning methods (e.g., Escherichia coli) can also be used to amplify DNA sequences. 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 using, for example, a conventional DNA sequencer or chip-based technology to identify the presence or absence of mutations or copy number variants.

[0159] In one aspect, provided herein are methods for identifying and treating subjects with a condition who may respond to a treatment regimen described herein. In one embodiment, the method comprises: (i) identifying whether a subject with a condition may respond to a treatment regimen described herein; and (ii) treating a subject determined to be likely to respond to the treatment regimen with a treatment regimen. In one embodiment, the subject has cancer or is at risk of developing cancer. In one embodiment, the treatment regimen comprises administering an effective amount of imiridone (e.g., ONC201) or an analog thereof. In one embodiment, the treatment regimen comprises administering an effective amount of compound (1). 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), for example, a compound of formula (45). In one embodiment, the compound of formula (10) is a compound of formula (50), for example, 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 structure of the analog of compound (1) is selected from the structure of formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31).

[0160] The level of the predetermined standard can be, for example, the average or median level measured in a sample from a subject. The level of the predetermined standard can be measured under the same or substantially similar experimental conditions as measuring a sample from a subject. The level of the predetermined standard can be obtained from a subject who responds to treatment with imiridone (such as ONC201) or an analog thereof. In one embodiment, the predetermined standard is obtained from a subject who responds to treatment with a 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 who does not respond to treatment with a compound. In one embodiment, the predetermined standard is obtained from a subject who responds to treatment with a compound, and if the level in the sample from the subject is different from the level in the predetermined standard (e.g., upregulated or downregulated), 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 measure protein levels in samples.

[0161] In one aspect, provided herein are methods for treating and assessing the effectiveness of a treatment in a subject having a condition. In one embodiment, the method comprises (i) treating the subject according to the methods of treatment described herein and (ii) assessing the effectiveness of the treatment as described herein. In one embodiment, the subject has cancer or is at risk of having cancer. In one embodiment, the treatment regimen comprises administering an effective amount of imiridone (such as ONC201) or an analog thereof. In one embodiment, the treatment regimen comprises administering an effective amount of compound (1). 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), for example, a compound of formula (45). In one embodiment, the compound of formula (10) is a compound of formula (50), for example, 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 structure of the analog of compound (1) is selected from formula (25), formula (26), formula (27), formula (28), formula (29), formula (30) or formula (31).

[0162] Other conditions that may be suitable for the methods described herein include attention deficit disorder; addiction; epilepsy; viral infection; inflammation; neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis; cardiovascular diseases, such as coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, cor pulmonale, arrhythmia, inflammatory heart disease, endocarditis, inflammatory cardiac hypertrophy, myocarditis, valvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, rheumatic heart disease; diabetes; and light chain amyloidosis.

[0163] V. Composition

[0164] In one aspect, a pharmaceutical composition is provided, comprising formula (10): or a compound of formula (1): Compound and pharmaceutically acceptable salt thereof. In one embodiment, the salt is a pharmaceutically acceptable monosalt of the compound. In one embodiment, the salt is a pharmaceutically acceptable disalt of the compound. In one embodiment, the salt is a pharmaceutically acceptable monosalt or polysalt (e.g., disalt or trisalt) selected from the following: hydrochloride, hydrobromide, bisulfate, sulfate, phosphate, fumarate, succinate, oxalate and lactate, acid sulfate, hydroxyl, tartrate, nitrate, citrate, acid tartrate, carbonate, malate, maleate, fumarate sulfonate, methanesulfonate, formate, acetate and carboxylate. In one embodiment, the salt is a salt 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 a salt with a counter ion such as methylamino, dimethylamino, diethylamino and triethylamino counter ions. In one embodiment, the salt is a dihydrochloride or a dihydrobromide.

[0165] Compound (1) (ONC201) has the same chemical structure as compound NSC 350625 as revealed by structural analysis (eg, NMR, X-ray diffraction), which is available from the National Cancer Institute's Developmental Therapeutics Program Repository.

[0166] In one embodiment, the pharmaceutical composition comprises a di-salt (e.g., dihydrochloride) of ONC201 or an analog thereof (e.g., emilidon). Salts (e.g., di-salts or tri-salts) of ONC201 analogs can be prepared from ONC201 analogs, which can be synthesized as described herein or using standard chemical synthesis methodologies known to those of ordinary skill in the art.

[0167] 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, 7th edition, edited by Raymond C. Rowe et al., American Pharmaceutical Association, Washington, USA and Pharmaceutical Press, London; and earlier versions. Exemplary pharmaceutically acceptable carriers, methods of preparing pharmaceutical compositions and various dosage forms, and modes of administration are well known in the art, for example, as described in detail in Pharmaceutical Dosage Forms: Tablets, ed. Larry L. Augsburger & Stephen W. Hoag., London: Informa Healthcare, 2008; and LV Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins, 2004; ARGennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed., 2005, particularly Chapter 89; and J. Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 10th ed., 2001.

[0168] 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, ointments or liquids. In one embodiment, the pharmaceutical composition includes conventional pharmaceutical carriers, such as aqueous, powdered or oily bases, thickeners.

[0169] 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 a compound or its salt 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 a compound or its salt 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 its salt. In one embodiment, the intravenous formulation comprises about 0.05%, 0.5% or 5% of the compound or its salt. In one embodiment, the intravenous formulation comprises a higher or lower concentration of the compound or its salt.

[0170] In one embodiment, the pH of the intravenous formulation is about 3. In one embodiment, the formulation is adjusted to a pH of 3 with a phosphate buffer. In one embodiment, the intravenous formulation comprises dextrose or sodium chloride. In one embodiment, the intravenous formulation comprises a compound or its salt at a concentration of about 5 mg / mL and a pH of 3 and forms a stable solution. In one embodiment, the intravenous formulation comprises a compound or its salt at a concentration of about 5 mg / mL and a pH<5 and forms a stable solution. In one embodiment, the intravenous formulation comprises a compound or its salt and one or more antioxidants. In one embodiment, the intravenous formulation comprises a mixture of a monohydrochloride and a dihydrochloride salt of the compound. In one embodiment, the intravenous formulation comprises a compound or its salt in the form of a 1% solution at a concentration of about 10 mg / mL. For example, the intravenous formulation is a solution at a pH of about 3.3. In one embodiment, the pH is less than 4.0.

[0171] In one embodiment, suitable pharmaceutically acceptable carriers include aqueous carriers. 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 oil.

[0172] In one embodiment, the intravenous formulation comprises ONC201 or its analog or its dihydrochloride salt dissolved in water at 25 mg / mL. In one embodiment, the formulation is adjusted to a pH of 3 with a phosphate buffer. In one embodiment, the formulation comprises dextrose, sodium chloride, or both. In one embodiment, the formulation comprises a dihydrochloride salt of ONC201 or its analog at a higher or lower concentration. In one embodiment, the formulation comprises ONC201 or its analog or its dihydrochloride salt at a concentration of about 5 mg / mL. In one embodiment, a formulation of about 5 mg / mL forms a stable solution and has a pH of 3. In one embodiment, a formulation of about 5 mg / mL has a pH of <5 and forms a stable solution. In one embodiment, the intravenous formulation comprises ONC201 or its analog or its dihydrochloride salt and one or more antioxidants. In one embodiment, the intravenous formulation comprises a mixture of monohydrochloride and dihydrochloride salts of ONC201 or its analog. In one embodiment, the intravenous formulation comprises ONC201 or its analog or its dihydrochloride salt in the form of a 1% solution at a concentration of about 10 mg / mL. For example, the intravenous formulation is a solution with a pH of about 3.3. In one embodiment, the pH is less than 4.0.

[0173] In one embodiment, the intravenous formulation comprises about 0.5% to about 10% (or about 5 mg / mL to about 100 mg / mL) of ONC201, or an analog thereof, or a di-salt thereof. In one embodiment, the formulation comprises about 5% (or about 50 mg / mL) of ONC201, or an analog thereof, or a di-salt thereof. In one embodiment, the intravenous infusion rate can be slowed to reduce the side effects of ONC201, or an analog thereof, or a di-salt thereof.

[0174] In one embodiment, the pharmaceutical composition comprises about 0.1% to 99% of ONC201 salt or its analog; and a pharmaceutically acceptable carrier, for example, 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 its analog in the range of about 5% to about 50%.

[0175] In one embodiment, the pharmaceutical composition comprises an antioxidant. Suitable antioxidants include: ascorbic acid derivatives, such as ascorbic acid, isoascorbic acid, sodium ascorbate; thiol derivatives, such as thioglycerol, cysteine, acetylcysteine, cystine, dithioerythritol, dithiothreitol, glutathione, tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT); sulfites, such as sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium sulfite and sodium thiosulfate; nordihydroguaiaretic acid. It should be noted that antioxidants for aqueous preparations generally include: sodium sulfite, sodium metabisulfite, sodium formaldehyde sulfoxylate or ascorbic acid and combinations thereof, while antioxidants for oil-based solutions and organic solvents include BHT, BHA or propyl gallate and combinations thereof. In yet other embodiments, the antioxidant may be one or more of the following: flavonoids, isoflavones, thioglycerol, L-cysteine, thioglycolic acid, alpha-tocopherol, ascorbyl 6-palmitate, dihydrolipoic acid, BHT, BHA, vitamin E, propyl gallate, beta-carotene, and ascorbic acid. Antioxidants may typically be used at about 0.1% to 1.0%, more typically about 0.2%, by weight.

[0176] In one embodiment, the pharmaceutical composition comprises emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent. For example, the other therapeutic agent is selected from the group consisting of hormone analogs and anti-hormones, 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; microtubule 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 analogs, BTK inhibitors, CRM1 inhibitors (e.g., KPT185), P53 modulators (imidazoline analogs (Nutlin)), anti-angiogenic agents (e.g., axitinib, afliberce pt), sorafenib and regorafenib), amifostin, anagrelid, clodronate, filgrastin, interferon, interferon alfa, folinic acid, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, 2-chlorodeoxyadenosine, 2-fluorodeoxy-cytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allavectin-7, hexamethylmelamine, avocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose, arglabin, arzoxifene, atamestane, atrasentan, auristatin, PE,ABT-199 (Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886 / AZD-6244 (selumetinib), ARRY-704 / AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (pimasertib), Avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic acid 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, ceflatonin, ceftriaxone, celecoxib,Celmoleukin, Cemadotin, CH4987655 / RO-4987655, chlorfenestrol, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicine, Compretin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3, Candida albicans 52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine, doxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diflomotecan, DIDOX, DMDC, dolastatin 10 10), doranidazole, DS-7423, E7010, E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3, ethynylcytidine, ethinyl estradiol yloestradiol), exatecan, exatecan mesylate, exemestane, exisulind, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, galliummaltolate, gefinitib, 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 trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, herceptin, hexomethionine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, etoposide ibrutinib, ibritumomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitors, IMC-1C11, IMC-A12 (cixutumumab), IMMUNOL, indisulam, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha-2b, interleukin- 2. INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B, isoflavones, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, kahalid F F), ketoconazole, KW-2170, KW-2450, chloroplatin, leflunomide, lenograstim, leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat,dichloromethyl diethylamine, 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 (daratumumab), MLN518, motexaf ingadolinium), MS-209, MS-275, MX6, neridronate, neratinib, nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, nolatrexed, norelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oncophage , oncoVEXGM-CSF, ormiplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, P D-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, methyl pivalate butyrate, pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, polyporous acid, porfiromycin, prednisone,prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY 869766, RDEA-436, butterfly mycin analogs, receptor tyrosine kinase (RTK) inhibitors, regorafenib, revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim, satraplatin, SB 408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tacedinaline, talaporfin, tarceva, tariquitar, tasisulam, taxotere, taxoprexin, tazarotene, fluoxetine, temozolamide, tesmilifene, testosterone, testosterone propionate, tesmilifene, tetraplatinum, tetrodotoxin, tezacitabine, thalidomide, theralux, therarubicin, thymalfasin,Thymectacin, tiazofurin, tipifarnib, tomudex, tirapazamine, tocladesine, toremofin, trabectedin, TransMID-107, trans-retinoic acid, tretinoin, trastuzumab, tremelimumab, triacetylguanylate, triapine, triciribine, trimetrexate, TLK-286, TXD 258, Tykerb / Tyverb, urocidin, valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1, WX-554, vectibix, xeloda, XELOX, 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.

