HK40052182B - Hexahydroimidazopyridopyrimidinone, analogs and salts thereof and their use in therapy - Google Patents

Description

Hexahydroimidazole, pyridopyrimidine ketone, its analogues and salts, and their therapeutic uses

This application is a divisional application of Chinese Patent Application No. 201680008476.7, filed on January 29, 2016, entitled "7-benzyl-4-(2-methylbenzyl)-2,4,6,7,8,9-hexahydroimidozono[1,2-A]pyrido[3,4-E]pyrimidine-5(1H)-one, its analogues, its salts, and methods thereof for treatment".

Technical Field

This application relates to compounds of formula (10) or pharmaceutically acceptable salts thereof, and their pharmaceutical uses.

Background Technology

TNF-associated apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that selectively induces apoptosis in cancer cells. TRAIL is a potent inducer of apoptosis in a wide range of human cancer cell lines—via pro-apoptotic death receptor 4 (DR4; TRAIL-R1) and death receptor 5 (DR5; TRAIL-R2) on the cell surface, through involvement in extrinsic or intrinsic apoptotic pathways. TRAIL plays a direct role in tumor suppression during immune surveillance, but this anti-tumor mechanism disappears during disease progression. TRAIL's ability to selectively induce apoptosis in cancer cells has driven ongoing clinical trials administering recombinant TRAIL, as well as long-lived TRAIL agonist antibodies targeting either of its two pro-apoptotic death receptors.

Despite their effectiveness, recombinant TRAILs possess properties that limit their potency, such as short serum half-life, stability, cost, and delivery. Delivery of recombinant TRAILs or TRAIL agonist antibodies to the brain is limited because they cannot cross the blood-brain barrier. Therefore, there remains a persistent need for anticancer compositions and methods.

Summary of the Invention

In one aspect, this document provides compounds of formula (10): wherein _R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroaryl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, thioaralkyl and acyl radical, wherein when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph). In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, _C1-4 alkylpyridinyl, _C1-4 alkylisoxazoalkyl, _{C1-4 alkylmorpholino, C1-4} alkylthiazolyl, and _C1-4 alkylpyrazinyl, wherein _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, _C1-4 alkylpyridinyl, _C1-4 alkylisoxazoalkyl, _C1-4 alkylmorpholino, _C1-4 alkylthiazolyl, and _C1-4 alkylpyrazinyl are optionally replaced by _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, or perhalogenated _C1-4 alkyl _-2- alkyl-3-alkyl-4 ... _1-4 alkyl or halogen substitution. In some embodiments, _R1 and/or _R2 are substituted or unsubstituted aralkyl or heteroaralkyl. In some embodiments, the heteroaryl group is selected from _C1-4 alkylpyrrole, C1-4 alkylfuranyl, _C1-4 alkylpyridyl, _C1-4 alkyl-1,2,4-thiadiazolyl, _C1-4 alkylpyrimidinyl, _C1-4 alkylthiophenyl, _C1-4 alkylisothiazolinyl, _C1-4 alkyl imidazolinyl, _C1-4 alkyltetrazolinyl, _C1-4 alkylpyrazinyl, _C1-4 alkylpyrimidinyl, _C1-4 alkylquinolinyl, C1-4 alkylisoquinolinyl, _C1-4 thioalkylphenyl, _C1-4 alkylbenzothiophenyl, _C1-4 alkylisobenzofuranyl, _C1-4 alkylpyrazolyl, _C1-4 alkylindolyl, _C1-4 alkylpurine, _C1-4 alkylcarbazole, _C1-4 alkylbenzimidazolinyl _, and _C1-4 alkylimidazolinyl. _1-4 alkylisoxazolyl. In some embodiments, _R1 and/or _R2 are substituted or unsubstituted benzyl or phenethyl. In some embodiments, _R1 and/or _R2 is a benzyl group on the benzyl ring that is optionally substituted with one or more of the following substituents: X, _{-CH3 ,} -NO2, _-OCH3 , _-CN , -CXH2, -CX2H, _{C2- C4 alkyl} , -CX3, _-CH2 (CX3), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p + 1} , -OCX3, _{-OCpH2p +1} , _{-OCpX2p +1 ,} ^ORm , _SRm , ^NRmRn , ^NRmC (O) ^{Rn , SORm} , _SO2Rm , C(O) ^{Rm and} C(O ^{) ORm} ;

^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl groups;

Where p is an integer from 2 to 20 and X represents halogen.

In one embodiment, compound (10) has the structure of compound (90), wherein _R2 is as defined above, and wherein _Rb1 , _Rb2 , _Rb3 , _Rb4 and _Rb5 are each independently selected from hydrogen, X, _-CH3 , _-NO2 , _-OCH3 , -CN, _-CXH2 , _-CX2H , _C2 - _C4 alkyl, _-CX3 , _-CH2 ( _CX3 ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _-CpX2p+1 , -OCX3 _{, -OCpH2p +1} , _{-OCpX2p +1} , ^ORm , _SRm , ^NRmRn , ^NRmC ( ^O ) ^Rn , ^SORm , ^SO2Rm , C(O) ^{Rm and} C(O) _ORm ^;

^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl groups;

Where p is an integer from 2 to 20 and X represents halogen.

In one embodiment, compound (10) has compound (40) wherein _R1 is as defined above, and wherein _Ra1 , _Ra2 , _Ra3 , _Ra4 and _Ra5 are each independently selected from _hydrogen , X, _-CH3 , -NO2, _-OCH3 , -CN, _-CXH2 , _-CX2H , _C2 - _C4 alkyl, _-CX3 , -CH2( _CX3 ), -CH( _{CX3 ) 2} , -C( _{CX3 ) 3} , _{-CpX2p +1} , -OCX3, _-OCpH2p +1, _{-OCpX2p+1 ,} ^ORm , ^SRm , _NRmRn , ^NRmC (O) ^Rn , ^SORm , ^SO2Rm , _C (O) ^{Rm and} C(O) ^{ORm ;}

^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl groups;

Where p is an integer from 2 to 20 and X represents halogen.

In one embodiment, compound (10) has the structure of compound (50): wherein _R1 is as defined above, and wherein _Rb is selected from X, _-CH3 , _-NO2 , _-OCH3 , -CN, _-CXH2 , _-CX2H , _{C2- C4} alkyl, _-CX3 , _-CH2 ( _CX3 ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1} , _-OCX3 , _-OCpH2p +1, _{-OCpX2p + 1} , ORm, ^SRm , ^NRmRn , ^NRmC ₍ O) ^Rn , ^SORm , ^SO2Rm , C(O) ^{Rm and} C(O) ^ORm ; and wherein _Ra1 , _Ra2 , _Ra4 and _Ra5 are each independently selected ^from hydrogen, X, _-CH3 , _-NO2 , -OCH ₃ , -CN, -CXH ₂ , -CX ₂ H, C ₂ -C ₄ alkyl, -CX ₃ , -CH ₂ (CX ₃ ), -CH(CX _{3 ) 2} , _-C ( _{CX 3 ) 3 , -C p} , OR ^m , SR ^m , NR ^m R ⁿ , NR ^m C(O)R ⁿ , SOR ^m , SO ₂ R ^m , C(O)R ^m and C(O)OR ^m ;

^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl groups;

Where p is an integer from 2 to 20 and X represents halogen.

In one embodiment, compound (10) has the structure of compound (80), wherein _Ra1 , _Ra2 , _Ra3 , _Ra4 , _Ras , _Rb1 , Rb2, _Rb3 , _Rb4 and _Rb5 are each independently selected from hydrogen, X, _-CH3 , _-NO2 , _-OCH3 , -CN, _-CXH2 , _-CX2H , C2 _{- C4} alkyl, _-CX3 , _-CH2 ( _CX3 ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1 , -OCX3} , _-OCpH2p+1 ^, -OCpX2p ₊₁ , _ORm , ^SRm , ^NRmRn , _{NRmC (O)Rn, SORm, SO2Rm} ^{, C ( O} ) ^{Rm and} C(O) ^ORm ;

^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl groups;

Where p is an integer from 2 to 20 and X represents halogen.

In another aspect, the present invention provides pharmaceutical compositions comprising a compound of formula (10) or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable salt of a compound of formula (10). In one embodiment, the salt is a pharmaceutically acceptable monosalt of a compound of formula (10). In one embodiment, the salt is a pharmaceutically acceptable disalt of a compound of formula (10). In one embodiment, the pharmaceutically acceptable salt is selected from hydrochloride, hydrobromide, hydrogen sulfate, sulfate, phosphate, fumarate, succinate, oxalate and lactate, hydrogen sulfate, hydroxyl group, tartrate, nitrate, citrate, hydrogen tartrate, carbonate, malate, maleate, fumarate, sulfonate, methanesulfonate, formate, acetate and carboxylate. In one embodiment, the pharmaceutically acceptable salt is selected from p-toluenesulfonate, benzenesulfonate, methanesulfonate, oxalate, succinate, tartrate, citrate, fumarate, glucuronide, ascorbate and maleate. In one embodiment, the pharmaceutically acceptable salt is selected from ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/or other counterions, such as methylamino, dimethylamino, diethylamino, and triethylamino counterions. In one embodiment, the pharmaceutical composition comprises a dihydrochloride or dihydrobromide of a compound of formula (10). In some embodiments, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition of the present invention comprises a second therapeutic agent. In one embodiment, the second therapeutic agent is an anticancer agent. In one embodiment, the anticancer agent is a mitotic inhibitor. In one embodiment, the anticancer agent is selected from: paclitaxel, docetaxel, and combinations thereof. In alternative embodiments, the second therapeutic agent is an antiangiogenic agent. In one embodiment, the antiangiogenic agent is bevacizumab. In one embodiment, the second therapeutic agent is administered as part of a combination therapy to treat a patient. In one embodiment, details of the combination therapy are included in the packaging insert of the compound of formula (10).

In some implementations, the pharmaceutical composition is formulated for oral administration.

In another aspect, the present invention provides a treatment method. In one embodiment, the treatment method involves administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof.

In one embodiment, the treatment method includes administering a pharmaceutical composition to a subject, comprising a pharmaceutically effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof. In another embodiment, the treatment method includes administering a pharmaceutical composition to a subject, comprising a pharmaceutically effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In some embodiments, the treatment method further includes the administration of additional therapeutic agents. In one embodiment, the additional therapeutic agent includes an anticancer agent. In one embodiment, the additional anticancer agent includes an antimitotic agent. In one embodiment, the additional anticancer agent includes paclitaxel, docetaxel, bevacizumab, or a combination thereof.

In one embodiment, the treatment method further includes testing for tumor necrosis factor (TNF)-associated apoptosis-inducing ligand (TRAIL) in a sample obtained from a subject undergoing treatment. In one embodiment, the sample is a blood sample.

In one implementation of the treatment method, the subject undergoing treatment has cancer or is at risk of developing cancer. In one implementation, the cancer is selected from colon cancer, breast cancer, glioblastoma pleomorphic, endometrial lymphoma, and colorectal cancer. In another implementation, the cancer is selected from actinic keratosis, Barrett's esophagus, atrophic gastritis, congenital parakeratosis, subarachnoid dysphagia, lichen planus, oral submucosal fibrosis, solar elastosis, cervical dysplasia, leukoplakia, and erythroplakia.

In one embodiment of the treatment method, the pharmaceutical composition is administered via an oral route. In another embodiment, the pharmaceutical composition is administered via a route selected from the following: intravenous, rectal, nasal, pulmonary, epidural, ocular, ear, intra-arterial, local, intracardiac, intraventricular, intradermal, intramuscular, intraperitoneal, intraosseous, intrasheath, intrabladder, subcutaneous, transdermal, transmucosal, sublingual, oral, vaginal, and inhalation routes.

In one embodiment, the present invention provides a method for treating a subject with brain cancer or at risk of brain cancer, the method comprising: administering to the subject a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides a treatment method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising: a pharmaceutically effective amount of a compound of formula (10) or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for treating a subject requiring such treatment, the method comprising:

(i) administering a first therapeutic agent to the subject, including a compound of formula (10) or a pharmaceutically acceptable salt thereof;

(ii) After administering the first treatment to the subject, wait until the predetermined waiting period has elapsed; and

(iii) When administering a second therapeutic agent to a subject, a predetermined waiting time is selected to obtain the delayed therapeutic effect of the first therapeutic agent without increasing the risk of increased risk of the first and second therapeutic agents or reducing the risk of possible combined toxicity.

In another aspect, the present invention provides a method for treating a subject requiring such treatment, the method comprising:

(i) administering a first therapeutic agent to the subject, including a compound of formula (10) or a pharmaceutically acceptable salt thereof;

(ii) Using pharmacokinetic analysis to monitor the levels of compound (10) or its salts or metabolites in subjects; and

(iii) Administer the second therapeutic agent according to the condition of the level of the first therapeutic agent in the subject.

In another aspect, the present invention provides a method comprising:

(i) administering a first therapeutic agent, including a compound of formula (10) or a pharmaceutically acceptable salt thereof, to the subject; and

(ii) The second therapeutic agent is administered based on the expected half-life of the compound of formula (10) in the subject who has undergone treatment.

On the other hand, this document provides analogues of compounds of formula (10): formula (1): and pharmaceutically acceptable salts thereof, and methods for their preparation. In one aspect, this document provides a treatment method. In one embodiment, the treatment method administers a pharmaceutical composition to a subject, the composition comprising: a pharmaceutically effective amount of a compound of formula (10) or formula (1) or an analogue thereof, or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In one embodiment, the subject undergoing treatment has cancer or is at risk of having cancer. In one embodiment, the subject is a human being.