[0177] In one embodiment, the additional therapeutic agent comprises a hormone analog, an anti-hormone, or both selected from the group consisting of tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxy-progesterone, octreotide, and combinations thereof. In one embodiment, the other therapeutic agents include one or more LHRH agonists selected from the group consisting of goserelin acetate, leuprolide acetate, triptorelin pamoate, and combinations thereof, and wherein the LHRH antagonists are selected from the group consisting of degarelix, cetrorelix, abarelix, ozarelix, degarelix, and combinations thereof. In one embodiment, the other therapeutic agents include one or more growth factor inhibitors selected from the group consisting 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 agents include one or more inhibitors of human epidermal growth factor selected from HER2, HER3, and HER4. In one embodiment, the other therapeutic agents include one or more tyrosine kinase inhibitors selected from the group consisting of cetuximab, gefitinib, imatinib, lapatinib, and trastuzumab, and combinations thereof. In one embodiment, the other therapeutic agents include one or more aromatase inhibitors selected from the group consisting of anastrozole, letrozole, liarozole, vorozole, exemestane,Atamestane and combinations thereof. In one embodiment, other therapeutic agents include one or more antimetabolites selected from the following antifolates: methotrexate, raltitrexed and pyrimidine analogs. In one embodiment, other therapeutic agents include one or more antimetabolites selected from the following pyrimidine analogs: 5-fluorouracil, capecitabine and gemcitabin. In one embodiment, other therapeutic agents include one or more antimetabolites selected from the following purine and / or adenosine analogs: mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine, fludarabine and combinations thereof. In one embodiment, other therapeutic agents include one or more antitumor antibiotics selected from the following: anthracyclines (doxorubicin, daunorubicin, epirubicin and idarubicin), mitomycin-C, bleomycin, actinomycin, plicamycin, streptozocin and combinations thereof. In one embodiment, other therapeutic agents include one or more platinum derivatives selected from the group consisting of cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment, other therapeutic agents include one or more alkylating agents selected from the group consisting of estramustin, dichloromethyldiethylamine, melphalan, chlorambucil, busulfan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, and combinations thereof. In one embodiment, other therapeutic agents include nitrosoureas selected from the group consisting of carmustin, lomustin, thiotepa, and combinations thereof. In one embodiment, other therapeutic agents include antimitotic agents selected from the group consisting of vinca alkaloids and taxanes. In one embodiment, the other therapeutic agent comprises one or more taxanes selected from the group consisting of paclitaxel, docetaxel, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more vinca alkaloids selected from the group consisting of vinblastine, vindesin, vinorelbin,Vincristine and combinations thereof. In one embodiment, other therapeutic agents include one or more topoisomerase inhibitors, which are epipodophyllotoxins. In one embodiment, other therapeutic agents include one or more epipodophyllotoxins selected from the following: etoposide (etoposide) (etopophos), teniposide, amsacrin, topotecan, irinotecan, mitoxantrone and combinations thereof. In one embodiment, other therapeutic agents include one or more serine / threonine kinase inhibitors selected from the following: PDK 1 inhibitors, B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTOR / PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1 inhibitors, CDK inhibitors, Aurora kinase inhibitors and combinations thereof. In one embodiment, other therapeutic agents include one or more tyrosine kinase inhibitors, which are PTK2 / FAK inhibitors. In one embodiment, the other therapeutic agents include one or more protein-protein interaction inhibitors selected from the group consisting of IAP, Mcl-1, MDM2 / MDMX, and combinations thereof. In one embodiment, the other therapeutic agents include one or more rapamycin analogs selected from the group consisting of everolimus, temsirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment, the other therapeutic agents include one or more therapeutic agents selected from the group consisting of amifostine, anagrelide, clodronate, filgrastim, interferon, interferon alpha, folinic acid, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, and combinations thereof. In one embodiment, the other therapeutic agents include one or more therapeutic agents selected from the group consisting of 2-chlorodeoxyadenosine, 2-fluorodeoxy-cytidine, 2-methoxyestradiol, 2C4, 3-orexin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A 105972, A 204197, Abiraterone, Aldesleukin, Alitretinoin, Alevetin-7, Hexamethylmelamine, Avosidix, Aminofide, Anthrapyrazole, AG-2037, AP-5280, Apaziquin, Apomin, Aranos, Agaleb, Arzoxifene, Atamestane, Atrasentan, Auristatin, PE, ABT-199 (Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (Ganitozumab), ARRY 162, ARRY 438162,ARRY-300, ARRY-142886 / AZD-6244 (selumetinib), ARRY-704 / AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (pimacitinib), Avastin, AZD-2014, azacytidine, azaepotinib, azonafidine, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, bilicodar citrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomoxicillin), BIBF 1120 (vegatinib), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycin, bleomycin A, bleomycin B, brivanib, bryostatin 1, bortezomib, bromocriptine, busulfan, BYL-719, CA-4 prodrug, CA-4, casai, calcitriol, canertinib, canfluamine, capecitabine, carboxyplatinum phthalate, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1 cefixime, homoharringtonine, ceftriaxone sodium, celecoxib, simulleukin, simulant Dotin, CH4987655 / RO-4987655, chlorfenestrol, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, Compretin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 Candida albicans 52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, chlorfenapyr, cytarabine, D 24851, decitabine, doxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepotin B, dexamethasone, dexrazoxane, diethylstilbestrol, diflutecan, DIDOX, DMDC, Aplysia 10, doladazol, DS-7423, E7010, E-6201, edatrexamate, edotreotide, efoxirox, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, esamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib,ET-18-0CH3, cytidine, ethinyl estradiol, exitecan, exitecan mesylate, exemestane, exesulin, retinoic acid phenamide, fentolimumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, garubicin, gallium maltolate, gefitinib, gemtuzumab, gematine, glucosfamide, GCS-100, GDC-0623, GDC-0941 (pitrilisib), GDC-0980, GDC-003 2. 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-2141 795, GW2016, granisetron, Herceptin, hexamethylenetetramine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibrutinib, ibritumomab tiuxetan, idatrexone, idestrone, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (citumumab), IMMUNOL, indesulan, interferon α-2a, interferon α-2b, pegylated interferon α-2b, interleukin-2, INK-1117 , INK-128, INSM-18, yonafarnib, ipilimumab, iproplatin, ilofofen, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, kahalide F, ketoconazole, KW-2170, KW-2450, levoplatin, leflunomide, linoglalast, leuprolide, leuprolide, lexidrone samarium, LGD-1550, linezolid, texafin lutetium, lometrexol, loxantrone, LU 223651, lutetotecan, LY-S6AKT1, LY-2780301, mafosfamide, mamastat, dichloromethyl diethylamine, 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 (daratumumab), MLN518, motesafungin, MS-209, MS-275, MX6, neridronate, neratinib, resova, nevasostat, nilotinib, nimesulide, nitroglycerin, noratript, norrelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oxafungin, oncoVEXGM-CSF,Omistim, Ortasai, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, pyrroloxanone, estrogen, panitumumab, patupirone, 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, Paetuzumab, 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, Pivalic acid methyl butyrate, Pixantrone, Bentotil O, PKI166, Pletrexed, Plicamycin, Muscarinic acid, Porfiramycin, Prednisone, Prednisolone, Quinmid, Quinupristin, R115777, RAF-265, Ramosetron, Ranopus enzyme, RDEA-119 / BAY 869766, RDEA-436, butterfly mycin analogs, receptor tyrosine kinase (RTK) inhibitors, REVLIMID, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizobactam, rhu-MAb, linfibeb, risedronate, rituximab, rotuximab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, benzohydrazide, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalbicin, SAHA, sargramostim, satraplatin, SB408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustine, ciocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, nilinyl hydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tekdinalin, talaporfin, tasiva, taquita, tassolan, taxotere, taspocin, tazarotene, fluoxetine, temozolomide, tesmilifen, testosterone, testosterone propionate, tesmilifen, tetraplatinum, tetrodotoxin, tezacitabine, thalidomide, cerules, pirarubicin, thymofasin, simikercin, thiazofurine, tipifarnib, toudex, tirapazamine, toradesin, toremofin, trabectedin, TransMID-107, trans-retinoic acid,Retinoic acid, trastuzumab, tremelimumab, triacetylguanylate, trastuzumab, traciribine, trimetrexate, TLK-286, TXD 258, Tykerb / Tavipo, Urocetin, Valrubicin, Vatalanib, Vincristine, Vinflunine, Virulin, WX-UK1, WX-554, Vectibix, Xeloda, XELOX, 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, Azoquinoline and combinations thereof.

[0178] In one embodiment, the additional therapeutic agent comprises a steroid including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazole. In one embodiment, the additional therapeutic agent comprises an antiemetic, including but not limited to 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), The invention relates to drugs that can be used to treat schizophrenia, ...

[0179] In one embodiment, the other therapeutic agent comprises an anticancer agent, and the anticancer agent comprises a mitotic inhibitor. In one embodiment, the mitotic inhibitor comprises a taxane. In one embodiment, the mitotic inhibitor comprises a taxane selected from paclitaxel and docetaxel.

[0180] In one embodiment, the pharmaceutical composition comprises emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof; and at least one anticancer agent, the at least one anticancer agent comprising one or more of the following: acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, hexamethylmelamine, ambomycin, ametrine, metantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene, binasafide dimesylate dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, cristalin mesylate,mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate, eflornithine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine , fadrozole, fazarabine, retinamide, floxuridine, fludarabine, fluorouracil, flurocitabine, fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, including recombinant interleukin II or rIL 2), interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-Ia, interferon γ-Ib, iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine hydrochloride hydrochloride), megestrol acetate, melengestrolacetate, melphalan, menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid,acid, nelarabine, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride hydrochloride), plicamycin, promestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine, simtrazene, sparlfosate, sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin ), streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone, triciribine, trimetrexate, triptorelin, tubulozole, uracil mustardmustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, zorubicin, and combinations thereof.

[0181] In one embodiment, the pharmaceutical composition comprises emiridone (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof; and at least one anticancer agent, the at least one anticancer agent comprising one or more of the following: lorlatinib (LORBRENA), pembrolizumab (KEYTRUDA), talazoparib (TALZENNA), emicizumab-kxwh injection (HEMLIBRA), cemiplimab-rwlc (LIBTAYO), dacomitinib tablets (VIZIMPRO), duvelisib (COPIKTRA), moxetumomab-tdfk (moxetumomab pasudotox-tdfk (LUMOXITI), nivolumab (OPDIVO), mogamulizumab-kpkc (Poteligeo), lusutrombopag (Mulpleta), iobenguane I-131 (iobenguane I-131 (AZEDRA), ivosidenib (Tibsovo), ribociclib (Kisqali), enzalutamide (XTANDI), ipilimumab (YERVOY), encorafenib and binimetinib (BRAFTOVI and MEKTOVI), bevacizumab (Avastin), venetoclax (VENCLEXTA), methoxy polyethylene glycol-epoetin beta (Mircera), Fulphila (pegfilgrastim-jmdb), avatrombopag (Doptelet), Retacrit (epoetin alfa-epbx (epoetin alfa-epbx), tisagenlecleucel (KYMRIAH), dabrafenib (TAFINLAR), trametinib (MEKINIST), fostamatinib disodium hexahydrate tablets (tablets (TAVALISSE), everolimus tablets for oral suspension (AfinitorDisperz), rucaparib, nilotinib (TASIGNA), abemaciclib (VERZENIO), apalutamide, abiraterone acetate (Zytiga), lutetium Lu 177dotatate (LUTATHERA), afatinib (Gilotrif), olaparib tablets ( tablets (olaparib (Lynparza)), pertuzumab (PERJETA), bosutinib (BOSULIF), cabozantinib (Cabometyx), Ogivri (trastuzumab-DKST), sunitinib malate (sunitinib), obinutuzumab (GAZYVA), emicizumab-kxwh (HEMLIBRA), dasatinib (SPRYCEL), brentuximab vedotin (ADCETRIS), alectinib (ALECENSA), vemurafenib (ZELBORAF), acalabrutinib (Calquence), axicabtagene sildenafil (Avastinib), and sutuzumab-dkst (SUTZ). ciloleucel (YESCARTA), pomacicillin (VERZENIO), copanlisib (ALIQOPA), Mvasi (bevacizumab-awwb), gemtuzumab ozogamicin (Mylotarg), inotuzumab ozogamicin (BESPONSA), liposome-encapsulated daunorubicin and cytarabinecombination (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), durvalumab (IMFINZI), brigatinib (ALUNBRIG), midostaurin (RYDAPT), regorafenib (STIVARGA), palbociclib (IBRANCE), osimertinib (TAGRISSO), niraparib (ZEJULA), avelumab (BAVEN CIO), ribociclib (KISQALI), lenalidomide (Revlimid), rucaparib (RUBRACA), daratumumab (DARZALEX), olaratumab (LARTRUVO), atezolizumab (TECENTRIQ), erlotinib (TARCEVA), cabozantinib (CABOMETYX), defibrotide sodium (Defitelio), crizotinib capsules (Xalkori), everolimus (Afinitor), eribulin (HALAVEN injection), and combinations thereof.

[0182] In one embodiment, the pharmaceutical composition comprises emetrinone (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof; and at least one anticancer agent, the at least one anticancer agent comprising one or more of the following: elotuzumab (EMPLICITI), necitumumab (PORTRAZZA), ixazomib (NINLARO), cobimetinib (COTELLIC tablets), talimogene laherparepvec (IMLYGIC), trabectedin (Yondelis Injection), irinotecan lipid injection (ONIVYDE), idarucizumab (Praxbind Injection), 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), belinostat (BELEODAQ), ceritinib (ZYKADIA), mercaptopurine (Purixan), siltuximab (Sylvant Injection),Injection), ofatumumab (ofatumumab injection (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.

[0183] Examples of suitable anticancer agents include those described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, eds. Laurence Brunton, Bruce Chabner, Bjorn Knollman, McGraw Hill Professional, 2010.

[0184] In some exemplary embodiments, the pharmaceutical composition comprises a salt (e.g., monosalt or disalt) of emilione (e.g., ONC201) or an analog thereof and at least one other therapeutic agent, wherein the other therapeutic agent comprises an anti-angiogenic agent, e.g., bevacizumab. In one embodiment, the anti-angiogenic agent is selected from aflibercept, axitinib, angiostatin, endostatin, 16 kDa prolactin fragment, laminin peptide, fibronectin peptide, tissue inhibitor of metalloproteinase (TIMP 1, 2, 3, 4), plasma plasminogen activator inhibitor (PAI-1, -2), tumor necrosis factor α (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-methoxyoestradiol, proliferative protein-related protein, suramin, sorafenib, regorafenib, thalidomide, cortisone, linomide, fumagillin (AGM-1470; TNP-470), tamoxifen, retinoic acid, CM101, dexamethasone, leukemia inhibitory factor (LIF), hedgehog inhibitor and combinations thereof.

[0185] The drug combination can include any desired ratio of the first therapeutic agent and the second therapeutic agent, as long as a synergistic or cooperative effect still occurs. The ratio of the first therapeutic agent and the second therapeutic agent of the synergistic drug combination is preferably about 1:9 to about 9:1. In one embodiment, the ratio of the first therapeutic agent and the second therapeutic agent of the synergistic combination is 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 ratio of the synergistic combination therapeutic agent is about 1:1.

[0186] 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, Retinoic Acid, Vandetanib, Vismodegib, Vorinostat, and combinations thereof.

[0187] In one embodiment, the second therapeutic agent comprises a small molecule multikinase inhibitor, such as sorafenib or regorafenib. In one embodiment, the second therapeutic agent comprises a hedgehog pathway inhibitor, such as vismodegib. In one embodiment, the second therapeutic agent comprises a drug selected from Table 2 below.

[0188] Table 2: Drug categories

[0189]

[0190]

[0191]

[0192] In one embodiment, the second therapeutic agent includes a drug targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor. In one embodiment, the second therapeutic agent includes a recombinant TRAIL or agonist antibody that activates one or more TRAIL receptors. In one embodiment, the second therapeutic agent includes one or more antibodies or recombinant TRAIL that activate signal transduction through DR4, DR5 or both. In one embodiment, the second therapeutic agent includes one or more of the following: AMG-655, LBY-135, mapatumumab, lexatumumab, Apobab and rhApo2L / TRAIL. In one embodiment, the second therapeutic agent includes an active agent selected from the group consisting of 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, ganituzumab, interferon gamma, sorafenib, XIAP antagonists, Bcl-2 antagonists, and Smac mimetics.

[0193] VI. Dosage

[0194] In one embodiment, the pharmaceutical composition comprises the following dosage range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 40, 50, 60 or 100 mg to about 2000 mg; about 4, 5, 6 or 10 mg to about 200 mg; about 0.4, 0.5, 0.6 or 1 mg to about 20 mg, wherein the weight can be based on the compound in its free base form. In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 50 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or about 5 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200 mg; or about 0.5 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mg. In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 40 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or more. 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 about 0.4 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 mg. In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 60 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or more. from about 6 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 from about 0.6 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg.In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 100 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 mg. g or 2000 mg; about 10 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 about 1 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg. In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof: about 200 mg to about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg. from about 20 mg to about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg; or from about 2 mg to about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg, based on the compound in its free base form. In one embodiment, the pharmaceutical composition comprises the following dosage level range of emilione (such as ONC201) or an analog thereof, or a pharmaceutically acceptable salt thereof: about 400 mg to about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 mg; about 40 mg to about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg; or about 4 mg to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg based on the compound in its free base form.In one embodiment, the pharmaceutical composition comprises the following dosage level ranges of emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof: about 50 mg to about 60, 70, 80, 90 or 100 mg; about 60 mg to about 70, 80, 90 or 100 mg; about 70 mg to about 80, 90 or 100 mg; about 80 mg to about 90 or 100 mg; about 90 mg to about 100 mg; about 5 mg to about 6, 7, 8, 9 or 10 mg; about 6 mg to about 7, 8, 9 or 10 mg; about 7 mg to about 8, 9 or 10 mg; 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.

[0195] In one embodiment, the pharmaceutical composition comprises the following dosage range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: 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 the following dosage level range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: about 1, 2, 3, 4, 5, 6, 7, 8 or 9 mg / kg to about 10, 20, 30 or 40 mg / kg; about 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mg / kg to about 20, 30 or 40 mg / kg; about 20, 21, 22, 23, 24, 25, 26, 27 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 2.0, 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 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 4.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9 mg / kg to about 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 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.

[0196] In one embodiment, the pharmaceutical composition comprises the following dosage range of emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof: about 37.5 mg / m 2 About 1500 mg / m 2 ; about 3.75mg / m 2About 150 mg / m 2 ; or about 0.4 mg / m 2 About 15 mg / m 2In one embodiment, the pharmaceutical composition comprises the following dosage range of emilione (such as ONC201) or its analog or its pharmaceutically acceptable salt: 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, 460 55, 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, 61 0, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765 、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895、900、905、910、915、920、 925、930、935、940、945、950、955、960、965、970、975、980、985、990、995、1000、1005、1010、1015、1020、1025、1030、1035、1040、1045、1050、1055、1060、 1065、1070、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、1340、13 45, 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 1500 mg / m 2 ; about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 8 6, 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 150 mg / m 2 or 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 15 mg / m2 .

[0197] VII. Dosage Form

[0198] The pharmaceutical composition used in the methods described herein is formulated into a dosage form that can be applied to a patient. In one embodiment, the composition is in the form of an oral or parenteral dosage unit. In one embodiment, it is in the form of an oral dosage unit. In one embodiment, the oral dosage unit is graded into several smaller doses that are applied to a subject within a predetermined time period to reduce the toxicity of the therapeutic agent applied. In one embodiment, the oral dosage unit is applied as a tablet or capsule containing a controlled release formulation, which can include multiple particles, granules, pills, 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) and intramuscular (M), rectal (PR) or transdermal. In one embodiment, the dosage form is selected from sterile solutions, suspensions, suppositories, tablets and capsules. In one embodiment, the oral dosage form is selected from tablets, caplets, capsules, lozenges, syrups, liquids, suspensions and elixirs. In one embodiment, the oral dosage form is selected from tablets, hard shell capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solids and semisolids.

[0199] In one embodiment, the pharmaceutical composition used in the methods described herein comprises a dermatological composition suitable for topical application to the skin. For example, the dermatological composition comprises a pharmaceutically or cosmetically acceptable medium. Dermatological compositions for topical application include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In some cases, it may be necessary or desirable and therefore conventional pharmaceutical carriers (aqueous, powder or oily bases, skin enhancers, thickeners) may be used. Suitable enhancers include ethers, for example, diethylene glycol monoethyl ether (which can be used as obtained) and diethylene glycol monomethyl ether; surfactants, for example, sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and lecithin; alcohols, for example, ethanol, propanol, octanol, benzyl alcohol; polyethylene glycol and its esters, for example, polyethylene glycol monolaurate; amides and other nitrogen-containing compounds, for example, urea, 2-pyrrolidone, l-methyl-2-pyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF), ethanolamine, diethanolamine and triethanolamine; terpenes; alkanones; and organic acids, especially citric acid and succinic acid. It is also possible to use and sulfoxides, e.g., DMSO and C10MSO, but are less preferred.

[0200] In one embodiment, the dosage form is selected from sustained release, controlled release, delayed release, and responsive release.

[0201] VIII. How to use

[0202] The compositions and methods described herein have utility in treating many disease conditions, including cancer (e.g., colorectal 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 tumors, chondrosarcomas, leptomeningeal disease, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal or rectal cancer, appendix cancer, astrocytomas, and atypical teratoid / rhabdoid tumors. 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 tumors, breast cancer, bronchial tumors, Burkitt's lymphoma, and spinal cord tumors. In one embodiment, the compositions and methods described herein are used to treat diseases such as carcinoid tumors, malignant tumors of unknown primary focus, atypical teratoid / rhabdoid tumors of the central nervous system, leptomeningeal diseases, embryonal tumors of the central nervous system, lymphomas of the central nervous system, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelocytic blood disease, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, and cutaneous T-cell lymphoma (including Sezary syndrome and mycosis fungoides (MF)). In one embodiment, the compositions and methods described herein are used to treat diseases such as embryonal tumors of the central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing's sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, and eye cancer (including intraocular melanoma and retinoblastoma). In one embodiment, the compositions and methods described herein are used to treat diseases such as gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic tumors, and gliomas. In one embodiment, the compositions and methods described herein are used to treat cancers selected from the group consisting of hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, and hypopharyngeal cancer. In one embodiment, the compositions and methods described herein are used to treat diseases such as Kaposi's sarcoma and kidney (renal cell) cancer. In one embodiment, the compositions and methods described herein are used to treat diseases such as Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), non-Hodgkin 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 neck cancer with occult primary, multiple endocrine neoplasia syndrome, oral cancer, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, complex karyotype blast phase leukemia, myelodysplastic / myeloproliferative neoplasms, 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 carcinoma and neuroblastoma. In one embodiment, the compositions and methods described herein are used to treat diseases such as oral cancer, lip and oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian germ cell tumors, ovarian epithelial cancer and ovarian low malignant potential tumors. In one embodiment, the compositions and methods described herein are used to treat diseases such as pancreatic cancer, papillomatosis, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumors, pineoblastomas and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blastomas, pregnancy and breast cancer, primary central nervous system lymphomas and prostate cancer. In one embodiment, the compositions and methods described herein are used to treat cancers selected from the group consisting of: rectal cancer, renal pelvis and ureteral renal cell (kidney) cancer, respiratory cancer involving the NUT gene on chromosome 15, retinoblastoma and rhabdomyosarcoma. In one embodiment, the compositions and methods described herein are used to treat high-grade 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 low-grade 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.

[0203] Compound (1) (ONC201) has broad anticancer activity, low toxicity (including few side effects, if any), low genotoxicity, and high bioavailability, including oral bioavailability, in in vitro models, animal models, and human clinical trials. These characteristics allow ONC 201 and various analogs to be particularly suitable for pediatric patients. These characteristics also make ONC201 and various analogs particularly suitable for chronic therapy, high-risk patients, and to ensure long-term response or stabilization of the disease or to prevent disease recurrence.

[0204] In another aspect, provided herein are methods for treating or preventing cancer in a subject in need thereof, the methods comprising: administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of compound (1)

[0205]

[0206] Or a pharmaceutically acceptable salt thereof, wherein the cancer involves the midline structure of the brain and has a histone H3 K27M mutation. 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 H3.3K27M or H3.1 K27M. In one embodiment, the histone H3 K27M mutation is in one or more histone genes selected from the group consisting of: H3F3A, H3F3B, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I or HIST1H3J. In one embodiment, in cancer tissue, DRD2 is overexpressed, 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.

[0207] In another aspect, provided herein are methods for treating or preventing cancer in a subject in need thereof, the methods 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. In one embodiment, the cancer is selected from the group consisting of: a central nervous system tumor, a brain tumor, a peripheral nervous system tumor, a pheochromocytoma, a paraganglioma, an adrenocortical carcinoma, an adrenal tumor, and a neuroendocrine tumor. In one embodiment, the cancer is from a glial cell and is selected from the group consisting of: a meningioma, an ependymoma, an oligodendroglioma, an astrocytoma, an optic glioma, a pinealoma, a rhabdoid tumor, and a diffuse intrinsic pontine glioma. In one embodiment, the cancer is from a neural cell and is selected from the group consisting of: a medulloblastoma, a neuroblastoma, a ganglioma, a primitive neuroectodermal tumor, and a 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 K27M mutation of the cancer 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, in cancer tissue, DRD2 is overexpressed, 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.

[0208] In another aspect, provided herein are methods for treating or preventing cancer in a subject in need thereof, the methods 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 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, wherein the histone H3 mutation is H3.3 K27M or H3.1 K27M. In one embodiment, the histone H3 K27M mutation of the cancer 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, in cancer tissue, DRD2 is overexpressed, 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.

[0209] In one embodiment, the compositions and methods described herein are used to treat pediatric cancers (e.g., pediatric solid tumors, pediatric sarcomas, pediatric Ewing's sarcoma, pediatric gliomas, pediatric central nervous system cancers, pediatric neuroblastomas, pediatric leukemias, and pediatric lymphomas).

[0210] 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, Sezary syndrome, skin cancer, eye cancer, skin cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, 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, laryngeal cancer, thymoma and thymic cancer, 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: cancer of unknown primary site, cancer of unknown primary site, childhood abnormal cancer, 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 female cancers.

[0211] In one embodiment, the compositions and methods described herein are used as first-line therapy (sometimes referred to as first-line 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" means a therapeutic agent that can be used with any regimen after the failure of the subject's primary treatment regimen or after the subject's condition does not respond to the primary treatment. In one embodiment, the compositions and methods described herein are used as rescue therapy. In one embodiment, the compositions described herein are used as rescue agents to offset the effects of the initial treatment. In one embodiment, the compositions described herein are used as rescue agents for subjects who are resistant to standard or initial treatment. In one embodiment, the compositions and methods described herein are used as neoadjuvant therapy. In one embodiment, neoadjuvant therapy includes administering one or more therapeutic agents described herein to the subject before the main or first-line treatment. In one embodiment, before administering the main or first-line treatment to the subject receiving treatment, the neoadjuvant therapy reduces the size or extent of the cancer being treated. In one embodiment, the compositions and methods described herein are used as adjuvant therapy. In one embodiment, adjunctive therapy comprises administering to a subject one or more therapeutic agents described herein, wherein the one or more therapeutic agents modify the effect of other therapeutic agents that have been administered to the subject or that are administered concurrently or subsequently to the subject.

[0212] In one embodiment, the compositions and methods described herein show a reduced chance of drug-drug interactions. In one embodiment, emilione (such as ONC201) or an analog thereof is eliminated from a patient's body before emilione can interact with another pharmaceutically active agent.

[0213] In one embodiment, the compositions and methods described herein exhibit toxicity levels that facilitate combination with other pharmaceutical agents.

[0214] The compositions and methods described herein are not limited to specific animal species. In one embodiment, the subject treated according to the methods described herein 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, cattle, 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 sex and 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.

[0215] The compositions and methods described herein are not limited to subjects of a particular age. In one embodiment, subjects treated according to the methods described herein and using the compositions described herein are over 50 years old, over 55 years old, over 60 years old, or over 65 years old. In one embodiment, subjects treated according to the methods described herein and using the compositions described herein are under 50 years old, under 55 years old, under 60 years old, or under 65 years old.

[0216] In one embodiment, the subject treated according to the methods described herein and using the compositions described herein is a pediatric patient. In one embodiment, the pediatric patient is less than 18 years old, less than 17 years old, less than 16 years old, less than 15 years old, less than 14 years old, less than 13 years old, less than 12 years old, less than 11 years old, less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old. In one embodiment, the pediatric patient is less than 12 months old, less than 11 months old, less than 10 months old, less than 9 months old, less than 8 months old, less than 7 months old, less than 6 months old, less than 5 months old, less than 4 months old, less than 3 months old, less than 2 months old, less than 1 month old. In one embodiment, the pediatric patient is less than 4 weeks old, less than 3 weeks old, less than 2 weeks old, less than 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.

[0217] 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.

[0218] 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, folinic acid, or lenalidomide.

[0219] In one embodiment, the subject has been treated with radiation therapy. In one embodiment, the subject has been treated with surgery. In one embodiment, the subject has been treated with adoptive T cell therapy.

[0220] In one embodiment, the cancer is no longer responsive to treatment with ibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, folinic acid, lenalidomide, radiation, surgery, or a combination thereof.

[0221] In one embodiment, the dose response relationship of the compositions and methods described herein in cancer cells is different from the dose response relationship of the same compositions and methods in normal cells. After being treated with different concentrations of ONC201 for 72 hours, the dose response relationship of ONC201 on proliferation and cell death in normal cells and tumor cells was determined by measuring cell viability. The tumors tested included human colon cancer cell lines (HCT116), breast tumor cell lines (MDA-MB-231), and human primary glioblastoma cell lines (U87). And the normal cells tested included human foreskin fibroblasts (HFF), human fetal lung fibroblasts (MRC-5), and human lung fibroblast cell lines (WI-38). Doxorubicin was used as a positive control at 1 μg / mL in normal fibroblasts. The cell viability of the normal cells tested when ONC201 was about 1 to 5 mg / mL was at least about 75%, while at the same ONC201 concentration, the viability of the tumor cells was significantly lower (e.g., equal to or less than 50%). In addition, when ONC201 concentrations were increased above about 5 mg / mL, the viability of tumor cells dropped below 25%, while the viability of normal cells remained at about 75%. Cell viability assays were performed in human fetal lung fibroblasts (MRC-5) after 72 hours of treatment with compound (1) (5 μM) or DMSO and during the recovery period after treatment in a completely drug-free medium. Cell recovery was seen with ONC201, but not with DMSO.

[0222] In one embodiment, the compositions and methods described herein can be used to treat cancer in a subject. In one embodiment, the compositions and methods described herein can be used to treat cancer in a human subject. In one embodiment, the method of treatment comprises administering a pharmaceutically effective amount of emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier to a subject in need of such treatment.

[0223] In one embodiment, the method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent in combination with (ii) a second therapeutic agent, the first therapeutic agent comprising emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof, wherein the first therapeutic agent and the second therapeutic agent 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 following dihydrochloride salts:

[0224]

[0225] It is understood that the dihydrochloride salt of ONC201 or its analogs (including compounds of formula (10)) or alternative di-salts thereof apparent from the teachings of this disclosure may be substituted for ONC201 or its analogs in the compositions or dosing regimens described herein.

[0226] In one embodiment, the method of treatment comprises administering a synergistic drug combination to a subject in need of such treatment, either simultaneously or sequentially, wherein the synergistic drug combination comprises (i) a first therapeutic agent comprising emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof; and (ii) a second therapeutic agent. In one embodiment, the method of treatment comprises administering a therapeutically synergistically effective amount of the first therapeutic agent and the second therapeutic agent in combination to a subject in need of such treatment, either simultaneously or sequentially. In one embodiment, the method of treatment comprises administering an effective amount of the first therapeutic agent in combination with an effective amount of the second therapeutic agent to a subject in need of such treatment, wherein the combination provides a synergistic effect in the in vivo treatment of a cancer sensitive to the combination, and wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially. In one embodiment, the method of treatment comprises administering an effective amount of the first therapeutic agent in combination with an effective amount of the second therapeutic agent to a subject in need of such treatment, wherein the combination provides a synergistic effect in the in vivo treatment of minimal residual disease sensitive to the combination, and wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially. In one embodiment, the second agent is administered before the first agent.

[0227] 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.

[0228] In one embodiment, the treatment targets a solid tumor, wherein the solid tumor is selected from the group consisting of: cervical cancer, endometrial cancer, extracranial germ cell tumors; extragonadal germ cell tumors; germ cell tumors; gestational trophoblastic tumors; ovarian cancer, ovarian germ cell tumors, ovarian epithelial cancer, and ovarian low-grade potential tumors; penile cancer, prostate cancer; pregnancy and breast cancer; high-grade prostate cancer; intermediate-grade prostate cancer; low-grade prostate cancer; castration-resistant prostate cancer; breast cancer; bile duct cancer; extrahepatic bile duct cancer; gallbladder cancer; hepatocellular (liver) cancer; renal (renal cell) cancer; liver cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer; basal cell carcinoma; basal cell nevus syndrome, Guillain-Nevus syndrome, melanoma, Merkel cell carcinoma, papillomatosis, multiple endocrine neoplasia syndrome; pancreatic cancer, parathyroid cancer, ocular melanoma; eye cancer; retinoblastoma; malignant fibrous histiocytoma; Ewing's sarcoma family; desmoplastic round cell tumor; chondrosarcoma, Kaposi's sarcoma, rhabdomyosarcoma; spinal cord tumors, soft tissue tumors Meningeal diseases, embryonal tumors of the central nervous system, chordoma, embryonal tumors of the central nervous system, ependymoma, ependymoma, neuroblastoma; moderately differentiated pineal parenchymal tumor, pinealoblastoma; adrenal cortical carcinoma; bone cancer, osteosarcoma; malignant fibrous histiocytoma of bone and osteosarcoma; osteosarcoma and malignant fibrous histiocytoma of bone; carcinoid tumor, tumor of unknown primary, bronchial tumor, lung cancer, pleuropulmonary blastoma; respiratory cancer involving the NUT gene on chromosome 15, stellate cell carcinoma CNS atypical teratoid / rhabdoid tumor, craniopharyngioma, glioma, brain cancer, medulloblastoma, medullary epithelioma, supratentorial primitive neuroectodermal tumor, pituitary tumor, gastric 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 cancer, cervical squamous cell carcinoma with occult metastasis of primary lesion, oral cancer, nasal and paranasal sinus cancer, nasopharyngeal cancer, oral cancer, lip and oral cancer, oropharyngeal cancer, paranasal sinus and nasal cancer, pharyngeal cancer, head and neck cancer and mesothelioma.