In another aspect, the present invention provides a method for treating a subject requiring such treatment, the method comprising:

(i) administering to the subject a first therapeutic agent, including an analogue of compound (1) (e.g., a compound of formula (10)) or a pharmaceutically acceptable salt thereof; and

(ii) Administer the second therapeutic agent based on the condition of an adverse event of the first therapeutic agent that has been resolved or is being resolved. In some implementation protocols, the adverse event of the first therapeutic agent is related to the blood level of the agent or metabolite in the treated subject.

On the other hand, the present invention provides a kit for monitoring compound (1) (e.g., compound (10) analogues, pharmaceutically acceptable salts, or metabolites thereof) in individuals treated with compound (1) or its analogues, pharmaceutically acceptable salts thereof, or metabolites thereof, using pharmacokinetic analysis. The kit includes multiple point-of-care devices or point-of-use devices capable of quantifying the drug in at least two samples or matrices suitable for storing those samples prior to laboratory quantification. In some embodiments, the kit also includes instructions for collecting and/or storing at least two samples.

In some embodiments, the treatment method administers a pharmaceutical composition to a subject comprising a pharmaceutically effective amount of a compound of formula (1) or (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) monitors the levels of the compound or a pharmaceutically acceptable salt thereof, or a metabolite thereof, in the subject using pharmacokinetic analysis, wherein the dose of the compound or a salt thereof is selected to maintain a concentration of the compound or a metabolite thereof at least about 400 ng/mL in the subject's whole blood, plasma, serum, or cerebrospinal fluid for 4 hours following treatment. In some embodiments, the subject undergoing treatment has cancer or is at risk of cancer. In some embodiments, the treatment method further includes a step of waiting for a predetermined waiting time between one or more repetitions of step (i) (e.g., the waiting time is the same length as the first time interval). In some embodiments, the subject is a human being.

In some embodiments, the treatment method administers a pharmaceutical composition to a subject, the pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (1) or formula (10), or an analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) monitors the level of the compound or its metabolites in the subject using pharmacokinetic analysis, wherein the dose of the compound or its salt is selected such that the AUC of the compound or its metabolites obtained in the subject’s whole blood, plasma, serum or cerebrospinal fluid over a period of 2, 6, 12, 24, 48, 72 or more, or extrapolated to infinity, is at least about 3,500 hr-ng/mL.

In some embodiments, the treatment method administers a pharmaceutical composition to a subject, the pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (1) or formula (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) monitors the level of compound (1), or a pharmaceutically acceptable salt thereof, or a metabolite thereof in the subject using pharmacokinetic analysis, wherein the dose of the compound or its salt is selected to result in an undetectable concentration of the compound or its metabolite in the subject's whole blood, plasma, serum, or cerebrospinal fluid at 3, 4, 5, 6, or 7 days after treatment.

In some embodiments, the treatment method includes: (i) administering multiple doses of a therapeutic agent, including a compound of formula (1) or formula (10), or an analogue thereof, or a pharmaceutically acceptable salt thereof, during a first time interval (e.g., 7 days), wherein multiple doses are administered during a second time interval within the first time interval (e.g., a first time interval consisting of two to five consecutive days per week); and (ii) repeating step (i) once or more. In some embodiments, the subject undergoing treatment has cancer or is at risk of having cancer. In some embodiments, the treatment method further includes a step of waiting for a predetermined waiting time (e.g., the waiting time is the same length as the first time interval) between one or more repetitions of step (i). In some embodiments, the subject is a human being.

In some embodiments, the pharmaceutical composition is administered to the subject once daily. In some embodiments, the pharmaceutical composition is administered to the subject according to an infrequent dosing regimen (e.g., once weekly or less frequently). In some embodiments, the pharmaceutical composition is administered to the subject according to a frequent dosing regimen (e.g., more than once weekly). In some embodiments, the pharmaceutical composition is administered to the subject once weekly. In some embodiments, the pharmaceutical composition is administered to the subject once every four weeks. In some embodiments, the pharmaceutical composition is administered to the subject twice weekly. In some embodiments, the pharmaceutical composition is administered to the subject three times weekly. In some embodiments, the pharmaceutical composition is administered to the subject four times weekly. In some embodiments, the pharmaceutical composition is administered to the subject once every two weeks. In some embodiments, the pharmaceutical composition is administered to the subject once every three weeks. In some embodiments, the pharmaceutical composition is administered to the subject on repeated cycles of once weekly, once every two weeks, once every three weeks, once every four weeks, or combinations thereof.

In one aspect, this document provides methods for identifying whether a subject with a disease is likely to respond to a treatment regimen. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the RNA modification level and/or expression or activity level of one or more proteins of Tables 1 and 3 in the sample, and/or the presence or absence of one or more mutations in at least one gene expressing one or more proteins of Tables 1 and 3; (iii) comparing the measured levels and/or mutations found in the sample with those of predetermined criteria; and (iv) determining whether the subject is likely to respond to the treatment regimen based on the measured and/or found levels of mutations in the sample with those of predetermined criteria. In one aspect, this document provides methods for providing prognosis for subjects with a disease. In some embodiments, the method includes: (i) obtaining a biological sample from the subject; (ii) measuring the RNA modification level and/or expression or activity level of one or more proteins of Tables 1 and 3 in the sample, and/or the presence or absence of one or more mutations in at least one gene expressing one or more proteins of Tables 1 and 3; (iii) comparing the measured levels and/or mutations found in the sample with those of predetermined criteria; and (iv) determining the subject's prognosis based on the measured and/or found levels of mutations in the sample with those of predetermined criteria.

In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or an analogue thereof. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is miRNA. In one embodiment, the RNA modification is methylation. In one embodiment, the RNA modification is ^N6 -methyladenosine ( ^m6A ). In one embodiment, the RNA modification is ^m6AmRNA methylation. In one embodiment, the subject is a human. In one embodiment, the subject is a domesticated pet, such as a cat or dog. In one embodiment, the protein is an RNA reader, such as YTHDF3. In one embodiment, the protein is an RNA writer. In one embodiment, the protein is an RNA wiper.

In one aspect, this document provides methods for identifying and treating subjects who have a condition and are likely to respond to a treatment regimen described herein. In some embodiments, the methods include (i) identifying whether the subject has a condition that is likely to respond to a treatment regimen described herein; and (ii) treating the subject identified as likely to respond to the treatment regimen. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or an analogue thereof.

In one aspect, this article provides a method for evaluating the effectiveness of monitoring subjects with a condition and undergoing treatment according to the treatment regimen described herein. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the RNA modification level and/or expression or activity level of one or more proteins of Tables 1 and 3 in the sample, and/or the presence or absence of one or more mutations in at least one gene expressing one or more proteins of Tables 1 and 3; (iii) comparing the measured levels and/or mutations found in the sample with those of predetermined criteria; and (iv) determining whether the subject responded to the treatment regimen based on the level of mutations measured and/or found in the sample with those of predetermined criteria.

In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or an analogue thereof. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is miRNA. In one embodiment, the RNA modification is methylation. In one embodiment, the RNA modification is ^N6 -methyladenosine ( ^m6A ). In one embodiment, the RNA modification is ^m6AmRNA methylation. In one embodiment, the subject is a human. In one embodiment, the subject is a domesticated pet, such as a cat or dog. In one embodiment, the protein is an RNA reader, such as YTHDF3. In one embodiment, the protein is an RNA writer. In one embodiment, the protein is an RNA wiper.

In one aspect, this document provides a method for screening potential treatments for a disease. In some embodiments, the method includes (i) contacting the proteins of Tables 1 and 3 with a test compound suspected of being a treatment for the disease; (ii) measuring the binding affinity or interaction between the test compound and the protein; and (iii) comparing the binding affinity or interaction between the test compound and a predetermined threshold, wherein an affinity or interaction of the test compound that is comparable to or greater than the threshold would indicate treatment for the disease. In some embodiments, the disease is cancer. In some embodiments, the predetermined threshold is the affinity or interaction between compound (1) or its analogue and the protein. In some embodiments, the predetermined threshold is the affinity or interaction between compound (1) and the protein. In some embodiments, the protein is an RNA reader, such as YTHDF3. In some embodiments, the protein is an RNA writer. In some embodiments, the protein is an RNA wiper.

In one aspect, this document provides a method for screening potential treatments for a disease. In some embodiments, the method includes (i) contacting the proteins of Tables 1 and 3 with a reference compound of the disease, wherein the reference compound binds to or interacts with the protein; (ii) contacting the proteins of Tables 1 and 3 with a test compound suspected of being a treatment for the disease; (iii) measuring the binding affinity or interaction between the reference and test compounds and the protein; and (iv) comparing the binding affinity or interaction between the reference and test compounds, wherein a comparable or stronger affinity or interaction of the test compound relative to the reference compound would indicate treatment for the disease. In one embodiment, the reference compound is compound (1) or an analogue thereof. In one embodiment, the reference compound is compound (1). In one embodiment, the disease is cancer. In one embodiment, the protein is an RNA reader, such as YTHDF3. In one embodiment, the protein is an RNA writer. In one embodiment, the protein is an RNA wiper.

In one aspect, this article provides a method for screening potential treatments for a disease. In some embodiments, the method includes (i) if any, using a processor to model the 3D structure of a test compound suspected to be a treatment for the disease and the 3D structure of the proteins in Tables 1 and 3 for binding or interaction; (ii) using the processor to determine the binding affinity or interaction between the test compound structure and the protein structure; and (iii) using the processor to compare the binding affinity or interaction of the test compound with a predetermined threshold, wherein a binding affinity or interaction of the test compound that is comparable to or greater than the threshold would indicate cancer treatment. In one embodiment, the predetermined threshold is the affinity or interaction between compound (1) or its analogue and the protein. In one embodiment, the predetermined threshold is the affinity or interaction between compound (1) and the protein. In some embodiments, the disease is cancer. In one embodiment, the protein is an RNA reader, such as YTHDF3. In one embodiment, the protein is an RNA writer. In one embodiment, the protein is an RNA wiper.

In one aspect, this article provides a method for screening potential treatments for a disease. In some embodiments, the method includes (i) using a processor to model the 3D structure of a reference compound and the 3D structures of proteins in Tables 1 and 3, combining or interacting them; (ii) if so, using a processor to model the 3D structure of a test compound suspected of being a treatment for the disease and the 3D structure of a protein, combining or interacting them; (iii) using a processor to determine the binding affinity or interaction between the reference and test compounds and the protein; and (iv) using a processor to compare the binding affinity or interaction between the reference and test compound structures, wherein a comparable or stronger affinity or interaction of the test compound relative to the reference compound would indicate a treatment for the disease. In one embodiment, the reference compound structure is the structure of compound (1) or an analogue thereof. In some embodiments, the reference compound structure is the structure of compound (1). In some embodiments, the disease is cancer. In some embodiments, the protein is an RNA reader, such as YTHDF3. In some embodiments, the protein is an RNA writer. In some embodiments, the protein is an RNA wiper.

In one aspect, this document provides a method for treating a subject with a condition by administering an agent that regulates the transcription, translation, or biological activity of YTHDF3 or other proteins or nucleic acids encoding these proteins listed in Tables 1 and 3. The agent includes, but is not limited to, siRNA, antisense nucleic acid, ribozyme, triple-helix forming agent, antibody and peptide, and small molecule compounds. Preferably, the siRNA, antisense nucleic acid, ribozyme, or triple-helix forming agent inhibits the translation or transcription of one or more genes listed in Tables 1 and 3. To achieve this, the oligonucleotides used are designed based on uniquely relevant sequences of the target gene. Various gene therapy methods can be used to regulate the expression of genes listed in Tables 1 and 3 according to the invention. For example, antisense DNA molecules can be engineered and used to block the translation of YTHDF3 mRNA in vivo. Alternatively, ribozyme molecules may be designed to cleave and destroy YTHDF3 mRNA in vivo. Alternatively, oligonucleotides designed to hybridize with the 5′ region of the YTHDF3 gene (including the region upstream of the coding sequence) and form a triple-helix structure can block or reduce the transcription of the YTHDF3 gene. In another alternative, the nucleic acid encoding the full-length wild-type YTHDF3 message can be introduced into cells in vivo, otherwise insufficient amounts of the wild-type YTHDF3 gene product will not be produced, or no wild-type YTHDF3 gene product will be produced at all.

In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the subject has a viral infection or is at risk of having a viral infection. In some embodiments, the subject is a human. In some embodiments, the dose of compound (1) or a pharmaceutically acceptable salt thereof ranges from about 125 mg to about 625 mg. In some embodiments, a sample obtained from the subject is tested for lysed and/or all cytokeratin-18 to determine whether the treatment regimen should continue based on the results of the lysed and/or all cytokeratin-18 test. In some embodiments, the treatment regimen described herein further includes the step of administering a second therapeutic agent, wherein compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof, is administered before, simultaneously with, or after the second therapeutic agent.

In one aspect, this document provides a method for identifying whether a subject with a condition is likely to respond to a treatment regimen described herein. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the expression, post-translational modification, or activity level, or mutation of eIF2-α, ATF4, CHOP, or DR5 in the sample; (iii) comparing the levels of the measured and/or detected mutations in the sample with those of predetermined criteria; and (iv) determining whether the subject is likely to respond to the treatment regimen based on the levels of the measured and/or detected mutations in the sample with those of predetermined criteria. In another aspect, this document provides methods for evaluating the effects of the treatment regimens described herein, monitoring, or providing prognostic outcomes for subjects with a condition. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the expression, post-translational modification, or activity level, or mutation of eIF2-α, ATF4, CHOP, or DR5 in the sample; (iii) comparing the levels of the measured and/or found mutations in the sample with those of predetermined criteria; and (iv) determining prognosis or determining whether the subject responded to the treatment regimen based on the levels of the measured and/or found mutations in the sample with those of predetermined criteria. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the subject has a viral infection or is at risk of having a viral infection. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof.

In one aspect, this document provides methods for identifying whether a subject with a condition is likely to respond to a treatment regimen described herein. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the expression, post-translational modification, or activity level, or mutation of at least one dopamine receptor in the sample; (iii) comparing the level of the measured and/or found mutation in the sample with predetermined criteria; and (iv) determining whether the subject is likely to respond to the treatment regimen based on the level of the measured and/or found mutation in the sample with predetermined criteria. In another aspect, this document provides methods for evaluating the effects of the treatment regimens described herein, monitoring, or providing prognostic outcomes for subjects with a condition. In some embodiments, the method includes (i) obtaining a biological sample from the subject; (ii) measuring the expression, post-translational modification, or activity level, or mutation of at least one dopamine receptor in the sample; (iii) comparing the level of the measured and/or found mutation in the sample with predetermined criteria; and (iv) determining prognosis or determining whether the subject responds to the treatment regimen based on the level of the measured and/or found mutation in the sample with predetermined criteria. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administration of an effective amount of compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof. In some embodiments, the dopamine receptor is selected from DRD2, DRD2S, DRD2L, and DRD3. In some embodiments, the dopamine receptor is from the D2-like family of dopamine receptors.

In one aspect, this document provides a method for screening potential treatments for a condition. In some embodiments, the method includes: (i) contacting at least one G protein-coupled receptor (GPCR) with a test compound suspected to be a treatment for the condition; (ii) measuring the binding affinity, interaction, or GPCR signaling between the test compound and the GPCR; and (iii) comparing the binding affinity or interaction between the test compound and a predetermined threshold, wherein GPCR antagonism or GPCR signaling antagonism of the test compound is comparable to or greater than the threshold indicating cancer treatment. In one embodiment, the condition is cancer. In one embodiment, the predetermined threshold is GPCR antagonism or GPCR signaling antagonism of compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof.

In one aspect, this document provides a method for screening potential treatments for a condition. In some embodiments, the method includes (i) exposing at least one dopamine receptor to a test compound suspected as a treatment for the condition; (ii) measuring the binding affinity or interaction between the test compound and at least one dopamine receptor; and (iii) comparing the binding affinity or interaction between the test compound and a predetermined threshold, wherein inhibition of at least one dopamine receptor by the test compound is comparable to or greater than the threshold to indicate cancer treatment. In some embodiments, the condition is cancer. In some embodiments, the dopamine receptor is selected from DRD2, DRD2S, DRD2L, and DRD3. In some embodiments, inhibition of the D2-like family of dopamine receptors indicates treatment for the condition. In some embodiments, inhibition of DRD2 and DRD3 dopamine receptors indicates treatment for the condition. In some embodiments, the predetermined threshold is the inhibition of at least one dopamine receptor by compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof.

In one aspect, the present invention provides a method for screening potential treatments for a disease. In some embodiments, the method includes (i) if any, using a processor to model the binding or interaction between a 3D structure of a test compound suspected to be a treatment for the disease and a 3D structure of at least one dopamine receptor; (ii) using the processor to determine the binding affinity or interaction between the test compound structure and the at least one dopamine receptor structure; and (iii) using the processor to compare the binding affinity or interaction of the test compound with a predetermined threshold, wherein inhibition of at least one dopamine receptor by the test compound is comparable to or greater than the threshold, indicating cancer treatment. In some embodiments, the disease is cancer. In some embodiments, the dopamine receptor is selected from DRD2, DRD2S, DRD2L, and DRD3. In some embodiments, inhibition of the D2-like family of dopamine receptors indicates treatment for the disease. In some embodiments, inhibition of DRD2 and DRD3 dopamine receptors indicates treatment for the disease. In some embodiments, the predetermined threshold is the inhibition of at least one dopamine receptor by compound (1) or a pharmaceutically acceptable salt thereof, or an analogue thereof, with the dopamine receptor.

In one aspect, this document provides methods for treating and evaluating the effects of treatment in subjects with a condition. In some embodiments, the method includes (i) treating the subject according to the treatment methods described herein; and (ii) evaluating the effects of the treatment as described herein. In some embodiments, the subject has cancer or is at risk of having cancer. In one embodiment, the treatment regimen includes administering an effective amount of a compound (1) or a pharmaceutically acceptable salt thereof or an analogue thereof.

The foregoing overview and the following detailed description of embodiments of the compositions and treatment methods will be better understood when read in conjunction with the appended claims. However, it should be understood that the invention is not limited to the precise arrangements and means described herein.

Attached Figure Description

The foregoing overview and the following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings of exemplary embodiments. However, it should be understood that the invention is not limited to the precise arrangements and means shown.

In the attached diagram:

Figure 1 shows the dose-response relationship, demonstrating the effect of various concentrations of compound (1) on the viability of tumor and normal cells;

Figure 2 illustrates the cell viability test in human fetal lung fibroblasts (MRC-5) cells after treatment with compound (1) for 72 hours.

Figure 3 illustrates the antagonistic effect of ONC201 on dopamine receptors (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5).

Figure 4 shows the levels of soluble prolactin detected by ELISA in peripheral blood of tumor patients at baseline and in real-time, following a single ONC201 dose (PO 125–625 mg). Post-sampling treatments included late treatments at 6 hours, 1, 2, 7 days, and 21 days.

Figures 5A and 5B show the relationship between mean ONC201 plasma concentration and time after the first dose of ONC201. In Figure 5A, plasma concentrations are shown as the mean for each dose cohort, and in Figure 5B, plasma concentrations for individuals treated with 625 mg are shown. Error bars represent standard deviations.

Figures 6A and 6B show individual measurements of AUC and _Cmax of ONC201 relative to dose. *The 625 mg dose group is based on the expected dose ratio line for males.

Figures 7A and 7B show the M30 test rate relative to four patients at time after a single dose and the M30 test rate relative to tumor type, respectively.

Figure 8 illustrates the in vitro sensitivity of ONC201 to multiple tumor types, as determined in a large number of cell lines from the Cancer Protocol Drug Sensitivity Genome (GDSC).

Detailed Implementation

The scientific terms used in this article are intended to have the meaning commonly understood by one of ordinary skill in the art. These terms are found to be defined in various standard references and used illustratively, including J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al. ., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L.Nelson and M.M.Cox, Lehninger Principles of Biochemistry, 4th Ed., V.H. Freeman & Company, 2004; Engelke, D.R., RNA Interference (RNAi): Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, P a., 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in MolecularBiology, Humana Press, 2 004; A.Nagy, M.Gertsenstein, K.Vintersten, R.Behringer, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold S pringHarbor Laboratory Press; Dec. 15, 2002, ISBN-10: 0879695919; Kursad Turksen (Ed.), Embryonic stem cells: methods and protocols in Methods Mol Biol. 2002; 185, Humana Press; Current Protocols in Stem Cell Biology, ISBN: 9780470151808, and U.S. Patent No. 8,673,923. The contents of the foregoing references are incorporated herein by reference in their entirety.

The singular terms “a,” “an,” and “the” are not restrictive and include plural indicators unless explicitly stated or indicated by the context.

The term “substitution,” if used herein, means that any one or more hydrogen atoms on a specified atom are replaced by the indicated group, provided that the normal valence of the specified atom does not exceed a certain value and the substitution results in a stable compound. When the substituent is a ketone (i.e., =O), two hydrogen atoms on the atom are substituted. Aromatic substituents are not present on the aromatic moiety of a ketone. A cyclic double bond is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N).

When any variable (e.g., _R4 ) appears more than once in any component or type of compound, its definition for each occurrence is independent of its definition for each other occurrence. Thus, for example, if a group is shown to be substituted by 0-3 _R4 moieties, that group may optionally be substituted by up to three _R4 moieties, and _R4 is independently selected from the definition of _R4 in each occurrence. Furthermore, combinations of substituents and/or variables are permitted, but only if such combinations result in a stable compound.

When an atom or chemical part is followed by a numerical range of subscripts (e.g., C _1-6 ), it is understood that this means covering all numbers within that range as well as all intermediate ranges. For example, "C _1-6 alkyl" refers to alkyl groups having 1, 2, 3, 4, 5, 6, 6-1, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

As used herein, "alkyl" is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, _C1-6 alkyl is intended to include _C1 , _C2 , _C3 , _C4 , _C5 , and _C6 alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl-sec-butyl, tert-butyl, n-pentyl, sec-pentyl, neopentyl, and n-hexyl. In some embodiments, straight-chain or branched alkyl groups have six or fewer carbon atoms in their main chain (e.g., straight-chain _C1 - _C6 , branched _C3 - _C6 ), and in other embodiments, straight-chain or branched alkyl groups have four or fewer carbon atoms. Similarly, cycloalkyl groups have three to eight carbon atoms in their ring structure, and in other embodiments, cycloalkyl groups have five or six carbon atoms in their ring structure. The most preferred are ( _C1 - _C6 ) alkyl groups, especially ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term "substituted alkyl" means an alkyl group as defined above, substituented by one, two, or three substituents selected from halogens, -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 , _-NO₂ , preferably containing one or two substituents selected from halogens, -OH, alkoxy, _-NH₂ , trifluoromethyl, -N( _CH₃ ) _₂ , and -C(=O)OH, more preferably selected from halogens, alkoxy, and -OH. Examples of substituted alkyl groups include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl, and 3-chloropropyl.

Unless otherwise specified, “lower alkyl” includes alkyl groups as defined above, but having 1 to 6 carbon atoms in their main chain structure, preferably 1 to 4. “Lower alkenyl” and “lower alkynyl” have a chain length of 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms.

The term "alkenyl" includes unsaturated aliphatic groups of similar length and possibly substituted alkyl groups, but containing at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g., vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched alkenyl groups, cycloalkenyl groups (e.g., alicyclic) (e.g., cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl- or alkenyl-substituted cycloalkenyl groups, and cycloalkyl- or cycloalkenyl-substituted alkenyl groups. In some embodiments, straight-chain or branched alkenyl groups have six or fewer carbon atoms in their backbone (e.g., straight chains with _C2 - _C6 , branched chains with _C3 - _C6 ). Similarly, cycloalkenyl groups may have three to eight carbon atoms in their ring structure, and in some embodiments, cycloalkenyl groups have five or six carbon atoms in their ring structure. The term " _C2 - _C6 " includes alkenyl groups containing 2-6 carbon atoms. The term " _C3 - _C6 " includes alkenyl groups containing 3 to 6 carbon atoms.

"Alynyl" includes unsaturated aliphatic groups of similar length and possibly substituted alkyl groups, but contains at least one triple bond. For example, "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentyynyl, hexynyl, heptyynyl, octyynyl, nonynyl, decanynyl), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl-substituted alkynyl groups. In some embodiments, the straight-chain or branched alkynyl group has six or fewer carbon atoms on its main chain (e.g., straight-chain _C2 - _C6 , branched _C3 - _C6 ). The term " _C2 - _C6 " includes alkynyl groups containing 2-6 carbon atoms. The term " _C3 - _C6 " includes alkynyl groups containing 3 to 6 carbon atoms.

As used herein, "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group, wherein each atom forming the ring (i.e., the skeleton atom) is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused to an aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following:

Monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl groups include, but are not limited to, tetrahydronaphthyl, indenyl, and tetrahydropentene. Polycyclic cycloalkyl groups include adamantane and norbornene. The term cycloalkyl includes "unsaturated non-aromatic carbocyclic group" or "non-aromatic unsaturated carbocyclic group," both of which refer to a non-aromatic carbocyclic ring as defined herein, containing at least one carbon-carbon double bond or one carbon-carbon triple bond.

As used in this article, "cycloalkylalkyl" refers to an alkyl group that has been substituted with a cycloalkyl group. Examples of cycloalkylalkyl groups include cyclopropylalkyl, cyclohexylalkyl, etc.

As used herein, "heterocyclic alkyl" refers to a non-aromatic heterocycle in which one or more cyclic atoms can be heteroatoms, such as O, N, or S atoms. Heterocyclic alkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) ring systems as well as spirocyclic systems. Examples of heterocyclic alkyl groups include morpholino, thiomorpholino, piperazine, tetrahydrofuran, tetrahydrothiophene, 2,3-dihydrobenzofuran, 1,3-benzodioxane, benzo-1,4-dioxane, piperidinyl, pyrrolidinyl, isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, imidazolyl, etc. Also included are portions that, in the definition of heterocyclic alkyl, can have one or more aromatic rings fused with a non-aromatic heterocycle (i.e., having the same bonds as it), such as phthalimino, naphthylimino, and heterocyclic benzo derivatives. A heterocyclic alkyl group having one or more fused aromatic rings is linked by an aromatic or non-aromatic portion. Also included in the definition of heterocyclic alkyl is one in which one or more cyclic atoms can be substituted with one or two oxygen or sulfhydryl groups. In some embodiments, the heterocyclic alkyl group has 1 to about 20 carbon atoms, and in other embodiments, about 3 to about 20 carbon atoms. In some embodiments, the heterocyclic alkyl group contains 1 or more cyclic atoms. In some embodiments, the heterocyclic alkyl group has 1 or more heteroatoms. In some embodiments, the heterocyclic alkyl group contains 1 double bond. In some embodiments, the heterocyclic alkyl group contains 1 triple bond.