[0229] In one embodiment, the treatment targets a lymphoma selected from the group consisting of diffuse large B-cell lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, Sezary syndrome, mycosis fungoides (MF); histiocytosis; Burkitt's 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 treatment 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.

[0230] 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 disorders; hairy cell leukemia; acute myeloid leukemia (AML); chronic myeloid leukemia (CML); and Langerhans cell histiocytosis. In one embodiment, the treatment targets an acute leukemia selected from the group consisting of acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and myeloproliferative diseases.

[0231] 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 crisis leukemia; multiple myeloma / plasma cell neoplasm, multiple myeloma, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, and myeloproliferative disorder.

[0232] In some cases, treatment targets a peripheral nervous system tumor. In some cases, treatment targets a paraganglioma. In some cases, treatment targets a pheochromocytoma.

[0233] In one embodiment, the treatment of cancer comprises preventing tumor growth in a subject with cancer. In one embodiment, the treatment of cancer comprises preventing the formation of cancer metastases in a subject with cancer. In one embodiment, the treatment of cancer comprises targeted therapy of minimal residual disease in a subject with cancer known to have minimal residual disease in cancer or a subject at risk of having minimal residual disease.

[0234] This may be indicated after the primary tumor has been treated surgically and / or after chemotherapy (radiotherapy) has begun or has been determined to be effective. Disseminated tumor cells may be in their dormant state and are usually not attacked by chemotherapy (radiotherapy). Patients treated in this way appear to be in a state of recovery and are referred to as "minimal residual disease". Nevertheless, dormant tumor cells have the potential to form metastases if they become metastatic cells due to growth stimulation after a longer dormant state.

[0235] The term "minimal residual disease" refers to a small amount of cancer cells that remain in a subject's body during or after treatment when the subject is in remission (not showing symptoms or signs of the disease). The methods described herein are preferably applied to the disease forms listed herein, including adult and pediatric forms of these diseases.

[0236] In one embodiment, the method of treatment can be used to treat autoimmune diseases. Autoimmune diseases include, but are not limited to, alopecia areata, antiphospholipids, autoimmune hepatitis, celiac disease, type 1 diabetes, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, inflammatory myopathy, multiple sclerosis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, and vitiligo.

[0237] In one embodiment, the method of treatment can be used to treat autoimmune and inflammatory disorders of the peripheral nervous system such as amyotrophic lateral sclerosis (Lou Gehrig's disease) which are based on various causes such as metabolic disorders (including diabetes, B12 and folate vitamin deficiencies), chemotherapy drugs and drugs used to treat HIV, toxins that cause peripheral nerve damage, cancers that develop peripheral neuropathy and paraneoplastic syndromes, alcohol abuse, chronic kidney disease, injuries that cause compression and other pathology of the nerves, infections such as Lyme disease, Guillain-Barre syndrome, connective tissue diseases: rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, certain inflammatory conditions such as sarcoidosis, celiac disease, genetic diseases such as charcot marie tooth syndrome, friedreich's ataxia, and / or idiopathic types where a specific cause has been found but inflammatory and / or autoimmune mechanisms are the cause of the disease.

[0238] In one embodiment, the method of treatment can be used to treat autoimmune and inflammatory disorders with ocular manifestations. Such ocular manifestations include, but are not limited to, ocular cicatricial pemphigoid, Moren's corneal ulcer, various forms of uveitis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, relapsing polychondritis, Wegener's granulomatosis, scleroderma, Behcet'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 ulcers, and less common entities such as choroiditis, retinal vasculitis, episcleral nodules, retinal detachment, and / or macular edema.

[0239] In one embodiment, the method of treatment can be used to treat acute allograft rejection in a transplant patient. In one embodiment, the method of treatment can be used to treat ischemic stroke. In one embodiment, the method of treatment can be used to treat inflammatory diseases including arthritis, psoriasis, asthma, and colitis.

[0240] In one embodiment, the therapeutic agent comprises a pharmaceutically acceptable monosalt of ONC201 or an analog thereof (e.g., a compound of formula (10)). In one embodiment, the therapeutic agent comprises a pharmaceutically acceptable disalt of ONC201 or an analog thereof (e.g., a compound of formula (10)). As described herein, some analogs may be trisalts. 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 monosalt or disalt selected from the group consisting of hydrochloride, hydrobromide, bisulfate, sulfate, phosphate, fumarate, succinate, oxalate and lactate, acid sulfate, hydroxy salt, tartrate, nitrate, citrate, acid tartrate, carbonate, malate, maleate, fumarate sulfonate, methane sulfonate, formate, acetate and carboxylate. In one embodiment, the therapeutic agent comprises ONC201 or its analog 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 its analog 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, lithium, and / or having a counterion such as methylamino, dimethylamino, diethylamino and triethylamino counterions and combinations thereof. In one embodiment, the therapeutic agent comprises a compound described herein in the form of a halogenated di-salt such as a dihydrochloride or dihydrobromide.

[0241] In one embodiment, the second therapeutic agent comprises an anticancer agent. In one embodiment, the second therapeutic agent is selected from acivicin, aclarubicin, acodazole, acrolin, adolesin, aldesleukin, alitretinoin, allopurinol, hexamethylmelamine, ambromycin, ametrine, amifostine, aminoglutethimide, acridine, anastrozole, anthramycin, arsenic trioxide, asparaginase, troline, azacitidine, azatep, azomycin, batimastat, benztepa, bevacizumab, bicalutamide, bisantrene, binefad dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, actinomycin, capecitabine, carbamazepine, carbetimide, carboplatin, carmustine, carrubicin, carzelesin, cedifingol, celecoxib, chlorambucil, siromycin , cisplatin, cladribine, crestofol mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin, daunomycin, decitabine, dextromaplatin, dezaguanine, dezaguanine mesylate, serquinone, docetaxel, doxorubicin, droloxifene, drostanolone, dazomib, edatrexate, eflornithine, esamitrucin, enloplatin, enprobamate, epipipridine, epirubicin, erbulazole, esorubicin, estramustine, etanidazole, etoposide, etobranin, fadrozole, fazarabine, retinamide, floxuridine, fludarabine, fluorouracil, flucitabine, fosquinone, fostramoxine, fulvestrant, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imofosine, interleukin II (IL-2,including recombinant interleukin II or rIL2), interferon α-2a, interferon α-2b, interferon α-n1, interferon α-n3, interferon β-Ia, interferon γ-Ib, iproplatin, irinotecan, lanreotide, letrozole, leuprolide, lirazo, lometrexol, lomustine, losoxantrone, masorol, maytansine, nitrogen mustard hydrochloride, megestrol acetate, melphalan, menolide, mercaptopurine, meprobamate Aminopterin, chlorpheniramine, metodepa, mibutidamide, mitokacin, mitolomine, mitogillin, mitomacin, mitomycin, mitospe, mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogamycin, omnaplatin, oxysulen, paclitaxel, pegaspargase, pelinomycin, pentamidine, peclomycin, perfosfamide, pipobroman, piposulfan, pyroxantrone hydrochloride, plicamycin, plometane, porfil sodium, Methyl mitomycin, prednimustine, methyl procarbazine, puromycin, pyrazofuranoside, liboadenosine, roglulimumide, safinol, semustine, cintrazine, phosphatase, sparomycin, spirogermanium, spiromustine, spiroplatin, streptomycin, streptozotocin, sulfachlorpyrifos, tamoxifen, teicogalan, fluazifop, tiloxantrone, temoporphine, teniposide, tiroxilon, testolactone, thioguanine, thioguanine , thiotepa, thiazolamide nucleoside, tirapazamine, topotecan, toremifene, triptolon, triciribine, trimetrexate, triptorelin, tobradazole, uramustine, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinpitidine, vinca glyceride, vinrosin, vinorelbine, vinprodine, vinralidine, vorozole, zeniplatin, ningstatin, zoledronic acid, zorubicin, and combinations thereof. ,

[0242] In one embodiment, the second therapeutic agent is selected from hormone analogs and anti-hormones, 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; microtubule 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, amifostin, anagrelid ), clodronate, filgrastin, interferon, interferon alpha, folinic acid, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porfimer, 2-chlorodeoxyadenosine, 2-fluorodeoxy-cytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, allavectin-7, hexamethylmelamine, avocidib, amonafide, anthrapyrazole, AG-2037, AP-5280, apaziquone, apomine, aranose, arglabin, arzoxifene, atamestane, atrasentan, auristatin, PE, ABT-199 (Venetoclax), ABT-263 (Navitoclax), AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886 / AZD-6244 (selumetinib), ARRY-704 / AZD-8330, AR-12, AR-42, AS-703988,AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (pimasertib), Avastin, AZD-2014, azacytidine, azaepothilone B, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, biricodardicitrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (afatinib, tomtovok), BIBF 1120 (vargatef), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycinic 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, CB T-1 cefixime, ceflatonin, ceftriaxone sodium, celecoxib, celmoleukin, cemadotin, CH4987655 / RO-4987655, chlorfenestrol, cilengitide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicine, Compretin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 candidin 52, CTP-37, CTLA-4 monoclonal antibody,CP-461, CV-247, cyanomorpholinodoxorubicin, cytarabine, D 24851, decitabine, doxorubicin, deoxyrubicin, deoxycoformycin, depsipeptide, deoxyepothilone B, dexamethasone, dexrazoxanet, diethylstilbestrol, diflomotecan, DIDOX, DMDC, dolastatin 10 10), doranidazole, DS-7423, E7010, E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, elsamitrucin, epothilone B. epratuzumab, ER-86526, erlotinib, ET-18-0CH3, ethynylcytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenretinide, figitumumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, fotemustine, galarubicin, galliummaltolate, gefitinib, gemtuzumab, gimatecan, glufosfamide ufosfamide), 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, hexamethylenetetramine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibritumomab, idatrexate, idenestrol, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (Western cixutumumab), IMMUNOL, indisulam, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha-2b, interleukin-2, INK-1117, INK-128, INSM-18, ionafarnib, ipilimumab, iproplatin, irofulven, isohomohalichondrin-B, isoflavones, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, kahalid F F), ketoconazole, KW-2170, KW-2450, chloroplatin, leflunomide, lenograstim, leuprolide, leuporelin, lexidronam, LGD-1550, linezolid, lutetium texaphyrin, lometrexol, losoxantrone, LU 223651, lurtotecan, LY-S6AKT1, LY-2780301, mafosfamide, marimastat, dichloromethyl diethylamine, 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 (daratumumab), 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, 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-04691502, PHT-4 27, 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 , methyl pivalate butyrate, pixantrone, phenoxodiol O, PKI166, plevitrexed, plicamycin, muscarinic acid, porfiromycin, prednisone, prednisolone, quinamed, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY 869766, RDEA-436, butterfly mycin analogs, receptor tyrosine kinase (RTK) inhibitors, revimid, RG-7167, RG-7304, RG-7421, RG-7321,RG 7440, rhizoxin, rhu-MAb, rinfabate, risedronate, rituximab, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, rubidazone, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabarubicin, SAHA, sargramostim, satraplatin, SB 408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustin, seocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, suberanilohydroxamic acid, sutent, T 900607, T 138067, TAK-733, TAS-103, tacedinaline, talaporfin, tarceva, tariquitar, tasisulam, taxotere, taxoprexin, tazarotene, fluazifop, temozolamide, tesmilifene, testosterone, testosterone propionate, tesmilifene, tetraplatinum, tetrodotoxin, tezacitabine, thalidomide, cerules theralux, therarubicin, thymalfasin, thymectacin, tiazofurin, tipifarnib, tomudex, tirapazamine, tocladesine, toremofin, trabectedin, TransMID-107, trans-retinoic acid, tretinoin, trastuzumab, tremelimumab,triacetylguanylate, triapine, triciribine, trimetrexate, TLK-286, TXD 258, tykerb / tyverb, urocidin, valrubicin, vatalanib, vincristine, vinflunine, virulizin, WX-UK1, WX-554, vectibix, xeloda, XELOX, 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.

[0243] 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, medroxy-progesterone, octreotide, and combinations thereof. In one embodiment, the second therapeutic agent is selected from LHRH agonists and LHRH antagonists. In one embodiment, the LHRH agonist is selected from goserelin acetate, luprolactone acetate, triptorelin pamoate, and combinations thereof. In one embodiment, the second therapeutic agent comprises an LHRH antagonist selected from the group consisting of degarelix, cetrorelix, abarelix, ozarelix, degarelix, and combinations thereof. In one embodiment, the second therapeutic agent comprises a growth factor inhibitor. In one embodiment, the growth factor inhibitor is selected from the group consisting 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, human epidermal growth factor (HER) is selected from HER2, HER3, and HER4.

[0244] 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.

[0245] 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, pyrimidine analogs 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 and 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, actinomycin, 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 estramustin, dichloromethyl diethylamine, melphalan, chlorambucil, busulfan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas and combinations thereof. In one embodiment, the second therapeutic agent comprises nitrosoureas. In one embodiment, nitrosoureas are selected from carmustin, lomustin, thiotepa and combinations thereof. In one embodiment, the second therapeutic agent comprises an antimitotic agent. In one embodiment, the antimitotic agent is selected from vinca alkaloids and taxanes. In one embodiment, taxanes are selected from paclitaxel, docetaxel and combinations thereof. In one embodiment, vinca alkaloids are 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, amsacrin, topotecan, irinotecan, mitoxantron, 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 PDK 1 inhibitors,B-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTOR / PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1 inhibitors, CDK inhibitors, Aurora kinase inhibitors, 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, rufolimus, sirolimus, and combinations thereof. In one embodiment, the second therapeutic agent is selected from amifostine, anagrelide, clodronate, filgrastim, interferon, interferon alpha, folinic acid, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, and combinations thereof. In one embodiment, the second therapeutic agent is selected from 2-chlorodeoxyadenosine, 2-fluorodeoxy-cytidine, 2-methoxyestradiol, 2C4, 3-orexin, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epothilone B, A 105972, A 204197, abiraterone, aldesleukin, alitretinoin, alevetin-7, hexamethylmelamine, avosidide, aminafide, anthrapyrazole, AG-2037, AP-5280, apaziquinone, apomin, alanos, agaleb, arzoxifene, atamestane, atrasentan, auristatin, PE, ABT-199 (venetoclax), ABT-263 (navitoc), AVLB, AZ10992, ABX-EGF, AMG-479 (ganituzumab), ARRY 162, 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, ARQ 680, AS-703026 (pimacitinib), Avastin, AZD-2014, azacytidine, azaepotinib, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, bilicodar citrate, BCX-1777, BKM-120, bleocin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291,BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW 2992 (Afatinib, Tomtovok), BIBF 1120 (Vegat), BI 836845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, bleomycin, bleomycin A, bleomycin B, brivanib, bryostatin 1, bortezomib, bromocriptine, busulfan, BYL-719, CA-4 prodrug, CA-4, casai, calcitriol, canertinib, canfluamine, capecitabine, carboxyplatinum phthalate, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1 cefixime, homoharringtonine, ceftriaxone sodium, celecoxib, simulleukin, simadotin, CH4987655 / RO-4987655, chlorfenapyr, cilengitide, Cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabine, colchicine, Compretin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 Candida albicans 52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, chloromorpholino doxorubicin, cytarabine, D24851, decitabine, doxorubicin, deoxydoxorubicin, desphenytoin, desphenytoin, deoxyepotyl B, dexamethasone, dexrazoxane, diethylstilbestrol, diflutecan, DIDOX , DMDC, Aplysia 10, doradazol, DS-7423, E7010, E-6201, edatrexa, edotreotide, iprexapro, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalutamide, esamitrucin, epothilone B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3, cytidine, ethinyl estradiol, exitecan, exitecan mesylate, exemestane, eximesulin, retinoic acid phentermine, fentolimumab, floxuridine, folic acid, FOLFOX, FOLFOX4, FOLFIRI, formestane, Fotemustine, garubicin, gallium maltol, gefitinib, gemtuzumab, gematinib, glufosfamide, GCS-100, GDC-0623, GDC-0941 (pitrilixib), 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, hexamethylenetetramine, histamine, homoharringtonine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibandronate, ibritumomab tiuxetan, idatrexone, idestrone, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (citumumab), IMMUNOL, indesulan, interferon α-2a, interferon α-2b, pegylated interferon α-2b, interleukin-2, INK-1117, INK-128, INSM-18, yonafarnib, ipilimumab, iproplatin, ilofofen, isoflavone, isotretinoin, ixabepilone, JRX-2, JSF-154, J-107088, conjugated estrogens, kahalide F, ketoconazole, KW-2170, KW-2450, levoplatin, leflunomide, linoglalast, leuprolide, leuprolide, lexidrone samarium, LGD-1550, linezolid, texafin lutetium, lometrexol, loxantrone, LU 223651, lutetotecan, LY-S6AKT1, LY-2780301, mafosfamide, mamastat, dichloromethyl diethylamine, MEK inhibitor, MEK-162, methyltestosterone, methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX-447, MDX-1379, MGV, midostinib, minodronic acid, mitomycin, mivobulin, MK-2206, MK-0646 (daratumumab), MLN518, motexafin gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, resovag, nevostat, nilotinib, nimesulide, nitroglycerin, noratript, norrelin, N-acetylcysteine, 06-benzylguanine, oblimersen, omeprazole, oxafungin, oncoVEXGM-CSF, omeprazole Ting, Ortasai, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, pyrroloanthrone, estrogen, panitumumab, patupirone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-0 5197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, paetuximab, perifosin, 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, methyl pivalate butyrate, pixantrone, phentothil O, PKI166, pletrexed, plicamycin, polyporus acid, porfibrinocin, prednisone, prednisolone, quimid, quinupristin, R115777, RAF-265, ramosetron, ranpirnase, RDEA-119 / BAY 869766, RDEA-436, butterfly mycin analogs, receptor tyrosine kinase (RTK) inhibitors, REVLIMID, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, rhizobactin, rhu-MAb, linfipa, risedronate, rituximab, rotuximab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, chlorphenhydramine, rubitecan, R-flurbiprofen, RX-0201, S-9788, sabalibixin, SAHA, sargramostim, seraplatin, SB 408075, Se-015 / Ve-015, SU5416, SU6668, SDX-101, semustine, ciocalcitol, SM-11355, SN-38, SN-4071, SR-27897, SR-31747, SR-13668, SRL-172, sorafenib, spiroplatin, squalamine, nilinyl hydroxamic acid, sutent, T900607, T 138067, TAK-733, TAS-103, tekdinine, talaporfin, tasiva, taquita, tassolan, taxotere, taspocin, tazarotene, fluoxetine, temozolomide, tesmilifen, testosterone, testosterone propionate, tesmilifen, tetraplatinum, tetrodotoxin, tezacitabine, thalidomide, cerules, pirarubicin, thymofasin, simikertin, thiazofurine, tipifarnib, tirapazamine, toradesin, toruudex, toremofin, trabectedin, TransMID-107, trans-retinoic acid, trastuzumab, tremelimumab, retinoic acid, triacetylguanylate, tropine, trociribine, trimetrexate, TLK-286 、TXD258、Tekpo / Tavipo、Urosetine、Valrubicin、Vatalanib、Vincristine、Vinflunine、Virulin、WX-UK1、WX-554、Vectibix、Xeloda、XELOX、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、Zoloft and their combinations. 、

[0246] In one embodiment, the other therapeutic agent comprises a steroid, including dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and cortivazole. In one embodiment, the other therapeutic agent comprises an antiemetic. Antiemetics include, but are not limited to, 5-HT3 receptor agonists (such as dolasetron, granisetron, ondansetron, tropisetron, palonosetron, and mirtazapine), dopamine agonists (such as domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine, and metoclopramide), NK1 receptor antagonists (such as aprepitant and casopitant), t)), antihistamines (such as cyclizine, diphenhydramine, dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine), cannabinoids (such as cannabis, dronabinol, nabilone, and Sativex), benzodiazepines (such as midazolam and lorazepam), anticholinergics (such as hyoscine), trimethoprim, ginger root, emetrol, propofol, mint, muscimol, and ajwain.

[0247] The pharmaceutical composition can be administered to the subject by any suitable route of administration. In one embodiment, the pharmaceutical composition is administered to the subject orally, parenterally, percutaneously or transmucosally. In one embodiment, the pharmaceutical composition is administered to the subject parenterally. In one embodiment, the pharmaceutical composition is administered to the subject by a parenteral route of administration selected from intravenous (IV), subcutaneous (SC) and intramuscular (IM). In one embodiment, the pharmaceutical composition is administered to the subject via a route of administration selected from rectal and percutaneous. In one embodiment, the pharmaceutical composition is administered to the subject in a dosage form selected from the group consisting of: sterile solution, suspension, suppository, tablet and capsule. In one embodiment, the pharmaceutical composition is administered to the subject in an oral dosage form selected from the group consisting of: tablet, caplet, capsule, lozenge, syrup, liquid, suspension and elixir. In one embodiment, the pharmaceutical composition is administered to the subject in an oral dosage form selected from the group consisting of: tablet, hard shell capsule, soft gelatin capsule, pill, granule, aggregate, powder, gel, solid and semisolid.

[0248] In one embodiment, the pharmaceutical composition is administered to a subject in a dosage form selected from the group consisting of sustained release, controlled release, delayed release, and responsive release.

[0249] In one embodiment, the pharmaceutical composition is administered to a subject once daily. In one embodiment, the pharmaceutical composition is administered to (e.g., once a week or less frequently) a subject according to an infrequent dosing regimen. In one embodiment, the pharmaceutical composition is administered to (e.g., more than once a week) a subject according to a frequent dosing regimen. In one embodiment, the pharmaceutical composition is administered to a subject once a week. In one embodiment, the pharmaceutical composition is administered to a subject once every four weeks. In one embodiment, the pharmaceutical composition is administered to a subject twice a week. In one embodiment, the pharmaceutical composition is administered to a subject once every two weeks. In one embodiment, the pharmaceutical composition is administered to a subject once every three weeks. In one embodiment, the pharmaceutical composition is administered to a subject with a repetitive cycle of once a week, once every two weeks, once every three weeks, once every four weeks, or a combination thereof.

[0250] In one embodiment, the method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent in combination with (ii) a second therapeutic agent, the first therapeutic agent comprising a compound containing emilione (such as ONC201) or an analog thereof or a pharmaceutically acceptable salt thereof, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially; and further comprises determining the expression of endoplasmic reticulum (ER) stress response genes in a biological sample. In one embodiment, the endoplasmic reticulum stress response genes are 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 endoplasmic reticulum stress response genes are selected from the group including, but not limited to, ATF3, activating transcription factor 4 (ATF4), CHOP, IRE1, binding immunoglobulin (BiP), eukaryotic translation initiation factor 2A (eIF2a), X-box binding protein 1 (XBP1). The biological sample can be a tumor, peripheral blood mononuclear cells, or a skin biopsy. The biological sample can be obtained before, during, or after drug administration. 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 induction of 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 more than 600% of ER stress genes. In one embodiment, the method of treatment further comprises adjusting the dose of the first therapeutic agent to achieve induction of 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 more than 600% of ER stress genes.

[0251] In one embodiment, the method of treatment comprises administering to a subject in need of such treatment: (i) a first therapeutic agent in combination with (ii) a second therapeutic agent, the first therapeutic agent comprising a compound containing emilione (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof, wherein the first therapeutic agent and the second therapeutic agent are administered simultaneously or sequentially; and further comprises determining the expression of proteasome activity in a biological sample. In one embodiment, the proteasome activity may be chymotrypsin-like, trypsin-like, and / or caspase-like activity. In one embodiment, the biological sample may be a tumor, a peripheral blood mononuclear cell, or a skin cell. The biological sample may be obtained before, during, or after drug administration. In one embodiment, the method of treatment further comprises adjusting the dose to achieve an inhibition of proteasome activity of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In one embodiment, the method of treatment further comprises adjusting the dosage 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 comprises adjusting the dosage 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 inhibition of proteasome activity.

[0252] In one aspect, provided herein are methods of treatment comprising administering to a subject in need of such treatment a combination of a first therapeutic agent and a second therapeutic agent, the first therapeutic agent comprising emilione (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof (e.g., a di-salt or tri-salt), the method comprising:

[0253] (i) administering a first therapeutic agent to a subject;

[0254] (ii) waiting until a predetermined waiting period has passed after the time at which the first therapeutic agent is administered to the subject; and / or until the adverse event has resolved or is resolving; and

[0255] (iii) administering a second therapeutic agent to the subject, wherein a predetermined waiting time is selected so as to obtain a delayed therapeutic effect of the first therapeutic agent without increasing the risk of possible combined toxic effects of the first and second therapeutic agents. In one embodiment, the predetermined waiting time is determined based on the clearance rate of the compound of the first therapeutic agent or its metabolite. In one embodiment, the predetermined waiting time is determined by a quantitative assessment of renal function and kidney 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 level of the compound of the first therapeutic agent or its metabolite; clearance rate of the compound of the first therapeutic agent or its metabolite; 24-hour urine clearance of the compound of the first therapeutic agent or its metabolite.

[0256] In one embodiment, the predetermined waiting time is substantially equal to the time required for systemic clearance of the compound of the first therapeutic agent or its metabolite from the subject. In one embodiment, the predetermined waiting time is substantially equal to the time required for renal clearance of the compound of the first therapeutic agent or its metabolite from the subject. In one embodiment, the predetermined waiting time is substantially equal to the time required for hepatic clearance of the compound of the first therapeutic agent or its metabolite from the subject. In one embodiment, the predetermined waiting time is substantially equal to the time required for total clearance of the compound of the first therapeutic agent or its metabolite from the subject. In one embodiment, the predetermined waiting time is about 4 hours. In other embodiments, the waiting time is 1 day. In one embodiment, the waiting time is until the C of the compound of the first therapeutic agent is 最大值 In one embodiment, the waiting time is about 2 days, about 3 days, about 4 days, about 5 days, about 6 days or about 7 days. In one embodiment, the waiting time is about 1 to 7 days, about 1 to 6 days, about 1 to 5 days, about 1 to 4 days, about 1 to 3 days or about 1 to 2 days. In one embodiment, the waiting time is up to 3 weeks. The previous is considered to be the "treatment period".

[0257] When the order of administration is reversed, the first therapeutic agent or drug can be administered at a time point that is 2 hours after the second therapeutic agent (i.e., the first administered drug). 最大值 In one embodiment, the first therapeutic agent can be administered after most or substantially all of the first drug has been eliminated from the body or a toxic effect of the first drug has resolved or is resolving.

[0258] In one embodiment, the method of treatment further includes monitoring the level of the compound of the first therapeutic agent or its metabolite in the subject using a pharmacokinetic analysis. In some such embodiments, monitoring the level of the compound of the first therapeutic agent or its metabolite in the subject using a pharmacokinetic analysis includes using the concentration of the compound of the first therapeutic agent or its metabolite in at least two samples obtained from the subject at a time point suitable for constructing a pharmacokinetic curve to construct a pharmacokinetic curve of the compound of the first therapeutic agent or its metabolite in the subject. In one embodiment, the method of treatment includes monitoring the level of the compound of the first therapeutic agent or its metabolite in the subject using a pharmacokinetic analysis, and before laboratory quantification, the sample is collected from the subject at the point of care or point of use by sampling or self-sampling on a fixed-point care device or a fixed-point use device or on a matrix suitable for storing samples. In one embodiment, each device in the fixed-point care device or the fixed-point use device is capable of quantifying the compound of the first therapeutic agent or its metabolite. In one embodiment, the method of treatment includes monitoring the level of a compound or metabolite thereof of the first therapeutic agent in the subject, and collecting one or more samples from the subject at the point of care or point of use by a biopsy device for analysis or for storage at the point of care or point of use device before laboratory analysis. In one embodiment, the biopsy is performed after a time interval of 3 to 8 hours after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 3 to 24 hours after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 8 to 24 hours after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 2 days after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 3 days after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 4 days after the first therapeutic agent is administered to the subject. In one embodiment, the biopsy is performed after a time interval of 1 to 7 days after the first therapeutic agent is administered.

[0259] In one embodiment, the pharmacokinetic profile comprises pharmacokinetic parameters suitable for guiding administration of the first therapeutic agent to the treated subject. In one embodiment, during the treatment period, the C of the first therapeutic agent after administration to the subject is 最大值 The range of C is about 1000 ng / dL to 1500 ng / dL. In one embodiment, during the treatment period, C 最大值 Less than 1500 ng / dL and greater than 85 ng / dL. In one embodiment, during the treatment period, the C of the first therapeutic agent after it is administered to the subject is 最大值 The range of C is about 1000 ng / mL to about 1500 ng / mL. In one embodiment, during the treatment period, C最大值 Less than 1500ng / mL and greater than 85ng / mL.

[0260] In one embodiment, the maximum concentration (“C 最大值 ”) is about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng / dL to about 1500 ng / dL; about 1 00, 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, 1 dL to about 15 ng / dL; or about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng / dL to about 15 ng / dL. 最大值 .

[0261] In one embodiment, the maximum concentration (“C 最大值”) is about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng / mL to about 1500 ng / mL; about 1 00, 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, 1 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149 ng / mL to about 150 ng / mL; or about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng / mL to about 15 ng / mL of C 最大值 .

[0262] In one embodiment, the maximum concentration (“C 最大值 ”) is selected from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1401 80, 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 thereof is 最大值 (“C 最大值148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 最大值 Selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng / dL.

[0263] In one embodiment, the C of the first therapeutic agent after administration thereof 最大值 selected from about 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng / mL. In one embodiment, the C of the first therapeutic agent after administration thereof is 250, 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. 最大值 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 最大值 Selected from about 10, 10.5, 11, 11.5, 120, 12.5, 13, 13.5, 14 or 14.5 ng / mL.

[0264] In one embodiment, the C of the first therapeutic agent after administration thereof 最大值selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 8 65, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 12 1455, 1465, 1475, 1485, 1495, or 1500 ng / dL. In one embodiment, the C of the first therapeutic agent after administration thereof is 100 ng / dL or 1500 ng / dL. 最大值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, 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 thereof 最大值 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.

[0265] In one embodiment, the C of the first therapeutic agent after administration thereof 最大值selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 8 65, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 12 05, 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. In one embodiment, the C of the first treatment after its administration 最大值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, 134, 135, 136, 137, 138, 139, 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 thereof 最大值 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.