As used in this article, "heterocyclic alkyl alkyl" refers to an alkyl group substituted with a heteroalkyl group. Examples of heterocyclic alkyl alkyl groups include morpholinoalkyl and piperazineylalkyl groups.

As used herein, "aryl" refers to monocyclic or polycyclic aromatic hydrocarbons (e.g., those having 2, 3, or 4 fused rings), such as phenyl, naphthyl, anthracene, phenanthrene, etc. In some embodiments, the aryl group has one carbon atom.

As used herein, "aralkyl" refers to an alkyl group substituted with an aryl group. Examples of aralkyl groups include benzyl and phenethyl.

As used herein, “heteroaryl” refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3, or 4 fused rings) systems. Any cyclic nitrogen atom in a heteroaryl group can also be oxidized to form an N-oxygen moiety. Examples of heteroaryl groups include, but are not limited to, pyridinyl, N-oxopyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolinyl, isoquinolinyl, thiopheneyl, imidazolinyl, thiazolyl, indoleyl, pyrroleyl, oxazolyl, benzofuranyl, benzothiopheneyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indoleyl, 1,2,4-thiophene diazolyl, isothiazolyl, benzothiopheneyl, purine, carbazoleyl, benzimidazolinyl, dihydroindoleyl, etc. In some embodiments, the heteroaryl group has 1 to about 20 carbon atoms, and in other embodiments, about 3 to about 20 carbon atoms. In some embodiments, the heteroaryl group contains one or more cyclic atoms. In some embodiments, the heteroaryl group has one or more heteroatoms.

As used in this article, "heteroaryl" refers to an alkyl group substituted with a heteroaryl group. An example of a heteroaryl group is pyridylmethyl.

As used herein, "halogen" or "halogen" refers to a fluorine, chlorine, bromine, or iodine atom, preferably fluorine, chlorine, or bromine, and more preferably fluorine or chlorine. The term "per halogen" refers to the portion in which all hydrogen atoms are replaced by halogen atoms.

The term "halogenated alkyl" refers to an alkyl portion having a halogen atom replacing one or more hydrogen atoms on the hydrocarbon backbone. _C1 - _C6 haloalkyls are intended to include straight-chain or branched alkyl groups having six or fewer carbon atoms on their backbone, and halogen atoms replacing one or more hydrogen atoms on the hydrocarbon backbone.

The term "alkoxy" or "alkoxy group" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. _C1 - _C6 alkoxy groups refer to six moieties having a few carbon atoms in the hydrocarbon skeleton. Examples of alkoxy groups (or alkoxy compounds) include methoxy, ethoxy, isopropoxy, propoxy, butoxy, and pentoxy. Preferred are ( _C1 - _C3 ) alkoxy groups, particularly ethoxy and methoxy. Examples of substituted alkoxy groups include haloalkoxy groups.

The term "hydroxyl" or "hydroxyl group" includes groups having -OH or -O-.

This invention also includes pharmaceutically acceptable salts of the chemicals described herein. As used herein, “pharmaceutically acceptable salt” refers to a derivative of the disclosed compound whose parent compound is modified by converting an existing acid or base moiety into its salt form. Pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids, etc. The pharmaceutically acceptable salts of this invention include conventionally non-toxic salts of the parent compound from which they are formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this invention are synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Typically, these salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or a mixture of both; typically, non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of applicable 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 PH. Stahl and CGWermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, ^2nd Revised edition, Weinheim/Zürich: Wiley-VCH/VHCA (2011), the full contents of which are incorporated herein by reference.

Examples of suitable inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid, while examples of suitable organic acids may include carboxylic acids, sulfonic 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, benzopentanoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, citric acid, maleic acid, salicylic acid, trifluoroacetic acid, 3-aminosalicylic acid, ascorbic acid, aconitic acid, nicotinic acid, isonicotinic acid, oxalic acid, gluconic acid, amino acids, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, or naphthalene-2-sulfonic acid. Suitable examples of inorganic bases may include sodium hydroxide, potassium hydroxide, and ammonia, and suitable examples of organic bases are amines, such as tertiary amines, such as trimethylamine, triethylamine, pyridine, N,N-dimethylaniline, quinoline, isoquinoline, α-methylpyridine, β-methylpyridine, γ-methylpyridine, quinalidine, or pyrimidine.

I. Compound (1), its salts and their synthesis

The inventors have found in in vitro models, animal models, and human clinical trials that ONC201 (compound (1)) possesses broad-spectrum anticancer activity, low toxicity, including few (if any) side effects, low genotoxicity, and high bioavailability, including oral bioavailability. These properties make ONC 201 and its analogues particularly suitable for a wide range of applications.

In one aspect, the present invention provides compound (1): compound (1) can be prepared by the synthetic method shown in Scheme 1 below.

Option 1

In one embodiment, the synthesis (1) of the dihydrochloride of the compound begins with a commercially available intermediate, N-benzyl-3-methoxy-4-piperidinone hydrochloride, compound (3). In one embodiment, the synthesis method includes producing compound (4), a free radical, from the neutralized compound (3) together with a base (step 1). In one embodiment, the synthesis method includes producing compound (4) from the neutralized compound (3) and an inorganic base. In one embodiment, the synthesis method includes producing compound (4) from the neutralized compound (3) together with an organic matrix. In one embodiment, the intermediate compound (3) is neutralized in the presence of an alcohol. For example, intermediate compound (3) is neutralized in the presence of n-butanol. In one embodiment, intermediate compound (3) is neutralized in the presence of at least one organic solvent. For example, intermediate compound (3) is neutralized in the presence of n-butanol and/or ethyl acetate. In one embodiment, intermediate compound (3) is neutralized in the presence of a base and at least one organic solvent. For example, intermediate compound (3) is neutralized in the presence of _NaHCO3 and n-butanol. In one embodiment, the intermediate compound (3) is neutralized in the presence of n-butanol and triethylamine ( _Et3N ).

In one embodiment, the synthetic method includes reacting compound (4) and compound (5) (step 2) to prepare intermediate compound (1). In one embodiment, the reaction in step 2 includes heating compound (4) and compound (5). In one embodiment, the reaction in step 2 includes reflux heating of compound (4) and compound (5) in the presence of a solvent. In one embodiment, the reaction in step 2 includes removing water and/or methanol (MeOH) formed in the reaction using a Dean-Stark trap.

In one embodiment, the synthetic method includes forming a dihydrochloride salt of the compound (1) (step 3). In one embodiment, the reaction in step 3 includes treating the compound (1) with HCl in dioxane. In one embodiment, the reaction in step 3 includes treating the compound (3) with 4N HCl in dioxane. In one embodiment, the synthetic method optionally includes recrystallization (1) of the dihydrochloride of the compound.

In a preferred embodiment, the method for synthesizing the dihydrochloride salt of compound (1) is described in Scheme 2 below.

Option 2

II. TNF-associated virus-inducing ligands (“TRAIL”)

TRAIL proteins can be tested in test samples obtained from subjects to detect TRAIL expression induced by the compounds described herein and their salts. Immunoassays can be used to test for TRAIL in samples, including but not limited to enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELIFA), flow cytometry, Western blotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescent immunoassay (LIA), fluorescence immunoassay (FIA), and radioimmunoassay. Assay methods can be used to obtain qualitative and/or quantitative results. The specific details of appropriate test methods for qualitative and quantitative analysis of samples are described in the standard references, including E. Harlow & D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; F. Breitling & S. Diibel, Recombinant Antibodies, John Wiley & Sons, New York, 1999; H. Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Basics: From Background to Bench, BIOS Scientific Publishers, 2000; BKCLo, Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; F. MAusubel 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 Univetsity Press, 2000; Givan, AL, Flow Cytometry: first principles, Wiley, New York, 2001; Gorczyca, W., Flow Cytometry in Neoplastic Hematology: ologic morph-immunophenotypic eorrelation, Taylor & Francis, 2006; Crowther, JR, The ELISA Guidebook (Methods in Molecular Biology), Humana Press, 2000; Wild, D., The Immunoassay Handbook, ^3rd Edition, Elsevier Science, 2005, and J.Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Ptess, ^3rd ed., 2001.

Examples of methods for detecting TRAIL in samples used to detect the effect of a pharmaceutical composition are described in U.S. Patent 8,673,923 to Wafik S. El-deiry et al., the entire contents of which are incorporated herein by reference.

In one implementation, the TRAIL test is used to monitor the subject. Thus, for example, test samples are obtained from the subject once or multiple times during and/or after treatment with the pharmaceutical composition to assess the effectiveness of the treatment. In another instance, test samples are obtained from the subject at different times to assess the course or progression of the disease or healing. In one implementation, death receptors may also be analyzed from circulating tumor cells to see if the administration of the compound described herein or its salts increases the amount or type of death receptors.

Cancers treated using the methods and compositions described herein are characterized by abnormal cell proliferation, including but not limited to pretumoral hyperplasia, cancer in situ, tumors, and metastases. The methods and compositions described herein can be used to prevent and improve signs and/or symptoms of cancer. The terms "treatment" and "curative therapy" used to treat a subject's cancer include: preventing, inhibiting, or improving the subject's cancer, such as slowing cancer progression and/or reducing or improving symptoms or signs of cancer. Examples of cancers treated using the methods and compositions described herein include, but are not limited to, breast cancer, CNS cancers, colon cancer, ovarian cancer, prostate cancer, leukemia, lung cancer, and lymphoma.

III. Compound (10) and its salts

In one respect, this document provides compounds and their associated salts (10) and the same process for their preparation. Those skilled in the art will understand that the general principles and concepts described herein in conjunction with compounds (1) and their salts (including principles and concepts related to methods and pharmaceutical compositions) apply to compounds of formula (10) and their salts.

In some embodiments, the present invention provides compound (10): wherein _R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, heteroaryl, aralkyl, heteroaryl, alkoxyalkyl, alkoxycarbonyl, aralkoxy, thioaralkyl and acyl groups, wherein when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph.

In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, _C1-4 alkylpyridinyl, C1-4 alkylisoxazoalkyl, _C1-4 alkylmorpholino, _C1-4 alkylthiazolyl, and _C1-4 alkylpyrazinyl, wherein the _C1-4 alkyl, C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, _C1-4 alkylpyridinyl, _C1-4 alkylisoxazoalkyl _{, C1-4} alkylmorpholino, _C1-4 alkylthiazolyl, and _C1-4 alkylpyrazinyl are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, perhalogenated _{C1-4 alkyl} , or halogen. In some embodiments, _R1 and/or _R2 are substituted or unsubstituted, aralkyl or heteroaralkyl. In some embodiments, the heteroaryl group is selected from _C1-4 alkylpyrrole, _C1-4 alkylfuranyl, _C1-4 alkylpyridyl, _C1-4 alkyl- _1,2,4- thiadiazolyl, C1-4 alkylpyrimidinyl, _C1-4 alkylthiophenyl, C1-4 alkylisothiazolinyl, _C1-4 alkyltetrazolinyl, _C1-4 alkylpyrazinyl, _C1-4 alkylpyrimidinyl, _C1-4 alkylquinolinyl, _C1-4 alkylisoquinolinyl, _C1-4 thioalkylphenyl, _C1-4 alkylbenzothiophenyl, _C1-4 alkylisobenzofuranyl, _C1-4 alkylpyrazolyl _{, C1-4} alkylindolyl, _C1-4 alkylpurine, _C1-4 alkylcarbazole, _C1-4 alkylbenzimidazolinyl, _{C1-4 alkylalkyl} Isoxazolyl group.

In some embodiments, _R1 and/or _R2 is a benzyl group that is optionally substituted on the benzyl ring with one or more of the following substituents: X _{, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C2-C4 alkyl , -CX3 , -CH2 ( CX3} ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1 ,} -OCX3, -OCpH2p+ ₁ , _{-OCpX2p + 1} , ^ORm , _SRm , NRmRn, ^NRmC (O) ^Rn , ^SORm , ^SO2Rm , C(O) ^{Rm and} C ₍ O) ^{ORm ; Rm} _and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl; ^and wherein p is an integer from 2 to 20 and X represents a halogen, including Includes fluorine, chlorine, bromine or iodine atoms, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine.

In some embodiments, _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 , _CH2CH2 (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), CH ₂ CH ₍ OH)Ph, (4-F-Ph)-4- _oxybutyl , _{CH2CH2NHCOOC ( CH3 ) 3} , _{CH2CH2CH2NH2 and CD2C6D5 .} In some embodiments, _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-diF-Ph), _CH2- (2,4-diCl-Ph), _CH2- (3,4-diCl-Ph), _CH2- (3,4-diF-Ph), _CH2- (3,5-diF _{-Ph), CH2 -} ((2- _CH3 )-Ph), _CH2 (2- _CH3,4 -F-Ph), _CH2 -((4- _OCH3 )-Ph), _CH2 -(3-pyridyl), CH _2- (3-isooxazolyl), CH ₂ CH _2- (4-morpholinyl), CH _2- (2-F,4-CF ₃ -Ph), CH ₂ CH(OH)Ph, (CH ₂ ) ₃ CO-4F-Ph, (4-F-Ph)-4-oxobutyl, CH ₂ CH ₂ NHCOOC(CH ₃ ) ₃ , CH ₂ CH ₂ CH ₂ NH ₂ and CD ₂ C ₆ D ₅ .