[0266] In one embodiment, the C of the first therapeutic agent after administration to the subject 最大值 The range of C of the first therapeutic agent in the blood (whole blood, plasma or serum) after administration is 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 the blood (whole blood, plasma or serum) after administration is 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. 最大值Selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 485 75, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 12 15, 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;About 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 8, 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 06, 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 to about 150 ng / dL; 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 / dL to about 15 ng / dL.;

[0267] In one embodiment, the C of the first therapeutic agent after administration thereof 最大值 The range of C is 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 first treatment is administered after C 最大值Selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 485 75, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 12 15, 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;About 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 8, 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 06, 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 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. ;

[0268] In one embodiment, the total drug exposure over time, measured as the area under the curve ("AUC") of a plot of drug concentration in the blood (whole blood, plasma, or serum) of a subject after drug administration versus time after drug administration, ranges from about 150 ng 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 15 ng hr / mL. In one embodiment, the AUC is less than 80 ng hr / mL and greater than or equal to 1.5 ng hr / mL.

[0269] 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 ng hr / mL to about 80 ng hr / mL.

[0270] 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.

[0271] In one embodiment, the total drug exposure over time is an AUC selected from the group consisting of: 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 ng hr / 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 ng hr / 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 ng hr / mL.

[0272] In another aspect, provided herein is a method of treating or use of a composition for treating a disease state, the use 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:

[0273] (i) administering to a subject a first therapeutic agent comprising emilione (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof;

[0274] (ii) monitoring the level of a compound of the first therapeutic agent or a metabolite thereof in the subject using a pharmacokinetic assay; and

[0275] (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 compound of the first therapeutic agent or its metabolite in the subject using the concentration of the compound of the first therapeutic agent or its metabolite in multiple samples obtained from the subject at a time point suitable for constructing a pharmacokinetic profile. In one embodiment, before laboratory quantification of the compound or its metabolite, at least two samples are collected at a point of care or point of use by sampling or self-sampling on a point-of-care device or a point-of-use device or on a matrix suitable for storing samples. In one embodiment, each point-of-care device or point-of-use device is capable of quantifying a compound or its metabolite. In one embodiment, the pharmacokinetic profile includes pharmacokinetic parameters suitable for guiding the administration of a compound or its salt to a subject. In one embodiment, the sample includes 2 to 12 samples. In one embodiment, 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 at least one parameter selected from the group consisting of: AUC, AUC 无穷大 , T 最大值 , C 最大值 , time above a threshold, steady-state concentration, absorption rate, clearance rate, distribution rate, terminal T-1 / 2, or a parameter derived from non-compartmental pharmacokinetics (PK) or compartmental PK analysis (including compartmental PK analysis based on a physiological model). In one embodiment, the method of treatment further comprises generating a report comprising a pharmacokinetic profile of the subject. In one embodiment, the report comprises a recommendation for dosing based on the pharmacokinetic profile of the subject. In one embodiment, a dose reduction of ONC201, an analog thereof, or a pharmaceutically acceptable salt thereof is indicated based on one or more pharmacokinetic parameters to reduce the risk of toxicity. In one embodiment, a reduction in the dose of a compound or a salt thereof is indicated based on a time above a threshold, wherein the threshold is a drug concentration above which toxicity occurs, or one or more of the following: AUC, AUC 无穷大 , mean residence time (MRT), an index defining pharmacokinetic properties, volume of distribution at steady state (Vss), volume of distribution during the terminal phase (Vz), or a combination of pharmacokinetic variables that adequately describe the pharmacokinetic properties. In one embodiment, the dosage of the compound or its salt is indicated to increase efficacy based on one or more pharmacokinetic parameters. In one embodiment, the increase in the dosage of the compound or its salt is indicated based on one or more of the following: AUC, AUC 无穷大, MRT, an index defining a pharmacokinetic property, a steady-state distribution volume (Vss), a distribution volume during the terminal phase (Vz), or a combination of a set of pharmacokinetic variables that adequately describe the pharmacokinetic property. In one embodiment, the dose of the compound or its salt is adjusted to within 5% to 25% of the desired target value. In one embodiment, each of the samples is applied to a point-of-care device or a point-of-use device to determine the concentration of the compound or its metabolite, wherein the point-of-care device or the point-of-use device comprises a lateral flow strip having a structure and composition such that one or more of the samples are applied to the lateral flow strip causing a portion of the drug in the sample to bind to a component of the lateral flow strip, thereby generating a detectable signal proportional to the drug concentration in the applied sample. In one embodiment, the sample is applied to a matrix suitable for storing the sample prior to laboratory quantification. 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 LCMS. In one embodiment, the pharmacokinetic parameters include steady-state concentration, absorbance, and terminal T 1 / 2 In one embodiment, at least one of the samples is whole blood.

[0276] IX. Multimodal Approach to Treatment

[0277] In one aspect, multimodal treatment methods are provided herein, wherein the administration of imiridone (such as ONC201), its analogs, or pharmaceutically acceptable salts thereof to a subject in need thereof is supplemented by other treatment modes. In one embodiment, multimodal treatment comprises administering a pharmaceutical composition comprising imiridone (such as ONC201), its analogs, or its pharmaceutically acceptable salts to a subject in combination with radiotherapy or after radiation has been determined to be ineffective. In one embodiment, multimodal treatment comprises administering a pharmaceutical composition comprising imiridone (such as ONC201), its analogs, or its pharmaceutically acceptable salts to a subject in combination with radiotherapy, wherein the pharmaceutical composition comprising imiridone (such as ONC201), its analogs, or its pharmaceutically acceptable salts and radiotherapy are administered simultaneously or sequentially in any order. In one embodiment, multimodal treatment comprises administering a pharmaceutical composition comprising imiridone (such as ONC201), its analogs, or its pharmaceutically acceptable salts, and radiotherapy in combination with a sequential arrangement. In one embodiment, the multimodal treatment comprises administering a pharmaceutical composition comprising imiridone (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof, and radiation therapy simultaneously. In one embodiment, the multimodal treatment is used to treat cancer. In one embodiment, the multimodal treatment comprises administering a pharmaceutical composition comprising imiridone (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof to a cancer subject and irradiating cancer cells with a radiation beam. In one embodiment, the multimodal treatment uses conformal radiation therapy (CRT) to deliver a prescribed dose volume histogram (DVH) to a cancer subject. In one embodiment, the multimodal treatment method uses intensity modulated radiation therapy (IMRT) to deliver radiation to cancer cells. In one embodiment, the multimodal treatment method uses technology to compensate for tumor motion in the subject during treatment (e.g., where a radiation dose must be administered to a chest tumor that moves as the patient breathes). For example, the multimodal treatment uses four-dimensional computed tomography (4D CT) scanning technology to adjust the delivered radiation field to compensate for tumor motion throughout the respiratory cycle.

[0278] Any suitable type of radiation, including fractionated gamma radiation, IMRT (intensity modulated radiation therapy), gamma knife, proton therapy, and brachytherapy, can be used with multimodal therapy. Radiation therapy and administration of imiprazole (such as ONC201), its analogs, or pharmaceutically acceptable salts thereof can be used to treat brain tumors, for example, glioblastoma or diseases that have metastasized to the brain from lung cancer. Multimodal therapy can be used to treat lung cancer, pancreatic cancer, colorectal cancer, breast cancer, sarcoma, prostate cancer, gynecological malignancies, and lymphoma. Gamma knife is frequently used to treat brain metastases. In one embodiment, multimodal therapy includes the use of proton therapy to treat cancer, including brain tumors, prostate cancer, and any tumor close to a vital organ where toxicity to nearby normal tissue is very important.

[0279] In one embodiment, the multimodal treatment comprises administering to a cancer subject a pharmaceutical composition comprising emilione (such as ONC201), an analog thereof, or a pharmaceutically acceptable salt thereof, and 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)) simultaneously or in combination.

[0280] In one embodiment, the multimodal therapy eliminates minimal residual disease without increasing the toxicity produced by treatment with imiprazole (such as ONC201), its analogs or pharmaceutically acceptable salts thereof. In one embodiment, the multimodal therapy improves the prognosis of the subject receiving treatment and / or reduces adverse side effects associated with a disease state or condition.

[0281] X. Additional emilidon derivatives, analogs and salts

[0282] In one aspect, provided herein are analogs of compounds of formula (10) and methods for their preparation. Those skilled in the art will appreciate that the general principles and concepts described herein in conjunction with ONC201 and compounds of formula (10) and salts thereof (including principles and concepts related to methods and pharmaceutical compositions) are equally applicable to the following analogs and salts thereof.

[0283] In one embodiment, the analog has the structure of compound (25):

[0284] Wherein Y is NR4 or O, and wherein R1, R2, R3 and R4 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocyclic free radical. In one embodiment, R1, R2, R3 and R4 are optionally substituted. In one embodiment, some or all of the hydrogen in R1, R2, R3 and R4 are replaced by deuterium. In other embodiments, R1, R2, R3 and R4 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C 1-4 In still other embodiments, R1, R2, R3 and R4 are 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-bis-F-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh and (CH2)3CO-4F-Ph.

[0285] In one embodiment, the analog has the structure of compound (26):

[0286] wherein R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radical. In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted. In one embodiment, R1 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, 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.

[0287] 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 wherein X is a halogen, including F, Cl, Br or I; preferably, F, Cl or Br; more preferably, F or Cl. In one embodiment, R2 is a benzyl group 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 wherein X is a halogen.

[0288] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, for example, benzyl or phenethyl. In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0289] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, such as benzyl or phenethyl. In one embodiment, the arylalkyl group is C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated 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 substituted or unsubstituted heterocycloalkylalkyl, such as morpholinylalkyl or piperazinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, such as isoxazolidinylmethyl or pyridylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halo substituted. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3 and -OCH3.

[0290] In one embodiment, the analog has the structure of compound (27):

[0291] wherein R1 is H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic radical. In one embodiment, R1 is selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4Alkyl or halogen substituted. In one embodiment, R1 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.

[0292] 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 wherein X is a halogen, including F, Cl, Br or I; preferably, F, Cl or Br; more preferably, F or Cl. In one embodiment, R1 is H. In one embodiment, R1 is a substituted or unsubstituted arylalkyl, such as benzyl or phenethyl. In one embodiment, the arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0293] In one embodiment, the analog has the structure of compound (28): wherein R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radical. In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C1-4 Alkyl or halogen substituted. In one embodiment, R1 is selected from the group consisting of H, CH3, CH2Ph, CH2-((2-Cl)-Ph), CH2-(2-thienyl), CH2CH2Ph, CH2-(2,4-diF-Ph), CH2-((2-CH3)-Ph), CH2CHOHPh, CH2CH2(4-N-benzyl-piperazine), and (CH2)3CO-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).

[0294] 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 wherein X is a halogen, including F, Cl, Br or I; preferably, F, Cl or Br; more preferably, F or Cl. In one embodiment, R2 is a benzyl group 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 wherein X is a halogen.

[0295] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, for example, benzyl or phenethyl. In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0296] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, for example, benzyl or phenethyl. In one embodiment, the arylalkyl group is C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated 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 substituted or unsubstituted heterocycloalkylalkyl, for example, morpholinylalkyl or piperazinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, such as isoxazolidinylmethyl or pyridylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halo substituted. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3 and -OCH3.

[0297] In one embodiment, the analog has the structure of compound (29):

[0298] wherein R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radical. In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted. In one embodiment, R1 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, 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).

[0299] 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 wherein X is a halogen, including F, Cl, Br or I; preferably, F, Cl or Br; more preferably, F or Cl. In one embodiment, R2 is a benzyl group substituted with one or more of the following substituents, either 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 wherein X is a halogen.

[0300] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, for example, benzyl or phenethyl. In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0301] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, for example, benzyl or phenethyl. In one embodiment, the arylalkyl group is C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated 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 substituted or unsubstituted heterocycloalkylalkyl, for example, morpholinylalkyl or piperazinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, such as isoxazolidinylmethyl or pyridylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halo substituted. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3 and -OCH3.

[0302] In one embodiment, the analog has the structure of compound (30):

[0303] wherein R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radical. In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted. In one embodiment, R1 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, 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).

[0304] 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 wherein X is halogen, including F, Cl, Br or I, preferably F, Cl or Br, more preferably F or Cl. In one embodiment, R2 is benzyl substituted with one or more of the following substituents, alone or in combination, at the ortho, meta and / or para positions of the benzyl ring: -CH3, -NO2, -OCH3, -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 wherein X is a halogen.

[0305] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, for example, benzyl or phenethyl. In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0306] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, for example, benzyl or phenethyl. In one embodiment, the arylalkyl group is substituted with halogen, 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 substituted or unsubstituted heterocycloalkylalkyl, for example, morpholinylalkyl or piperazinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, for example, isoxazolidinylmethyl or pyridylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halo substituted. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3 and -OCH3.

[0307] In one embodiment, the analog has the structure of compound (31):

[0308] wherein R1 and R2 independently represent H, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio, alkanoyl, sulfhydryl, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radical. In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine and C 1-4 Alkylthienyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone and C 1-4 Benzyl-piperazine is optionally C1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted. In one embodiment, R1 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, 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).

[0309] 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 wherein X is a halogen, including F, Cl, Br or I; preferably, F, Cl or Br; more preferably, F or Cl. In one embodiment, R2 is a benzyl group 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 wherein X is a halogen.

[0310] In one embodiment, R1 is H. In one embodiment, R1 is substituted or unsubstituted arylalkyl, for example, benzyl or phenethyl. In one embodiment, arylalkyl is replaced by C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halogen substituted.

[0311] In one embodiment, R2 is a substituted or unsubstituted arylalkyl group, for example, benzyl or phenethyl. In one embodiment, the arylalkyl group is C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated 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 substituted or unsubstituted heterocycloalkylalkyl, for example, morpholinylalkyl or piperazinylalkyl. In one embodiment, R2 is substituted or unsubstituted heteroarylalkyl, for example, isoxazolidinylmethyl or pyridylmethyl. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 Alkyl or halo substituted. In one embodiment, the heterocycloalkylalkyl or heteroarylalkyl is substituted with one or more substituents selected from the group consisting of halo, -CH3, -CF3 and -OCH3.

[0312] In one embodiment, provided herein is a compound of formula (100): Wherein R1 and R2 are independently selected from H, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxycarbonyl, aralkyloxy, aralkylthio and acyl. In one embodiment, R1 is CH2Ph ​​and R2 is CH2-(2-CH3-Ph), which is a linear isomer of ONC201 It lacks anticancer 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 prevention or treatment of liver fibrosis.

[0313] In one embodiment, R1 and R2 are independently selected from the group consisting of: H, C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4Alkyl phenyl ketone, C 1-4 Benzyl-piperazine, C 1-4 Alkylthienyl, C 1-4 Alkylpyridyl, C 1-4 Alkylisoxazolidinyl, C 1-4 Alkylmorpholinyl, C 1-4 Alkylthiazolyl and C 1-4 Alkylpyrazinyl, where C 1-4 Alkyl, C 1-4 Alkylphenyl, C 1-4 Alkyl phenyl ketone, C 1-4 Benzyl-piperazine, C 1-4 Alkylthienyl, C 1-4 Alkylpyridyl, C 1-4 Alkylisoxazolidinyl, C 1-4 Alkylmorpholinyl, C 1-4 Alkylthiazolyl and C 1-4 The alkylpyrazinyl group is optionally substituted with C 1-4 Alkyl, C 1-4 Alkoxy, hydroxy, fully halogenated C 1-4 In one embodiment, R1 and / or R2 are substituted or unsubstituted arylalkyl or heteroarylalkyl. In one embodiment, heteroarylalkyl is selected from C 1-4 Alkyl pyrrolyl, C 1-4 Alkyl furanyl, C 1-4 Alkylpyridyl, C 1-4 Alkyl-1,2,4-thiadiazolyl, C 1-4 Alkyl pyrimidinyl, C 1-4 Alkylthienyl, C 1-4 Alkylisothiazolyl, C 1-4 Alkyl imidazolyl, C 1-4 Alkyl tetrazolyl, C 1-4 Alkylpyrazinyl, C 1-4 Alkyl pyrimidinyl, C 1-4 Alkylquinolyl, C 1-4 Alkyl isoquinolyl, C 1-4 Alkylphenylthio, C 1-4 Alkylbenzothiophene, C 1-4 Alkyl isobenzofuranyl, C 1-4 Alkylpyrazolyl, C 1-4 Alkyl indolyl, C 1-4 Alkyl purine, C 1-4 Alkylcarbazolyl, C 1-4 Alkylbenzimidazole and C 1-4 Alkylisoxazolyl.