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In one embodiment, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In one embodiment, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In one embodiment, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In one embodiment, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In one embodiment, _R2 is a substituted or unsubstituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridylmethyl group. In one embodiment, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In one embodiment, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

In one embodiment, compound (10) has the structure of compound (80): wherein _Ra1 , _Ra2 , _Ra3 , Ra4, _Ra5 , _Rb1 , _Rb2 , _Rb3 , _Rb4 and _Rb5 are each independently selected from X, _-CH3 , _-NO2 , _-OCH3 , _-CN , -CXH2, _-CX2H , C2 _-C4 alkyl, _-CX3 , _-CH2 (CX3 ₎ , -CH( _CX3 ) ₂ , -C _{( CX3} ) ₃ , _{-CpX2p +1 ,} -OCX3, -OCpH2p ₊₁ , -OCpX2p ⁺ ₁ , _ORm , _SRm , ^NRmRn , _{NRmC (O)Rn, SORm, SO2Rm} ^{, C ( O} ) ^{Rm and} C(O) ^{OR m} ; ^Rm and ^Rn are independently selected from hydrogen or _C1 - _C4 alkyl; and where p is an integer from 2 to 20 and X represents a halogen.

In one embodiment, compound (10) has the structure of compound (90) wherein _R2 is as defined above, and wherein _Rb1 , _Rb2 , _Rb3 , _Rb4 and _Rb5 are each independently selected from _X , _-CH3 , -NO2, _-OCH3 , _-CN , -CXH2, _-CX2H , _C2 - _C4 alkyl, _-CX3 , _-CH2 ( _CX3 ), -CH( _CX3 ) ₂ , -C( _{CX3 ) 3} , -CpX2p ₊₁ , -OCX3, _-OCpH2p +1, _{-OCpX2p +1 ,} ^ORm , ^SRm , _NRmRn , ^NRmC (O) ^Rn , ^SORm , ^SO2Rm , C(O) ^Rm _and C(O) ^ORm ; ^Rm and ^Rn are independently selected from ^hydrogen or _C1 - ^C ₄ -alkyl; and where p is an integer from 2 to 20 and X represents a halogen.

In one embodiment, compound (10) has the structure of compound (40) wherein _R1 is as defined above, and wherein _Ra1 , _Ra2 , _Ra3 , _Ra4 and _Ra5 are each independently selected from hydrogen, _X , _-CH3 , -NO2, _-OCH3 , _-CN , -CXH2, _-CX2H , _{C2 -C4 alkyl} , _-CX3 , -CH2( _CX3 ), -CH( _CX3 ) ₂ , -C( _{CX3 ) 3} , -CpX2p ₊₁ , -OCX3, _{-OCpH2p +1, -OCpX2p +} ¹ _, ^{ORm, SRm ,} _NRmRn , ^NRmC (O) ^Rn , ^SORm , _SO2Rm , C(O) ^Rm and C(O) ^{ORm ; Rm and Rn} are independently selected from hydrogen or _C1 -C ₄ alkyl; and where p is an integer from 2 to 20 and X represents a halogen. In one embodiment, R ₁ is hydrogen. In one embodiment, R ₁ is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In one embodiment, the aralkyl group is substituted with a C _1-4 alkyl, C _1-4 alkoxy, hydroxyl, fully halogenated C _1-4 alkyl, or halogen. 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 the following: halogenated, -CH ₃ , -CF ₃ , -OCH _3. In one embodiment, the benzyl group is substituted with one halogen substituent, such as a fluorine substituent, and two halogen substituents, such as fluorine substituents, are substituted at the two meta positions.

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

_Ra1 , _Ra2 , _Ra3 , _Ra4 , and _Ra5 are as defined above. In one embodiment, the benzyl group is substituted with one or more halogens. In some embodiments, the benzyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In one embodiment, _Ra1 or _Ra5 is a halogen, such as fluorine. In one embodiment, _Ra2 or _Ra3 is a halogen, such as a fluorine substituent.

In one embodiment, compound (10) has the structure of compound (50) wherein _R1 is as defined above, and wherein _Rb is selected from X, _-CH3 , _-NO2 , -OCH3, _-CN , _-CXH2 , _-CX2H , C2 _{- C4} alkyl, _-CX3 , _-CH2 ( _CX3 ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1} , _-OCX3 , _-OCpH2p+1 ^, _-OCpX2p+1 , _ORm , ^SRm , ^NRmRn , _NRmC (O) ^Rn , ^SORm , ^SO2Rm , C(O) ^Rm and C(O) ^ORm ; ^Rm and ^Rn are independently selected from ^hydrogen or _C1 - _C4 alkyl; and ^wherein p is an integer from 2 to 20 and X represents a halogen, and _wherein R _a1 , _Ra2 , _Ra4 , and _Ra5 are each independently selected from hydrogen, X, _-CH3 , -NO2, _-OCH3 , _-CN , _-CXH2 , _-CX2H , _C2 - _C4 alkyl, _-CX3 , -CH2( _CX3 ), -CH( _CX3 ) ₂ , -C( _CX3 ) ₃ , -CpX2p _{+1, -OCX3, -OCpH2p+1 , -OCpX2p + 1 , ORm} , ^SRm , _NRmRn , NRmC(O) ^Rn , ^SORm , ^SO2Rm , C(O) ^Rm , ^and C(O) ^{ORm ; Rm and Rn are} independently selected from hydrogen or _C1 - _C4 alkyl; ^and where p is an integer from 2 to ₂₀ and X represents a halogen. In one embodiment, _R1 is hydrogen. In one embodiment, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In one embodiment, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, perhalogenated _C1-4 alkyl, or halogen. In one embodiment, _Rb is selected from halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, one or more _Ra1 , _Ra2 , _Ra4 , _Ra5 are selected from halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, _Ra1 , _Ra2 , _Ra4 , _Ra5 are hydrogen, and _Rb is selected from halogenated, _-CH3 , _-CF3 , and _-OCH3 . In one embodiment, _Rb is a halogen, such as fluorine, and _Ra1 is methyl. In one embodiment, _Rb is fluorine or chlorine, and _Ra2 is fluorine or chlorine. In one embodiment, _Rb is _CF3 . In one embodiment, _Rb is _-OCH3 . In one implementation, _Rb is chlorine and _Ra1 is chlorine.

In one embodiment, compound (50) has the structure of compound (55): wherein _Ra1 , _Ra2 , _Ra4 , _Ra5 , and _Rb are as defined above. In one embodiment, _Rb is selected from halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, one or more _Ra1 , _Ra2 , _Ra4 , and _Ra5 are selected from halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, _Ra1 , _Ra2 , _Ra4 , and _Ra5 are hydrogen, and _Rb is selected from halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, _Rb is a halogen such as fluorine, and _Ra1 is methyl. In one embodiment, _Rb is fluorine or chlorine, and _Ra2 is fluorine or chlorine. In one embodiment, _Rb is _CF3 . In one embodiment, _Rb is _OCH3 . In one implementation, _Rb is chlorine and _Ra1 is chlorine.

In one embodiment, compound (10) has the structure of compound (60). In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, _R1 is a substituted or unsubstituted heterocyclic alkyl group or a substituted or unsubstituted heteroaryl group, such as _CH2- (2-thienyl), _CH2- (3-thienyl), _CH2-2 -pyridyl, _CH2-3 -pyridyl, _CH2-4 -methyl _- 2-thiazolyl, _CH2-2 -pyrazinyl, _CH2CH2 (4-N-benzyl-piperazine), _CH2- (3-isooxazolyl), and _CH2CH2- (4-morpholinyl). In one embodiment, the aralkyl group is substituted with _{a C1-4 alkyl} , _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In one embodiment, the benzyl group is substituted with one or more halogens. In some embodiments, the benzyl group is substituted with one or more substituents selected from the following: halogen (e.g., fluorine) _-CH3 , _-CF3 , _-OCH3 . In one embodiment, the benzyl group at the para position is halogenated, _-CH3 , _-CF3 , _-OCH3 . In one embodiment, _R1 is fluorophenyloxybutyl or hydroxyphenylethyl.

Synthesis of compound of formula (10) in scheme 3

Option 3

Compounds of formula (10) are synthesized from substituted piperidinones, which are converted by reaction with substituted aminoimidazolines to give core compound (10). There are two routes in which the R1 substituent is present in the piperidinone (e.g., 68). In this route, (68) is reacted with dimethyl carbonate in toluene using sodium hydride at 80 °C to form piperidinone ester (69). A commercially available methylthiohydrozoline HI salt (63) is reacted with an amine in dioxane at 70 °C to give an _R2 -substituted aminoimidazoline (64) as its HI salt. The reaction of (64) with piperidinone ester (69) in 1-butanol, with water removed by a Dean-Stark trap over 3–6 hours, gives trichlorocompound (10). In a variation of this scheme, N-BOC-protected piperidone (61) is converted to a BOC-protected compound (65) by the same method, which is then treated with HCl in dioxane to remove the BOC group, and then converted to 66) by extraction with 1NNaOH and methyl chloride. Subsequently, (66) is treated with a halide (67) or an epoxide (70) to obtain the desired compound (10).

The crude product can be purified by column chromatography elution with methyl chloride methanol, or by HPLC using acetonitrile:TFA:H2O, to obtain the final product as a free base or TFA salt. Treatment of the free base with HCl in dioxane, or lyophilization of the TFA salt, yields product (10) as an HCl or TFA salt. Alternatively, the free base can be treated with another inorganic or organic acid to form other salts, typically selected from salts known to be pharmaceutically acceptable. The salts of compound (10) are usually solids, and examples have been crystallized from ethanol or other solvents to obtain high-quality crystals. In the case of compound (1), the tricyclic structure was determined by X-ray crystal structure and NMR.

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 them in the cellular environment. Expression of these binding targets can be used to predict responses to compound (1) (ONC201) or its analogues (i.e., as biomarkers). Additionally, these compounds can be used to screen for competitive tests of structurally unrelated anticancer compounds known in the art to identify drugs capable of transcending target interactions with higher affinity. Furthermore, these molecules may possess pharmaceutical properties that produce therapeutic improvements or allow for additional therapeutic applications by altering drug properties, including but not limited to pharmacokinetics, potency, safety, biodistribution, or metabolism.

Examples of compound (10)

As described in Example 12 below, a compound (1) with an aminoalkyl linker (i.e., compound (33)) was used to identify proteins that interact with compound (1). Compound (1) was found to interact with proteins involved in the methylation of N6-methyl-adenosine ( ^m6A ) mRNA. Proteins involved in the epigenetic modification of ^m6A mRNA include those listed in Table 1. These proteins include methylated mRNAs (RNA writers), such as METTL3, METTL14, WTAP, and KIAA1429; those that demethylate and kill ^m6A mRNAs (RNA wipers), such as FTO and ALKBH5; and those that specifically recognize ^m6A RNAs (RNA readers), such as YTHDF3, YTHDF2, YTHDF1, YTHDC1, and YTHDC2.

Table 1: ^m6A mRNA methylation proteins

IV.x Assess the sensitivity and efficacy of treatment options.

Measuring expression, post-translational modification, or activity levels, or mutations in eIF2-α, ATF4, CHOP, DR5, or cleaved or whole cytokeratin 18 can be used to predict response or sensitivity, and to identify subjects who may respond to treatments described herein, such as those using compound (1) or its pharmaceutically acceptable salts or medicaments thereof. Additionally, measuring expression, post-translational modification, or activity levels, or mutations in eIF2-α, ATF4, CHOP, DR5, or cleaved or whole cytokeratin 18 can be used to assess or monitor the effectiveness of treatments described herein. Furthermore, measuring expression, post-translational modification, or activity levels, or mutations in eIF2-α, ATF4, CHOP, DR5, or cleaved or whole cytokeratin 18 can be used in vitro or in vitro to screen for structurally unrelated anticancer compounds. For example, competition and other tests can be used to identify drugs that can transcend target interactions with higher affinity, to compare changes in these levels with the respective changes produced by compound (1) or its analogues. The test can also be performed on living mammalian cells, which more closely approximate the effect of the drug at specific serum levels in the body, or on microsomal extracts prepared from cultured cell lines.

In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or compound (10) or an analogue thereof. In some embodiments, the treatment regimen includes administering an effective amount of compound (1). In some embodiments, the treatment regimen includes administering an effective amount of a compound of formula (10). In some embodiments, the compound of formula (10) is a compound of formula (40), for example, a compound of formula (45). In some embodiments, the compound of formula (10) is a compound of formula (50), for example, compound of formula (55). In some embodiments, the compound of formula (10) is a compound of formula (80). In some embodiments, the compound of formula (10) is a compound of formula (90). In some embodiments, the compound of formula (10) is a compound of formula (60). In some embodiments, the analogues of compound (1) have compounds (25), (26), (27), (28), (29), (30), or (31).

The predetermined standard level can be, for example, the mean or median level measured from a sample from a subject. The predetermined standard level can be measured under the same or substantially similar experimental conditions as those used to measure the sample from the subject. The predetermined standard level can be obtained from a subject responding to the chemical substance (1) or compound (10) or its analogues. In one embodiment, the predetermined standard is obtained from a subject who responds to chemical treatment, and if the level in a sample of the standard is similar to the level in the standard, the subject can be classified as likely to respond to treatment. The predetermined standard level can be obtained from a subject who does not respond to chemical treatment. In one embodiment, the predetermined standard is obtained from a subject who does not respond to compound treatment, and if the level in a subject's sample differs from the expected standard level (e.g., up- or down-regulated), the subject can be classified as likely to respond to treatment. The predetermined standard level can be obtained from a normally healthy subject.

Immunoassays can be used to determine the levels of protein or methyl chemistry in a sample, including but not limited to enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (ELIFA), flow cytometry, Western blotting, immunoprecipitation, immunohistochemistry, immunocytochemistry, luminescent immunoassay (LIA), fluorescent immunoassay (FTA), and radioimmunoassay. The methyl chemistry levels in ^6A mRNA samples can be obtained by methylRNA immunoprecipitation (Me-RIP) or other quantitative biochemical assays known in the art.

Nucleic acid mutations can be determined using any of many known methods. For example, a biological sample of an individual can be obtained. Such biological samples include, but are not limited to, bodily samples (such as urine, saliva, plasma, or serum) or tissue samples (such as buccal tissue samples or buccal cells). The biological sample can then be sequenced or scanned using known methods. For example, DNA arrays can be used to analyze at least a portion of a subject's genome sequence. Furthermore, complete or partial genome sequence information can be used. Such sequences can be determined using standard sequencing methods including chain termination (Sanger dideoxynucleotide), dye terminator sequencing, and SOLID™ sequencing (Applied Biosystems). The entire genome sequence can be sequenced by restriction enzyme cutting or shearing (mechanically) into shorter fragments. Known methods such as PCR and vector-based cloning methods (e.g., E. coli) can also be used to amplify DNA sequences. In one embodiment, at least a portion of a 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 a chip-based technology to determine the presence of mutations or copy number variations.

In one aspect, this document provides a method for identifying and treating a subject with a condition who can respond to a treatment regimen described herein. In one embodiment, the method includes (i) identifying whether a subject with a condition can respond to a treatment regimen described herein; and (ii) treating the subject with the treatment regimen that may respond to the treatment regimen. In one embodiment, the subject has cancer or is at risk of having cancer. In one embodiment, the treatment regimen includes administering an effective amount of compound (1) or compound (10) or an analogue thereof. In some embodiments, the treatment regimen includes administering an effective amount of compound (1). In some embodiments, the treatment regimen includes administering an effective amount of a compound of formula (10). In some embodiments, the compound of formula (10) is a compound of formula (40), such as compound of formula (45). In some embodiments, the compound of formula (10) is a compound of formula (50), such as compound of formula (55). In some embodiments, the compound of formula (10) is a compound of formula (80). In some embodiments, the compound of formula (10) is a compound of formula (90). In some embodiments, the compound of formula (10) is a compound of formula (60). In some embodiments, an analogue of compound (1) has a structure selected from the structures of compound (25), compound (26), compound (27), compound (28), compound (29), compound (30) or compound (31).

The predetermined standard level can be, for example, the mean or median level measured in a sample from a subject. The predetermined standard level can be measured under the same or substantially similar experimental conditions as those used to measure the sample from the subject. The predetermined standard level can be obtained from a subject who responds to the chemical substance (1) or compound (10) or its analogues. In one embodiment, the predetermined standard is obtained from a subject who responds to compound treatment, and if the subject's sample level is similar to the level in the standard, then the subject can be classified as having a responsiveness to treatment. The predetermined standard level can be obtained from a subject who does not respond to chemical treatment. In one embodiment, the predetermined standard is obtained from a subject who does not respond to compound treatment, and if the level in the subject's sample differs from the expected standard level (e.g., up- or down-regulated), then the subject can be classified as likely to respond to treatment. The predetermined standard level can be obtained from a normal healthy subject. An immunoassay can be used to determine the protein level in the sample.

In one aspect, this document provides methods for treating and evaluating the effectiveness of treatment in subjects with a condition. In some embodiments, the method includes (i) treating the subject according to the treatment methods described herein; and (ii) evaluating the effectiveness of the treatment as described herein. In some embodiments, the subject has cancer or is at risk of having cancer. In some embodiments, the treatment regimen includes administering an effective amount of compound (1) or compound (10), or an analogue thereof. In some embodiments, the treatment regimen includes administering an effective amount of compound (1). In some embodiments, the treatment regimen includes administering an effective amount of a compound of formula (10). In some embodiments, the compound of formula (10) is a compound of formula (40), such as compound (45). In some embodiments, the compound of formula (10) is a compound of formula (50), such as compound of formula (55). In some embodiments, the compound of formula (10) is a compound of formula (80). In some embodiments, the compound of formula (10) is a compound of formula (90). In some embodiments, the compound of formula (10) is a compound of formula (60). In some embodiments, the analogues of compound (1) have a structure selected from the structures of compound (25), compound (26), compound (27), compound (28), compound (29), compound (30) or compound (31).

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

V. Composition

In one aspect, a pharmaceutical composition is provided comprising a compound of formula (10): or formula (1): and a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable salt of the compound. 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 hydrochloride, hydrobromide, hydrogen sulfate, sulfate, phosphate, fumarate, succinate, oxalate and lactate, hydrogen sulfate, hydroxyl group, tartrate, nitrate, citrate, tartrate, carbonate, malate, maleate, fumarate sulfonate, methanesulfonate, formate, acetate and carboxyl group. In one embodiment, the salt is a pharmaceutically acceptable salt selected from p-toluenesulfonate, benzenesulfonate, citrate, methanesulfonate, oxalate, succinate, tartrate, fumarate and maleate. In one embodiment, the salt is a pharmaceutically acceptable salt selected from ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/or counterions such as methylamino, dimethylamino, diethylamino, and triethylamino. In one embodiment, the salt is a dihydrochloride or dihydrobromide salt of the compound.

Compound (1) has the same chemical structure as revealed by structural analysis (e.g., NMR, X-ray diffraction) of chemical substance NSC350625 obtained through the National Cancer Institute Development Therapy Program Repository.

In one embodiment, the pharmaceutical composition comprises a disalt (e.g., a dihydrochloride) of compound (1) or an analogue thereof (e.g., a compound of formula (10)). A salt (e.g., a disalt or trisalt) of an analogue of compound (1) may be prepared from an analogue of compound (1), which may be synthesized as described herein or using standard chemical synthesis methods known to those skilled in the art.

In one embodiment, the pharmaceutical composition includes at least one pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, 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 editions. Exemplary pharmaceutically acceptable carriers, pharmaceutical compositions, and methods of administration of various dosage forms are well known in the art, for example in: Pharmaceutical Dosage Forms: Tablets, edited by Larry L. Augsburger & Stephen W. Hoag., London: Informa Healthcare, 2008; and in L.V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, Pa. Lippincott, Williams&Wilkins, 2004; A.R. Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott Williams&Wilkins, 21st ed., 2005, part icularly chapter 89; and J.G. Hardman et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill Professional, 10th ed., 2001.

In one embodiment, the pharmaceutical composition is used for ocular administration. In one embodiment, the pharmaceutical composition is used for topical administration. In one embodiment, the pharmaceutical composition is formulated as oil drops, ointment, or liquid. In one embodiment, the pharmaceutical composition includes conventional pharmaceutical carriers such as aqueous, powdered, or oily bases, thickeners, etc.

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

In one embodiment, the pH of the intravenous preparation is about 3. In one embodiment, the intravenous preparation is adjusted to pH 3 with phosphate buffer. In one embodiment, the intravenous preparation comprises glucose or sodium chloride. In one embodiment, the intravenous preparation comprises a compound or a salt thereof at a concentration of about 5 mg/mL and pH 3, forming a stable solution. In one embodiment, the intravenous preparation comprises a compound or a salt thereof at a concentration of about 5 mg/mL and pH < 5, forming a stable solution. In one embodiment, the intravenous preparation comprises a compound or a salt thereof and one or more antioxidants. In one embodiment, the intravenous preparation comprises a mixture of mono- and dihydrochlorides of the compound. In one embodiment, the intravenous preparation comprises a 1% solution of the compound or a salt thereof at a concentration of about 10 mg/mL, for example, the intravenous preparation is a solution with a pH of about 3.3. In one embodiment, the pH is less than 4.0.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In one embodiment, a suitable pharmaceutically acceptable carrier includes an oil. In one embodiment, a suitable pharmaceutically acceptable carrier includes sterile water. In one embodiment, a suitable pharmaceutically acceptable carrier includes an aqueous carrier. In some embodiments, the intravenous preparation comprises glucose and/or sodium.

In one embodiment, the intravenous preparation comprises compound (1) or its analogue or dihydrochloride dissolved in 25 mg/mL water. In one embodiment, the intravenous preparation is adjusted to pH 3 with phosphate buffer. In one embodiment, the intravenous preparation comprises glucose or sodium chloride. In one embodiment, the intravenous preparation comprises a dihydrochloride of compound (1) or its analogue at a higher or lower concentration. In one embodiment, the intravenous preparation comprises compound (1) or its analogue or dihydrochloride at a concentration of about 5 mg/mL. In one embodiment, the intravenous preparation comprises compound (1) or its analogue or dihydrochloride at a concentration of about 5 mg/mL and pH 3 to form a stable solution. In one embodiment, the intravenous preparation comprises compound (1) or its analogue or dihydrochloride at a concentration of about 5 mg/mL and pH < 5 to form a stable solution. In one embodiment, the intravenous preparation comprises compound (1) or its analogue or dihydrochloride and one or more antioxidants. In one embodiment, the intravenous preparation comprises a mixture of monohydrochloride and dihydrochloride of compound (1) or its analogue. In one embodiment, the intravenous formulation comprises compound (1) or its analogue or its dihydrochloride, as a 1% solution with a concentration of about 10 mg/mL, for example, the intravenous formulation having a pH of about 3.3. In one embodiment, the pH is less than 4.0.

In one embodiment, the intravenous formulation comprises about 0.5% to about 10% (or about 5 mg/mL to about 100 mg/mL) of compound (1) or its analogues or their disalts. In one embodiment, the intravenous formulation comprises about 5% (or about 50 mg/mL) of compound (1) or its analogues or their disalts. In one embodiment, the intravenous infusion rate may be slowed to reduce the side effects of compound (1) or its analogues or their disalts.

In one embodiment, the pharmaceutical composition comprises about 0.1-99% of a salt of compound (1) or its analogues; and a pharmaceutically acceptable carrier, such as oil or sterile water or other aqueous carrier. In one embodiment, the pharmaceutical composition comprises about 5% to about 50% of a mono or disalt of compound (1) or its analogues in an oral dosage form.

In some embodiments, the pharmaceutical composition includes an antioxidant. Suitable antioxidants include: ascorbic acid derivatives, such as ascorbic acid, isoascorbic acid, sodium ascorbate, thioglycerol, cysteine, acetylcysteine, cystine, dithiored threitol, dithiothreitol, glucothiocyanate, tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sulfites such as sodium sulfate, sodium bisulfite, sodium acetone bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, and sodium thiosulfate, and dihydroguaiac acid. It should be noted that antioxidants used in aqueous formulations typically include: sodium sulfite, sodium metabisulfite, sodium formaldehyde sulfonate, and ascorbic acid and combinations thereof, while antioxidants used in oil-based solutions and organic solvents include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl propionate and combinations thereof. In other embodiments, the antioxidant may be one or more of the following: flavonoids, isoflavones, monothioglycerol, L-cysteine, thioglycolic acid, α-tocopherol, ascorbic acid 6-palmitate, dihydrolipoic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin E, propyl gallate, β-carotene, or ascorbic acid. The antioxidant may typically be used at about 0.1% to 1.0% by weight, more typically about 0.2%.