[0314] In one embodiment, R1 and / or R2 are 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 , SR m NR m R n NR m C(O)R n 、SOR m 、SO2R m 、C(O)R m and C(O)OR m ; R m and R n Independently selected from H or C1-C4 alkyl; and 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; more preferably, F or Cl.

[0315] XI. Reference Examples

[0316] It should be understood that the following description and examples are intended only for illustrative purposes and are not intended to limit the scope of this disclosure. The following examples are intended to illustrate the disclosed embodiments and should not be construed as limiting these embodiments. In addition to those compounds described below, additional compounds can be prepared by the following reaction scheme or its appropriate variation or modification. refer to Example 1. Synthesis of 2-chlorobenzylamino-2-imidazoline hydroiodide

[0317] 2-Chlorobenzylamine (141 mg, 1.0 mMol) was added to a stirred solution of 2-methylthio-2-imidazoline hydroiodide (244 mg, 1.00 mMol) in dry dioxane (2.0 mL). The reaction mixture was stirred at 70 ° C for 90 minutes under argon. The solution was cooled to room temperature, filtered on a sinter funnel, washed with cold dioxane (2 mL) and dried under vacuum. White solid compound 4·HI (R2 = 2-chlorobenzyl) (242 mg, 72%) was obtained and used without further purification.

[0318] Reference Example 2. Synthesis of 2-chlorobenzylamino-2-imidazoline

[0319] 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 at 7 ° C for 30 minutes under argon. Thereafter, methylene chloride (5 mL) was added and the mixture was stirred for another 5 minutes. The reaction mixture was extracted with methylene chloride (2×2.5 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The resulting free base (150 mg, 100%) was obtained as a viscous liquid and used in the next reaction without any further purification. MS (ESI) 210 (M+H).

[0320] Reference Example 3. Synthesis of 4-oxo-3-piperidinic acid methyl-1-benzyl ester (Compound (6)) .

[0321] Triethylamine (6 mL) was added to stirred 4-oxo-3-piperidinylcarboxylic acid methyl-1-benzyl ester (5.7 g, 20 mMol) in ethyl acetate (50 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 Na2SO4, filtered and evaporated. The resulting free base residue (5, R1=benzyl) in a viscous oily state was used for the next reaction without any further purification. MS (ESI) 248 (M+H)

[0322] Reference Example 4. Synthesis of ONC202 (Compound (14))

[0323] To a solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mMol), 4-oxo-3-piperidincarboxylic acid methyl 1-benzyl ester (5, R1 = 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 hours. Thereafter, the reaction mixture was refluxed at 125° C. to 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 purified by RP HPLC (10% to 40% acetonitrile / water) to give ONC202 TFA salt (228 mg, 50% yield) as a white solid MS (ESI) 407 (M+H).

[0324] 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 were prepared using the same method starting from different benzylamines.

[0325] Reference Example 5. Synthesis of ONC207 (Compound (19))

[0326] Under nitrogen at room temperature through 0.5 hour to 60% sodium hydride (3.5g, 88mMol) in the suspension of dry toluene (50mL) dropwise add dimethyl carbonate (4.32g, 48.0mMol).After adding a few drops of methanol, 1-tert-butyloxycarbonyl-4-piperidone (4.8g, 24mMol) dissolved in dry toluene (20mL) is added dropwise to the reaction mixture, while stirring at 80 ℃ for 1 hour.The reaction mixture is stirred at the same temperature for 3 hours and then cooled to 0 ℃ (ice bath), and adjusted to pH 6 to 6.5 with acetic acid.The resulting cold mixture is diluted with water (10mL) and adjusted to pH 8 with 5% sodium hydroxide solution.Separate the toluene layer and extract the aqueous layer with toluene (20mL).The combined organic layer is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The compound was dried in vacuum to obtain methyl 1-tert-butoxycarbonyl-4-oxo-3-piperidincarboxylate (5.0 g, 80%). The obtained compound was used in the next reaction without any further purification.

[0327] PPTS (10.0 mg) was added to 2-methylbenzylamino-2-imidazoline (190 mg, 1 mMol), 1-tert-butoxycarbonyl-4-oxo-3-piperidincarboxylate (315 mg, 1.1 mMol) in 1-butanol (2 mL) and the mixture was stirred at room temperature for 48 hours. Thereafter, the reaction mixture was refluxed at 125° C. to 130° C. for 2 hours. The solvent was removed under vacuum, extracted with ethyl acetate (10 mL), 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% to 40% acetonitrile / water) to give ONC207 (262 mg, 50%) TFA salt as a white solid MS (ESI) 297 (M+H).

[0328] Reference Example 6. Synthesis of ONC209 (Compound (21))

[0329] A mixture of ONC207 (100 mg, 0.2 mMol), phenethyl bromide (55.0 mg, 0.28 mMol) and potassium carbonate (150 mg, 1.0 mMol) in N,N-dimethylformamide (3 mL) was heated to 70 ° C for 12 hours. The solvent was removed under vacuum, extracted with ethyl acetate (10 mL), and washed with water (5 mL). The organic layer was dried over anhydrous Na2SO4, filtered and evaporated. The crude free base was purified by RP HPLC (10% to 40% acetonitrile / water) to give ONC209 (62 mg, 50%) TFA salt as a white solid MS (ESI) 401 (M+H).

[0330] The same procedure was used starting from different halides to obtain ONC215 and 214. Compounds 227, 228, 229, 230, 231, 232, 233, 234, 235 and 236 were prepared starting from different benzylamines using procedures similar to those of Examples 1 and 5. The intermediate compounds wherein R1 is H were then treated with different halides as above.

[0331] Compound ONC216 was prepared by treating ONC215 with TFA.

[0332] Compound (72) was prepared by reacting a precursor NH compound prepared similarly to Example 5 and treating it with styrene oxide.

[0333] Reference Example 7. Synthesis of ONC208 (Compound (20))

[0334] To a solution of 2-methylbenzylamino-2-imidazoline (190.0 mg, 1.0 mmol), 1-methyl 4-oxo-3-piperidincarboxylate (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 hours. Thereafter, the reaction mixture was refluxed at 125° C. to 130° C. for 2 hours. The solvent was removed under vacuum, extracted with ethyl acetate (10 mL), 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% to 40% acetonitrile and water to give ONC208 (270.0 mg, 50%) TFA salt MS (ESI) 311 (M+H) as a white solid. Reference Example 8. Synthesis of ONC201 (Compound (1))

[0335] Compound (3) (239.7 g, 0.845 mol, 1.6 eq.) was added in portions to 800 mL of saturated NaHCO3 stirred in a 2L round-bottom flask. n-Butanol (500 mL) was added to the resulting mixture, which was stirred for 30 minutes and then transferred to a separatory funnel. The organic phase containing compound (4) was separated and transferred to a 2L three-necked round-bottom flask equipped with mechanical stirring, N2 inlet, thermocouple, condenser and Dean-Stark water separator. Compound (5) (100 g, 0.528 mol, 1 eq.) and pyridinium p-toluenesulfonate (PPTS) (6.63 g, 0.026 mol, 5 mol%) were added to the contents of the flask. The resulting mixture was heated to reflux for 6 hours. The water in the reaction mixture was separated into the Dean-Stark water separator as needed. The reflux temperature was increased from 93° C. to 118° C. The reaction progress was monitored by HPLC. When the peak area of ​​compound (1) on HPLC remains constant over the reaction time, the reaction is stopped.

[0336] Reference Example 9. Synthesis of the di-salt of ONC201 (compound (1)·2HCl)

[0337] Without isolating compound (1), the reaction mixture from 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 3L three-necked round-bottom flask equipped with mechanical stirring, N2 inlet, thermocouple, condenser and Dean-Stark water separator. While stirring the reaction mixture, a dioxane-MTBE solution containing 1N HCl (dioxane containing 4N HCl: 300 mL, 1.2 mol, 2.27 equivalents; MTBE: 1200 mL) was added dropwise until no solid was precipitated in the reaction mixture after the addition of HCl. The reaction mixture was heated to reflux at 60° C. to 65° C. for 2 hours. Water was separated into the Dean-Stark water separator as needed. Once cooled to room temperature, the solid precipitate 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.

[0338] The above solid (200 g) was added to a 2L three-necked round-bottom flask equipped with mechanical stirring, N2 inlet, thermocouple and condenser, followed by ethanol (1000 mL). The mixture was heated to reflux at 78 ° C for 2 hours. Once cooled to room temperature, the solid was filtered through a sintered glass funnel and washed with ethanol (200 mL×3). The wet solid was dried in a vacuum oven at 85 ° C for 3 days until the residual solvent met the specifications. 120 g of compound (2) was obtained as a white solid with a yield of 49% and an HPLC purity of 99.7%.

[0339] Reference Example 10. Activity of imiridone

[0340] A number of emilidones were prepared based on the above synthesis. The viability of human cancer cells was measured 72 hours after treatment with each compound. The change in potency (relative to ONC201) was determined and is shown in Table 3.

[0341] Table 3: Relative potency of ONC201 analogs

[0342]

[0343]

[0344] *Relative to the potency of ONC201; A indicates an increase in potency >2-fold of ONC201; B indicates a potency within 2-fold of ONC201; and C indicates a decrease in potency >2-fold of ONC201.

[0345] ONC212

[0346]

[0347] Determination of the IC of ONC201 and ONC212 (5 nM to 5 μM, 72 h) after treatment of several acute myeloid leukemia (AML) cell lines (n=3) 50 And shown in Table 4.

[0348] Table 4

[0349] AML cell lines <![CDATA[ONC201 IC 50 (μM)]]> <![CDATA[ONC212 IC 50 (μM)]]> MV411 3.25 0.01 HL60 >5 0.21 MOLM14 3.92 0.01

[0350] Cell viability of MV411 AML cells treated with ONC212 and cytarabine (5 nM to 5 μM, 24 h) (n=3) was measured. Fig.10 A). In addition, the cell viability of MOLM14, MV411 AML cells, MRC5 lung fibroblasts and Hs27a bone marrow cells treated with ONC212 (5 nM to 5 μM, 72 hours) (n=3) was measured ( Fig.10 B). Cell viability of MOLM14 and MV411 AML cells treated with ONC212 (250 nM) for 4, 8, 24, 48, 72 and 96 hours was measured. ONC212 medium was replaced with fresh medium at these time points and cell viability of all samples was determined at 96 hours. (n=2) Fig.10 C).

[0351] Additionally, a single ONC212 dose administered orally or intraperitoneally to mice bearing human colon cancer xenografts resulted in a significant reduction in tumor volume compared to a vehicle-treated control cohort.ONC212 has a wide therapeutic window, as doses of ONC212 in mice were well tolerated at least up to 225 mg / kg.

[0352] Furthermore, ONC212 demonstrated efficacy in ONC201-resistant AML xenograft models ( Fig.11 MV411AML cells (5×10 6 ) were implanted subcutaneously into the flank of athymic nude mice. ONC212 and ONC201 were administered orally (PO) as indicated. Tumor volume (A and B) and body weight (C) were measured on the indicated days (n=10). * indicates p<0.05 relative to vehicle.

[0353] The efficacy of ONC212 in AML was evaluated in vitro and ONC212 was 400-fold more potent than ONC201 (Table 4). ONC212 was also effective in AML cells resistant to standard of care cytarabine ( Fig.10 A). Despite the robust improvement in efficacy, ONC212 still maintains a wide therapeutic window in vitro and is non-toxic to normal cells at effective concentrations ( Fig.10 B). Exposure to 250 nM ONC212 for 8 hr is sufficient to cause a robust reduction in cell viability in MOLM14 and MV411 AML cells ( Fig.10 C) ONC201 requires at least 24 to 48 hours of exposure to achieve efficacy.

[0354] ONC212 efficacy was determined in a leukemia xenograft model with MV411 AML cells resistant to standard of care cytarabine ( Fig.11 Once-weekly oral administration of ONC212 50 mg / kg significantly reduced leukemia xenograft tumor growth, whereas a similar dose of ONC201 was ineffective in this model ( Fig.11 A). Interestingly, ONC212 was ineffective at 25 mg / kg biweekly and 5 mg / kg weekly / biweekly. Fig.11 B). These ONC212 administration regimens were associated with weight loss ( Fig.11 C) or the overall observations are irrelevant.

[0355] ONC212 25 mg / kg represents the NOAEL in the mouse and rat non-GLP oral single dose studies, which is also the effective dose in the mouse xenograft study. ONC212 is approximately 10 times more toxic than ONC201 (NOAEL 225 mg / kg in the rat non-GLP oral single dose study).

[0356] ONC206

[0357]

[0358] ONC206 demonstrated efficacy in an Ewing sarcoma xenograft model. MHH-ES-1 Ewing sarcoma cells (5×10 6 ) were implanted subcutaneously into the flank of athymic nude mice. ONC206 (PO) and methotrexate (IV) were administered on days 1 and 13 as indicated. Tumor volumes were measured on the indicated days ( Fig.12 A) and body weight ( Fig.12 B) (n=4).

[0359] In addition, the IC values ​​of ONC201 and ONC206 (5 nM to 5 μM, 72 h) were determined for several cell lines (n=3) after treatment. 50 And are shown in Table 5 below.

[0360] Table 5

[0361] Cell lines <![CDATA[ONC201 IC 50 (μM)]]> <![CDATA[ONC206 IC 50 (μM)]]> MV411(AML) 3.25 0.2 K562(CML) >5 0.22 MOLM14(AML) 3.92 0.27 MHH-ES-1 (Ewing's sarcoma) 5.65 0.61 HFF (normal fibroblasts) >5

[0362] ONC206 showed a 20-fold increase in in vitro potency compared to ONC201 and was non-toxic to normal cells in vitro at therapeutic doses (Table 5). In a single non-GLP oral dose study in rats, it was noted that ONC206 had an overall increase of only 2-fold in toxicity (NOAEL 125 mg / kg) relative to ONC201 (NOAEL 225 mg / kg). In vivo efficacy in a non-toxic Ewing's sarcoma model ( Fig.12 ONC206 efficacy was comparable to methotrexate chemotherapy, but chemotherapy was associated with weight loss.

[0363] ONC213

[0364]

[0365] In vitro analysis of arrestin recruitment (GPCR activation hallmark) for GPCR activity using a heterologous reporter assay indicated that ONC213 selectively targets DRD2 / 3 and GPR132 / 91. Dual targeting of DRD2 / 3 and GPR132 / 91 represents a new 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 ONC201 but less than ONC206. The GPR132 potency of ONC213 is less than ONC212. Specifically, ONC213 exhibited in vitro anticancer potency similar to ONC212 in HCT116 / RPMI8226 cancer cells, but ONC213 had reduced in vitro toxicity to normal cells compared to ONC212 ( Fig.13 ). In a mouse MTD study with a NOAEL of 75 mg / kg, ONC213 demonstrated a safety profile three times that of ONC212 (25 mg / kg). ONC213's GPR91 agonist activity provides opportunities for immunology, immuno-oncology, and hematopoietic applications (Nature Immunology 9:1261 (2008); J Leukoc Biol. 85(5):837 (2009)).

[0366] ONC237

[0367]

[0368] In vitro analysis of arrestin recruitment (a hallmark of GPCR activation) to GPCR activity using a heterologous reporter assay indicated that ONC237 selectively targets GPR132 and DRD5. ONC237 is a GPR132 agonist and DRD5 antagonist and has reduced anticancer efficacy (IC) compared to ONC201. 50 31.2 μM). This data shows that combining GPR132 agonism and DRD5 (D1-like dopamine receptor) antagonism results in inferior anticancer effects compared to ONC213, which combines GPR132 agonism and DRD2 / 3 antagonism.

[0369] ONC236

[0370]

[0371] In vitro analysis of arrestin recruitment (a hallmark of GPCR activation) using a heterologous reporter assay for GPCR activity indicated that ONC236 is a highly selective GPR132 agonist. 5088nM) was comparable to ONC212 (10nM), better than ONC206 / ONC201, and the response completeness was better than ONC201 but not better than ONC212.

[0372] ONC234

[0373]

[0374] In vitro analysis of arrestin recruitment (a hallmark of GPCR activation) for GPCR activity using a heterologous reporter gene assay indicated that ONC234 is a broad-spectrum and potent GPCR targeting small molecule. ONC234 hits several GPCRs, including antagonist activity for adrenaline, histamine, serotonin, CHRM, CCR, DRD2 / 5 receptors, and CXCR7 agonist activity. Anticancer efficacy of ONC236 in HCT116 cells (IC 50 234 nM) was similar to ONC206, the completeness of response was the same as ONC212, and was superior to ONC201.

[0375] Reference Example 11. GPCR antagonism of ONC201

[0376] 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 an activated GPCR that serves as a reporter gene. For each dopamine receptor (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5), a cell line overexpressing the reporter construct was amplified from a frozen stock. A total volume of 20 μL of cells was seeded into a white-walled 384-well microplate and incubated at 37°C prior to testing, at 4 °C for 1 hr. 80 antagonist and then agonist at 1% concentration. Perform intermediate dilutions on sample stocks to generate assay buffer containing 5x samples. Add 3.5 μL of 5x samples to cells and incubate at 37°C or room temperature for 30 minutes; vehicle concentration is 1%. Add 5 μL of 6×EC 80 The agonist's assay buffer was added to the cells and incubated at 37°C or room temperature for 90 or 180 minutes prior to assay reading. % Antagonism was calculated using the following formula: Antagonism = 100% x (1 - (test sample mean RLU - vehicle control mean RLU) / (EC 80 Control mean RLU - vehicle control mean RLU).

[0377] Reference Example 12: Selective antagonism of DRD2 by ONC201.

[0378] 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 significant efficacy and excellent safety profile against aggressive and refractory tumors, oral ONC201 is being evaluated as a novel therapeutic agent in five early clinical trials targeting select advanced cancers.

[0379] In this example, prediction and validation of selective direct molecular interactions between ONC201 and specific dopamine receptor family members are reported. Experimental GPCR analysis indicated that ONC201 selectively antagonizes the D2-like but not D1-like dopamine receptor subfamily. Reporter gene assays in heterologous expression systems showed that ONC201 selectively antagonized the short and long isoforms of DRD2 and DRD3, with less potency for DRD4 and no antagonism for DRD1 or DRD5. Increased prolactin secretion is a clinical hallmark of DRD2 antagonism caused by several psychotropic drugs that potently target this receptor. In the first-in-human trial in advanced solid tumors, ELISA measurements of peripheral blood of patients treated with ONC201 determined that 10 / 11 patients evaluated showed prolactin induction (average 2-fold).

[0380] Using the TCGA database, the D2-like dopamine receptor subfamily (particularly DRD2) is ubiquitously and selectively overexpressed in several malignancies. Preclinical reports have shown that DRD2 inhibition confers anti-tumor efficacy through ATF4 / CHOP induction and inhibition of Akt and ERK signaling, both attributes of ONC201, without killing normal cells.

[0381] method

[0382] ONC201 dihydrochloride was obtained from Oncoceutics. Kinase inhibition assays for kinase panels were performed as described (see Anastassiadis et al., Nat Biotech 29: 1039 (2011)). GPCR arrestin recruitment and cAMP-regulated reporter gene assays were performed as described (see McGuinness et al., Journal of Biomolecular Screening 14: 49 (2009)). PathHunterTM (DiscoveRx) β-arrestin cells expressing one of several GPCR targets were seeded at 5000 cells per well in a 384-well white solid bottom assay plate (Corning 3570) in a volume of 20 μL in an appropriate cell plating reagent. Cells were incubated at 37° C., 5% CO2 for 18 to 24 hours. Samples were prepared in a buffer containing 0.05% fatty acid-free BSA (Sigma). For agonist mode testing, samples (5 μL) were added to pre-plated cells and incubated at 37°C, 5% CO2 for 90 minutes. For antagonist mode testing, samples (5 μL) were added to pre-plated cells and incubated at 37°C, 5% CO2 for 30 minutes, followed by the addition of EC 80 Agonist (5 μL) lasts for 90 minutes. For Schild analysis, samples (5 μL) were added to pre-plated cells and incubated at 37°C, 5% CO2 for 30 minutes, followed by addition of serially diluted agonist (5 μL) for 90 minutes at 37°C, 5% CO2. Control wells defining the maximum and minimum responses of each assay mode were tested in parallel. Inhibitor recruitment was measured by adding 15 μL PathHunter detection reagent and incubated at room temperature for 1 to 2 hours and read on a Perkin Elmer Envision plate reader. For agonist and antagonist testing, data were normalized for percentage efficacy using appropriate controls and fitted to a sigmoidal dose response (variable slope), Y = bottom + (top - bottom) / (1 + 10^((LogEC 50 =X)*slope)), where X is the logarithmic compound concentration. For Schild analysis, data were normalized to percent efficacy using appropriate controls and fitted to Gaddum / Schild EC using global fitting. 50 displacement, where Y = bottom + (top - bottom) / (1 + 10^((LogEC-X) * slope)), Antag = 1 + (B / (10^(-1*pA2)))^ Shield slope and LogEC = Log (EC 50*Antag). EC was performed in the CBIS Data Analysis Suite (Cheminnovation) 50 / IC 50 Analyses and Schild analysis were performed in GraphPad Prism 6.0.5.

[0383] result

[0384] ONC201 is a small molecule in Phase II clinical trials for selected advanced cancers. A p53-independent inducer of the pro-apoptotic TRAIL pathway was discovered in a phenotypic screen. Although ONC201-induced ATF4 / CHOP upregulation and Akt / ERK signaling inactivation (Allen et al., Science translational medicine 5, 171ra117-171ra117 (2013)) have been characterized for their contribution to anticancer activity, its molecular binding target remains elusive.

[0385] In vitro analysis of arrestin recruitment (a hallmark of GPCR activation) to GPCR activity using a heterologous reporter gene assay indicated that ONC201 selectively antagonizes the D2-like (DRD2 / 3 / 4) but not the D1-like (DRD1 / 5) dopamine receptor subfamily ( Figure 1 ). No antagonism of adrenoceptor alpha receptors or other GPCRs was observed under the conditions evaluated. Within the DRD2 family, ONC201 antagonizes both the short and long isoforms of DRD2 and DRD3, with DRD4 being less potent. 50Further characterization of the antagonism of DRD2L by ONC201-mediated arrestin recruitment was evaluated by displacement analysis, which determined that the dissociation constant of ONC201 was 2.9 μM, which is equivalent to its effective dose in many human cancer cells. Confirmed results of cAMP regulation in response to ONC201 were obtained, which is another measure of DRD2L activation. The abili...

Claims

1. A method for treating or preventing cancer in a subject in need thereof, the method comprising: According to a weekly regimen of oral administration, a pharmaceutical composition is administered to a subject in need of such treatment, the pharmaceutical composition comprising an amount of compound (1) or a pharmaceutically acceptable salt thereof, The weekly regimen cycle includes two doses per week on two consecutive days, Days 1 and 2, followed by a rest period, Days 3 to 7.

2. The method of claim 1, wherein the weekly regimen cycle is repeated.

3. The method of claim 1 or 2, wherein the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg.

4. The method of claim 3, wherein the dose is 625 mg.

5. The method of claim 3, wherein the selected dose is calculated based on the subject's body weight (kg) in mg / kg.

6. The method of any one of claims 3 to 5, wherein one or more subsequent doses are reduced from the initial dose.

7. The method of any one of claims 1 to 6, wherein each dose is administered to the subject on an empty stomach, without food, within two (2) hours before or after each dose.

8. The method of any one of claims 1 to 7, wherein the weekly regimen is modified after one or more cycles to include a dose once a week, day 1, followed by a rest period, days 2 to 7.

9. The method of any one of claims 1 to 8, further comprising performing radiation therapy prior to starting the weekly regimen cycle.

10. The method of claim 9, wherein the radiation is 54 to 60 Gy at 1.8 to 2.2 Gy / fraction.

11. The method of claim 8 or 9, wherein initiation of the weekly regimen cycle is performed within 6 weeks after completion of radiation.

12. The method of claim 8 or 9, wherein initiation of the weekly regimen cycle is performed one of 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after completion of radiation.

13. The method of any one of claims 9 to 12, further comprising administering one or more additional radiation treatments after initiating the weekly regimen cycle.

14. The method of any one of claims 1 to 13, wherein the cancer involves midline structures of the brain.

15. The method of any one of claims 1 to 14, wherein the cancer has a histone H3 K27M mutation.

16. The method according to any one of claims 1 to 15, wherein the cancer is a central nervous system tumor, a brain tumor, a glioma, a peripheral nervous system tumor, a pheochromocytoma, a paraganglioma, an adrenocortical carcinoma, an adrenal tumor, and a neuroendocrine tumor.

17. The method of any one of claims 1 to 16, wherein the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons, or spinal cord.

18. The method according to any one of claims 15 to 17, wherein the histone H3 K27M mutation is H3.3K27M or H3.1 K27M.

19. The method of any one of claims 1 to 18, wherein DRD2 is overexpressed in the tissue, DRD5 is underexpressed in the tissue, or both.

20. The method according to any one of claims 1 to 19, wherein the subject is a human.

21. The method of claim 20, wherein the subject is a pediatric subject and the dose is calculated based on the body weight of the pediatric subject.

22. The method of claim 21, wherein the dose is rounded to the nearest 125 mg increment.

23. The method of any one of claims 1 to 22, further comprising administering one or more doses of temozolomide.

24. The method of any one of claims 1 to 23, further comprising administering one or more doses of bevacizumab.

25. The method of any one of claims 1 to 24, further comprising performing one or more radiation treatments after initiating administration.

26. A compound (1) or a pharmaceutically acceptable salt thereof, For use in treating or preventing cancer in a subject in need thereof, the amount is according to a weekly regimen cycle for oral administration comprising two doses per week on two consecutive days, Day 1 and Day 2, followed by a rest period, Day 3 to Day 7.

27. The compound for use according to claim 26, wherein the weekly regimen cycle is repeated.

28. The compound for use according to claim 26 or 27, wherein the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg or 625 mg.

29. The compound for use according to claim 28, wherein the dose is 625 mg.

30. The compound for use according to claim 28, wherein the selected dose is calculated based on the subject's body weight (kg) in mg / kg.

31. A compound for use according to any one of claims 28 to 30, wherein one or more subsequent doses are reduced from the initial dose.

32. A compound for use according to any one of claims 26 to 31, wherein each dose is administered to the subject on an empty stomach, without food, within two (2) hours before or after each dose.

33. A compound for use according to any one of claims 26 to 32, wherein the weekly regimen is modified after one or more cycles to include a dose once a week, day 1, followed by a rest period, days 2 to 7.

34. The compound for use according to any one of claims 26 to 33, further comprising radiation therapy prior to starting the weekly regimen cycle.

35. The compound for use according to claim 34, wherein the radiation is 54 to 60 Gy at 1.8 to 2.2 Gy / fraction.

36. The compound for use according to claim 34 or 35, wherein initiation of the weekly regimen cycle is performed within 6 weeks after completion of radiation.

37. The compound for use according to claim 34 or 35, wherein initiation of the weekly regimen cycle is performed at one of 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks after completion of radiation.

38. A compound for use according to any one of claims 34 to 37, further comprising one or more additional radiation treatments after initiation of said weekly regimen cycle.

39. A compound for use according to any one of claims 26 to 38, wherein the cancer involves midline structures of the brain.

40. A compound for use according to any one of claims 26 to 39, wherein the cancer has a histone H3K27M mutation.

41. The compound for use according to any one of claims 26 to 40, wherein the cancer is a central nervous system tumor, a brain tumor, a glioma, a peripheral nervous system tumor, a pheochromocytoma, a paraganglioma, an adrenocortical carcinoma, an adrenal tumor and a neuroendocrine tumor.

42. A compound for use according to any one of claims 26 to 41, wherein the cancer involves the thalamus, medulla, hypothalamus, basal ganglia, pineal gland, midbrain, cerebellum, pons or spinal cord.

43. The compound for use according to any one of claims 40 to 42, wherein the histone H3 K27M mutation is H3.3 K27M or H3.1 K27M.

44. The compound for use according to any one of claims 26 to 43, wherein DRD2 is overexpressed in the tissue, DRD5 is underexpressed in the tissue, or both.

45. A compound for use according to any one of claims 26 to 44, wherein the subject is a human.

46. ​​The compound for use according to claim 45, wherein the subject is a pediatric subject and the dose is calculated based on the body weight of the pediatric subject.

47. The compound for use according to claim 46, wherein the dose is rounded to the nearest 125 mg increment.

48. A compound for use according to any one of claims 26 to 47, further comprising administering one or more doses of temozolomide.

49. A compound for use according to any one of claims 26 to 48, further comprising administering one or more doses of bevacizumab.

50. A compound for use according to any one of claims 26 to 49, further comprising one or more radiation treatments after the start of administration.

51. A pharmaceutical composition comprising compound (1) or a pharmaceutically acceptable salt thereof, The dosage is administered twice a week on two consecutive days, Day 1 and Day 2, followed by a rest period, Day 3 to Day 7.

52. The pharmaceutical composition of claim 51, wherein the dose is one or more of 125 mg, 250 mg, 375 mg, 500 mg, or 625 mg.

53. The pharmaceutical composition of claim 51 or 52, wherein the dose is 625 mg.

54. A pharmaceutical composition according to any one of claims 47 to 49, wherein the selected dose is calculated based on the subject's body weight (kg) in mg / kg.

55. The pharmaceutical composition of claim 54, wherein the subject is a pediatric subject and the dosage is calculated based on the body weight of the pediatric subject.

56. A pharmaceutical composition according to any one of claims 52 to 55, wherein the dose is rounded to the nearest 125 mg increment.

57. The pharmaceutical composition of any one of claims 51 to 55, further comprising radiation therapy prior to commencing the weekly regimen of administration.

58. The pharmaceutical composition of claim 56, wherein the radiation is 54 to 60 Gy at 1.8 to 2.2 Gy / fraction.

59. The pharmaceutical composition of claim 56 or 57, wherein initiation of the weekly regimen of administration is performed within 6 weeks after completion of radiation.

60. The pharmaceutical composition of claim 56 or 57, wherein initiation of the weekly regimen of administration is performed at one of 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after completion of radiation.

61. The pharmaceutical composition of any one of claims 56 to 59, further comprising one or more additional radiation treatments after initiation of the weekly regimen cycle.

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