In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, and at least one other therapeutic agent. Other therapeutic agents, for example, are 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; tubuline 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 (e.g., Nutlins), and anti-angiogenic agents. Factors (e.g., atezolizumab, afestin, sorafenib, and regorafenib), amifostine, anagrelide, clodronate, filgrastim, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porphyrin, 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epomycin B, A 105972, A 204197, abiraterone, interleukin, ivermectin, allovocin-7, altretamine, alvocidib, amonafide, anthraphenazole, AG-2 037, AP-5280, apaziquone, apomine, arianiline, atraxifen, atametadalafil, atestistein, aspirin PE, AVLB, AZ10992, ABX-EGF, AMG-479 (ganitomab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (selumetinib) 704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib) AZD-2014, azacytidine, azabeta-methyl-B, azonafide, BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, bevacizumab, BEZ-235, disodium docosanoate, BCX-1777, BKM-120, buclomisin, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtovok), BIBF1120 (vargatef), BI 836 845, BI 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, Bleomycin A, Bleomycin B, Brivanib, Bryostatin-1, Bortezomib, Brostallicin, Busulphan, BYL-719, CA-4 Prodrug, CA-4, CapCell, Calcitriol, Capecitabine, Canfosfamide, Capecitabine, Carboxyphthalimide, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1 Cefixime, Cefotaxime, Ceftriaxone, Celecoxib, Celmoleukin, Cema dotin, CH4987655/RO-4987655, chlorotriphenylamine, silengiptide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicine, cobustatin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholine doxorubicin, cytarabine, D 24851, decitabine, deoxyvitamin, deoxyvitamin, deoxyribonucleic acid, dephospholipid ascorbic acid B, dexamethasone, dexamethasone, diethylstilbestrol, diflomotec an, didox, DMDC, dolatasstatin 10, dorastinazole, DS-7423, E7010, E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFR inhibitor, EKB-569, EKB-509, enzastaurin, itramycin B, epremycin B, epremycin, epomefloxacin, ER-86526, erlotinib, ET-18-0CH3, ethinyl cytidine, ethinyl estradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenivelamine, pheneretan, pheneretan, folic acid, FOLFOX, FOLFO X4, FOLFIRI, Gefitinib, Gefitinib, Gefitinib, Gefitinib, Gefitinib, Gefitinib, GFC-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT Immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK- 2110183, GSK-2141795, GW2016, Granisetron, Herceptin, Hexamethylmelamine, Histamine, Homoharringtonine, Hyaluronic Acid, Hydroxyurea, Hydroxyprogesterone Caprate, Ibandronate, Ibumetabine, Itramycin, Itrastatin, Esstatin, IDN-5109, IGF-1R Inhibitor, IMC-1C11, IMC-A12 (cixutumumab), Immunoglobulin, Hemolysin, Interferon α-2a, Interferon α-2b, Pegylated Interferon α-2b, Interleukin-2, INK-1117, INK-128, INSM-18, Ionafamib, Ipilimumab, Isoflavones, Isotretinoin, Ixabepi Lone, JRX-2, JSF-154, J-107088, Conjugated Estrogen, Kahalid F, Ketoconazole, KW-2170, KW-2450, Lobaplatin, Leflunomide, Isoroxetine, Leuporelin, Lexidronam, LGD-1550, Linezolid, Thai Baht, Tylosin, Losartan, Ruxotanine, LU 223651, Lurtotecan, LY-S6AKT1, LY-2780301, Mafosfamide, Marimastat, MEChlorethylamine, MEK Inhibitor, MEK-162, Methyltestosterone, Methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX- 447, MDX-1379, MGV, midostaurin, minodromic acid, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexafin gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, noratrexate, noreline, N-acetylcysteine, O6-benzylguanine, oblimersen, omeprazole, oncophage, oncoVEXGM-CSF, ormiplatin, o rtataxel, OX44 antibody, OSI-027, OSI-906 (1 insitinib), 4-1BB antibody, oxantrazole, estrogen, panitumumab, patupilone, pegfilgrastim, PCK-3145, pEG-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 Agents, PG-TXL, PG2, PLX-4032/RO-5185426(vemurafenib)-3603/RO-5212054, PT-100, PWT-33597, PX-866, pyrplatin, neopentyloxymethylbutyrate, heptaprerenone, phenoxodiol O, PKI166, plevitrexed, sclerosingmycin, polymalonic acid, porphyrin, prednisone, prednisolone, quinolones, R115777, RAF-265, ramosetron, ranpimase, RDEA-119/BAY869766, RDEA-436, rebeccamycin analogs, receptor tyrosine kinase (RTK) inhibitors. Regorafenib, Revimid, RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, Rhizobacterium, Rhu-MAb, Relifatin, Risedronate, Rituximab, Rofanumab, Rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR109881A, Naringin, Erythromycin, R-Flurbiprofen, RX-0201, S-9788, Abarabine, SAHA, Sargramostim, Satraplatin, SB 408075, Se-015/Ve-015, SU5416, SU6668, SDX-101, Semustin, 407 1, SR-27897, SR-31747, SR-13668, SRL-172, Sorafenib, Spirocarboplatin, Casserole, Simvastatin Hydroxyxamic Acid, Sutent, T 900607, T 138067, TAK-733, TAS-103, Tylosin, Tapapalpenem, Tylosin, Tylosin, Tyrosartan, Tegafur, Temozolomide, Tessaprofen, Temoferol, Temoferolin, Testosterone, Propionate, Tesmyfen, Tetrachloroplatinic Acid, Tetrodotoxin, Tezazazepine, Thalidomide, Trastuzumab, Tiprazole, Tovaminamide, Tovaminin, Tramatinib, TransMID-107, Retinic Acid, Trastuzumab, Tremelimumab, Retinoic Acid, Triacetyluridine, Triapine Triciribine, Trimethoprim, TLK-286TXD 258, Tykerb/Tyverb, Urobilirubin, Psychosin, Varassini, Vincristine, Virulizin, WX-UK1, WX-554, Vectibix, Xeloda, XELOX, XL-147, XL-228, XL-281, XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, Zoledronat, Zosuquidar and combinations thereof.

In one embodiment, the other therapeutic agents include hormone analogs, anti-hormones, or those selected from tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutamylimide, cyproterone acetate, finasteride, busereline acetate, and fludrocortisone. In one embodiment, the other therapeutic agents comprise one or more LHRH agonists and/or antagonists selected from gosereline acetate, cloprostatin acetate, triptoreline pamoate, and combinations thereof, and wherein the LHRH antagonist is selected from gagarelix, cetroreline, abalex, ozarelix, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more growth factor inhibitors selected from inhibitors of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER), and hepatocyte growth factor (HGF). In one embodiment, the other therapeutic agent comprises one or more inhibitors of human epidermal growth factor, HER2, HER3, and HER4. In one embodiment, the other therapeutic agent comprises one or more tyrosine kinase inhibitors selected from cetuximab, gefitinib, imatinib, lapatinib, and trastuzumab, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more aromatase inhibitors selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more antimetabolites, which are antifolate agents selected from methotrexate, raltitrexed, and pyrimidine analogs. In one embodiment, the other therapeutic agent comprises one or more antimetabolites, which are pyrimidine analogs selected from 5-fluorouracil, capecitabine, and gemcitabine. In one embodiment, the other therapeutic agent comprises one or more antimetabolites, which are purine and/or adenosine analogs selected from mercaptopurine, thioguanine, cladribine, and pentostatin, cytarabine, fludarabine, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more antitumor drugs, doxorubicin, doxorubicin, epirubicin, and demethoxydaunorubicin, serine-C, bleomycin, actinomycin D, sclerosomycin, streptozocin, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more platinum derivatives selected from cisplatin, oxaliplatin, carboplatin, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more alkylating agents selected from estramustin, mecloreth amine, melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitsoureas, and combinations thereof. In one embodiment, other therapeutic agents include nitsoureas selected from carmustin, lomustin, thiotepa, and combinations thereof. In one embodiment, other therapeutic agents include antimitotic agents selected from vinca alkaloids and taxanes. In one embodiment, other therapeutic agents comprise one or more taxanes selected from paclitaxel, docetaxel, and combinations thereof. In one embodiment, other therapeutic agents comprise one or more vincristine alkaloids selected from vitamins, virincilin, virinex, vincristine, and combinations thereof. In one embodiment, other therapeutic agents comprise one or more topoisomerase inhibitors as epipodophyllotoxin. In one embodiment, other therapeutic agents comprise one or more epipodophyllotoxins selected from etoposide and vanbex, teniposide, amsacrin, topotecan, irinotecan, mitoxantron, and combinations thereof. One or more serine/threonine kinase inhibitors are selected from PDK1 inhibitors, β-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTOR/PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors, CDK inhibitors, Aurora kinase inhibitors, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more tyrosine kinase inhibitors, which are PTK2/FAK inhibitors. In one embodiment, the other therapeutic agent comprises one or more protein-protein interaction inhibitors selected from IAP, Mcl-1, MDM2/MDMX, and combinations thereof. In one embodiment, the other therapeutic agent comprises one or more rapamycin analogs selected from everolimus, sirolimus, ridaforolimus, sirolimus, and combinations thereof. In one embodiment, the other therapeutic agents include one or more selected from amivestin, anagrelide, clodronat, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, and combinations thereof. In one embodiment, other therapeutic agents include one or more selected from 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epomycin B, A 105972, A 204197, abiraterone, interleukin, retinoic acid, allolovectin-7, altretamine, alvocidib, amonafide, anthraquinone, AG-2037, AP-5280, azaquinone, aliquots, alicanidine, atraxifen, atametatan, atestan, aspirin PE AVLB, AZ10992, ABX-EGF, AMG-479 (ganitumab), ARRY 162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244 (cerumidine)-704/AZD-8330, AR-12, AR-42, AS-703988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), AZD-2014, azacytidine, azaprotectin B, azonafide BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, Bevacizumab, BEZ-235, Disodium docosanoate, BCX-1777, BKM-120, Bucosamide, 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, B I 6727, BI 836845, BI 847325, BI 853520, BUB-022, Bleomycin A, Bleomycin B, Brivanib, Bryostatin-1, Bortezomib, Brostallicin, Busulphan, BYL-719, CA-4 Prodrug, CA-4, CapCell, Calcitriol, Capecitabine, Canfosfamide, Capecitabine, Carboxyphthalimide, CCl-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1 Cefixime, Cefotaxime, Ceftriaxone, Celecoxib, Celmoleu Kin, cemadotin, CH4987655/RO-4987655, chlorotriphenylamine, silengiptide, cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, clofarabin, colchicine, cobustatin A4, COT inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 cryptophycin 52, CTP-37, CTLA-4 monoclonal antibody, CP-461, CV-247, cyanomorpholine doxorubicin, cytarabine, D 24851, decitabine, desorubicin, desogestrel B, dexamethasone, dexamethasone, hexamethylenetetramine Diethylstilbestrol, diflomotecan, didox, DMDC, dolalastatin 10, dorazoni, DS-7423, E7010, E-6201, edaraxamine, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, enzalool, efloxacin, epromycin B, epratuzumab, ER-86526, erlotinib, ET-18-0CH3, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulind, fenivel-Aminide, phenoxylate, fluorouracil, folic acid, FOLFO X, FOLFOX4, FOLFIRI, mesosulfanilamide, flutoxetine, camptothecin, maltol, gefinitib, gemutuzumab, gimatecan, glucosamide, GCS-100, GDC-0623, GDC-0941 (pictrelisib) 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) b) GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, Herceptin, hexamethylmelamine, histamine, homoserine, hyaluronic acid, hydroxyurea, hydroxyprogesterone caproate, ibuprofen ibandronate, itramycin, itratamivir, isostemma pentaphyllum, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (cixutumumab), immunoglobulin, hemolysin, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha-2b, interleukin-2, INK-1117, I NK-128, INSM-18, Ionafarnib, Isoproterenol, Isomalmin B, Isoflavones, Isotretinoin, Ixabepilone, JRX-2, JSF-154, J-107088, Conjugated Estrogen, Kahalid F, Ketoconazole, KW-2170, KW-2450, Lopaplatin, Leflunomide, Leuporelin, Lexidronam, LGD-1550, Linezolid, Lutexazol, Lomotrane, Loosxantrone, LU 223651, Lurtotecan, LY-S6AKT1, LY-2780301, Mafosfamide, Ma Rimastat, MEK inhibitors, MEK-162, methyltestosterone, methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronate, mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexat in gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimetazidine Serum, Nitroglycerin, Nortracene, Norreline, N-acetylcysteine, O6-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, PDO3 25901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillylalcohol, pertuzumab, PI3K inhibitor, PI3K/mTOR inhibitor, PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-52 12054, PT-100, PWT-33597, PX-866, Pyrapira, Neopentyloxymethylbutyrate, Heptaviridone, Phenoxydiol O, PKI166, Pertussis, Glucosinolate, Polylysine, Porphyromycin, Prednisone, Prednisolone, Quinine, Quinupordin, R115777, RAF-265, Ramosetron, Reboninase, RDEA-119/BAY 869766, RDEA-436, Rabecamycin Analog, Receptor Tyrosine Kinase (RTK) RG-7167, RG-7304, RG-7421, RG-7321, RG 7440, Rhizobacterium, rhu-MAb, rinfabate, Rifampicin, Rituximab Antibiotics, robatumumab, rofecoxib, RO-31-7453, RO-5126766, RO-5068760, RPR 109881A, pyridazolone, erythromycin, R-flurbiprofen, RX-0201, S-9788, doxorubicin, 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, Spirocycline, Saccharomethionine, Simvastatin Hydroxyxamic Acid, Sutent, T 900607, T 138067, TAK-733, TAS-103, Tacicaine, Tacaroamine, Tylosin, Texazone, Tegafur, Temozolomide, Texaxafen, Testosterone, Testosterone Propionate, Tesmyfen, Tetrachloroplatinic Acid, Tetrodotoxin, Tezazapine, Thalidomide, Trastuzumab, Enalapril, Topeminomin, Tiramozolamide, Tolvastatin, Tomitraz, Tomifenfen, Tramatinib, TransMID-107, Retinoic Acid, Trastuzumab, Tremelimumab, Retinoic Acid, Triacetyluridine, Triaziridine, TLK-286TXD 258, Tyk erb/tyverb, urocidin, valrubicin, vatalani, vincristine, vitamins, virulizin, WX-UK1, WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281, XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat, zosuquidar and combinations thereof.

In one embodiment, other therapeutic agents include steroids. Steroids include, but are not limited to, dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and corticosteroids. In one embodiment, other therapeutic agents include antiemetics. Dopamine agonists (e.g., domperidone, olanzapine, haloperidol, chlorpromazine, chlorpromazine, alisapride, atorvastatin, etc.), 5-HT3 receptor agonists (e.g., doxasetron, granisetron, ondansetron, tropisetron, proclopramide, and metoclopramide), NK1 receptor antagonists (e.g., aspirin and placebo), antihistamines (e.g., cimetidine, diphenylamine, xyleneamine, doxycycline, medelidazole, promethazine, hydroxyzine), cannabinoids (e.g., cannabis, tanshinone, and sativex), benzodiazepines (e.g., midazolam and lorazepam), anticholinergics (e.g., hyoscine), triphenyl ether carboxamide, ginger, emeffol, propofol, menthol, thymol, and ferulic acid.

In one embodiment, the other therapeutic agents include anticancer agents, including mitotic inhibitors. In one embodiment, the mitotic inhibitor includes taxane. In one embodiment, the mitotic inhibitor including taxane is selected from paclitaxel and docetaxel.

In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof; and at least one anticancer agent, including but not limited to one or more avermectin, ararubicin, atradazole, oxazoline, atefloxacin, retinoic acid, allopurinol, atrazine, onmexazol, ipratropium, amifostine, aminoglutamyl imide, acridine, anastrozole, citalopram, arsenic trioxide, asparaginase, aspirin, azacitidine, azetepa, azotomycin, batimastat, benzo[a]tellurium, bevacizumab, bicalutamide, bisanffene, bisnafide dimethylsulfonate, bizelesin, bleomycin, buquina, bropirimine, busulfan. Cactus cymoxanil, capecitabine, carsimycin, caroxetine, carboplatin, carmustine, carbimethin, carpiritonin, celecoxicon, celecoxicon, chlorambucil, siromoline, cisplatin, cladribine, sinoproxetine mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, donomycin, decitabine, dexamethasone, dezagurazine, dezagurine mesylate, quinaquine, docetaxel, doxorubicin, dextrin Loxifen, Drotahistamine, Duzomycin, Idatrixamine, Etesulin, Ixacin, Enloplatin, Ebuprofen, Etoposide, Etopostatin, Fadrolazole, Fazalabine, Fenivel Aamine, Fluorouracil, Fludarabine, Fluoxetine, Fluoxetine, Fostainine, Fluvitamin, Gemcitabine, Hydroxyurea, Demethoxydaunorubicin, Ifosfamide, Sarcosine, Interleukin II (IL-2, including recombinant interleukin II or rIL2), interferon α-2a, interferon α-2b, interferon α-1, interferon α-α3, interferon β-1a, interferon γ-1b, methadone, melantalin acetate, melphalan, megamol, mercaptopurine, methotrexate, metopine, meturedepa, mitindomide, mitocarcin, mitochocin, mitochocin, mitochocin, mitogenic agents, mitotane, mitoxantrone, mycophenolic acid, nelabine, nocodazole, nocaramycin, olanzapine, oxilagel, paclitaxel, polyethylene glycol, pomomycin, pentomustine, amide, pipobroman, piposulfan, piroxazone hydrochloride, safranin Promimipramine, Porphyrin, Porphyromycin, Prednimustine, Procarbazine, Puromycin, Pyrifuran, Glottisine, Rosichlor, Benzophenol, Ulinastatin, Pyprazole, Staphylococcus, Strychnine, Spiramycin, Spirilidine, Spirocycline, Zocin, Somonifen, Temozolomide, Temoxifen, Temopofen, Teniposide, Teniroride, Titolonolactone, Timoxane, Thioguanine Thionyl, Thio-Tapate, Thiazole Furan, Tilapasamine, Topotecan, Toremifene, Terliprizone, Triptorelin, Trastuzumab, Uracil, Vitapan, Verbitriptyline, Vitamins, Vincristine, Vincristine Sulfate, Vincristine, Vibixetine, Vodingaline, Vlincillin, Vinorelbine, Vonisolone, Vezoloxidine, Vorozolol, Zeniplatin, Zenostatin, Zoledronic Acid, Zorimethamine and combinations thereof.

Examples of suitable anticancer agents include, but are not limited to, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, edited by Laurence Brunton, Bruce Chabner, Bjom Knollman, McGraw Hill Professional, 2010.

In some exemplary embodiments, the pharmaceutical composition comprises a salt (e.g., a monosalt or disalt) of compound (1) or compound (10) or an analogue thereof, and at least one other therapeutic agent, wherein the other therapeutic agent includes an anti-angiogenic agent. For example, an anti-angiogenic agent is bevacizumab. In one embodiment, the anti-angiogenic agent is selected from apraxiber, axitinib, angiostatin, endostatin, a 16kDa prolactin fragment, laminin peptide, fibronectin peptide, tissue metalloproteinase inhibitors (TIMP 1, 2, 3, 4), plasminogen activator inhibitors (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, platelet-reactive protein (TSP), SPARC, 2-methoxyestradiol, proliferator-associated protein, suramin, sorafenib, tirofenib, thalidomide, cortisone, mitochondria, fumonisin (AGM-1470; TNP-470), tamoxifen, retinoic acid, CM101, dexamethasone, leukemia inhibitory factor (LIF), hedgehog inhibitors and combinations thereof.

The drug combination may include first and second therapeutic agents in any desired ratio, provided that synergy or a synergistic effect still occurs. The synergistic drug combination preferably contains first and second therapeutic agents in a ratio of about 1:9 to about 9:1. In one embodiment, the synergistic drug combination is provided in a ratio of about 1:7 to about 7:1, from about 1:6 to about 6:1, from about 1:5 to about 5:1, from about 1:4 to about 4:1, from about 1:3 to about 3:1, or from about 1:2 to about 2:1. In one embodiment, the synergistic drug combination comprises first and second therapeutic agents in a ratio of about 1:1.

In one embodiment, the second therapeutic agent is selected from allopurinol, arsenic trioxide, azazalidine, bortezomib, bevacizumab, capecitabine, carboplatin, celecoxib, chlorambucil, clafabulin, cytarabine, dacarbazine, docetaxel, doxorubicin HCl, fluorouracil, gemcitabine HCl, hydroxyurea, ifosfamide, imatinib mesylate, ixaprilone, lenatriuretic acid, megestrol acetate, methotrexate, mitotane, mitoxantrone hydrochloride, oxaliplatin, paclitaxel, pralidoxetine, rofexomil, sorafenib, streptozocin, tamoxifen citrate, topotecan hydrochloride, retinoic acid, vandetanib, vismodegib, vorinostat, and combinations thereof.

In one embodiment, the second therapeutic agent comprises a small molecule multi-kinase 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.

Table 2: Classification of Drugs

In some embodiments, the second therapeutic agent comprises a drug that targets the tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) receptor. In one embodiment, the second therapeutic agent comprises a recombinant TRAIL or an agonist antibody that activates one or more TRAIL receptors. In one embodiment, the second therapeutic agent comprises one or more antibodies or recombinant TRAILs whose signaling is activated by DR4, DR5, or both. In one embodiment, the second therapeutic agent comprises one or more of AMG-655, LBY-135, mapatumumab, lexatumumab, Apomab, and rhApo2L/TRAIL. In one embodiment, the second therapeutic agent comprises a selection from camptothecin, 5-FU, capecitabine, cisplatin, doxorubicin, irinotecan, paclitaxel, cisplatin, bortezomib, BH3I-2, rituximab, radiation, triterpenoids, sorafenib, gemcitabine e, HDAC inhibitors, carboplatin, T-101 (gossypol derivative), ABT-263, ABT-737 and GX-15-070 (obatoclax), vorinostat, cetuximab, panitumumab, bevacizumab, ganitumumab, interferon-gamma, sorafenib, XIAP antagonists, Bcl-2 antagonists and Smac mimics.

VI. Dosage

In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in doses ranging from about 40, 50, 60, or 100 mg to about 2000 mg; about 4, 6, 6, or 10 mg to about 200 mg; or about 0.4, 0.5, 0.6, or 1 mg to about 20 mg, wherein the weight may be based on the compound in its free base form. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in doses ranging from about 50 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg. 000 mg; from about 5 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 and 200 mg; or from about 0.5 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mg. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of about 40 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 20 mg. 00 mg; from about 4 mg to about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg; or from about 0.4 mg to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mg. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of about 60 mg to about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2 mg. 000mg; about 6mg; about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200mg; or about 0.6mg; about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20mg. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of 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; from about 10 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 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 compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of 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; 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 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 basis form. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in doses ranging from 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 free base form of the compound. In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or its analogues or pharmaceutically acceptable salts thereof, in dose ranges from about 50 mg to about 60, 70, 80, 90 or 100 mg; from about 60 mg to about 70, 80, 90 or 100 mg; from about 70 mg to about 80, 90 or 100 mg; from about 80 mg to about 90 or 100 mg; from about 90 mg to about 100 mg; from about 5 mg to about 6, 7, 8, 9 or 100 mg. 0 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; from 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.

In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of about 1 mg/kg to about 40 mg/kg; 0.1 mg/kg to about 4 mg/kg; or 0.01 mg/kg to about 0.40 mg/kg. In another embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, in a dose range of 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, 28 or 29 mg/kg to about 30 or 40 mg/kg; about 3 0, 31, 32, 33, 34, 35, 36, 37, 38 or 39 mg/kg to about 40 mg/kg; about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mg/kg to about 1, 2, 3 or 4 mg/kg; about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 or 1.9 mg/kg to about 2, 3 or 4 mg/kg; from about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 mg/kg to about 3 or 4 mg/kg; or from about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8 or 3.9 mg/kg to about 4 mg/kg; from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 mg/kg to about 0.10, 0.20, 0.30 or 0.40 mg/kg; from about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18 or 0.19 mg/kg to about 0.20, 0.30 or 0.40 mg/kg; about 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28 or 0.29 mg/kg to about 0.30 or 0.40 mg/kg; or about 0.30 mg/kg to about 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38 or 0.39 mg/kg to about 0.40 mg/kg.

In one embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or its analogues, or a pharmaceutically acceptable salt thereof, at doses ranging from about 37.5 mg/ ^m² to about 1500 mg/ ^m² ; about 3.75 mg/ ^m² to about 150 mg/ ^m² ; or from about 0.4 mg/m² to about 15 mg/ ^m² . In another embodiment, the pharmaceutical composition comprises compound (1) or compound (10) or its analogues, or a pharmaceutically acceptable salt thereof, at dose levels ranging from 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, 440, 405, 405, 315, 350, 350, 310, 315, 350, 350, 310, 315, 320, 325, 330, 315, 320, 325, 330, 335, 340, 345, 330, 335, 340, 345, 350, 335, 340, 345, 315, 350, 350, 310, 315, 350, 350, 310, 315, 340, 350, 330, 315, 340, 345, 330, 335, 340, 345, 350, 335, 340, 345, 350, 335, 340, 345, 350, 335, 340, 345, 390, 335, 340, 345, 390, 335, 340, 350, 340, 395, 340, 350, 405, 405, 405, 405, 405, 405, 405, 405, 405, 405, 405, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780 785, 790, 795, 8800, 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, 1345, 1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495mg/m ² to approximately 1500 mg/ ^m² ; from approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 or 149 mg/ ^m² to about 150 mg/ ^m² ; or about 0.5, 1, 1.5, ² , 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 111, 11.5, 12, 12.5, 13, 13.5, 14 or 14.5 mg/m² to about 15 mg/ ^m² .

VII. Dosage Form

Suitable pharmaceutical compositions that can be used with the methods described herein can be formulated into dosage forms that can be administered to patients. In one embodiment, the pharmaceutical composition is in the form of an oral dose unit or a parenteral dose unit. In one embodiment, the pharmaceutical composition is in the form of an oral dose unit. In some embodiments, the oral dose unit is divided into several smaller doses, which are administered to the subject over a predetermined time period to reduce the toxicity of the therapeutic agent being administered. In some embodiments, the oral dose unit is administered as a tablet or capsule comprising a controlled-release formulation that may contain multiple granules, pellets, pills, microtablets, or tablets. In one embodiment, the pharmaceutical composition is in the form of a parenteral dose unit. In one embodiment, the pharmaceutical composition is in the form of an intravenous (IV), subcutaneous (SC), intramuscular (M), rectal (PR), and transdermal delivery unit as an intestinal dose unit. In one embodiment, the pharmaceutical composition is in a dosage form selected from sterile solutions, suspensions, suppositories, tablets, and capsules. In one embodiment, the composition is an oral dosage form selected from tablets, pouches, capsules, lozenges, syrups, liquids, suspensions, and elixirs. In one embodiment, the composition is an oral dosage form selected from tablets, hard-shell capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solids, and semi-solids.

In some embodiments, the pharmaceutical composition used in the methods described herein comprises a dermatological composition suitable for topical administration. For example, dermatological ingredients include cosmetically or pharmaceutically acceptable media. Dermatological compositions for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. In some embodiments, conventional drug carriers, aqueous, powder, or oily bases, thickeners, skin enhancers, etc., may be necessary or desirable and can therefore be used. Examples of suitable reinforcing agents include, but are not limited to, ethers such as diethylene glycol monoethyl ether (commercially available) and diethylene glycol monomethyl ether; surfactants such as sodium lauryl sulfate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, poloxamer (231,182,184), Tween (20,40,60,80), and lecithin (US Patent No. 4,783,450); alcohols such as ethanol, propanol, octanol, benzyl alcohol, etc.; polyethylene glycol and its esters such as polyethylene glycol monolaurate; amides and other nitrogen-containing compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes; ketones; and organic acids, particularly citric acid and succinic acid. Sulfoxides, such as DMSO and C10MSO, may also be used, but are less preferred.

In some embodiments, the pharmaceutical composition is a dosage form selected from sustained-release, controlled-release, delayed-release, and responsive-release formulations.

VIII. Usage Instructions

The compositions and methods described herein are practically applicable in treating a wide range of disease conditions, including cancers such as colorectal, brain, and glioblastoma. In one embodiment, the compositions and methods described herein are used to treat conditions such as ocular melanoma, alopecia round cell tumors, chondrosarcoma, multi-irritant diseases, diffuse large cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, HIV-related cancers, HIV-related lymphoma, anorectal cancer, appendiceal cancer, astrocytoma, and atypical malformations/striated tumors. In one embodiment, the compositions and methods described herein are used to treat diseases such as basal cell carcinoma, basal cell nevus syndrome, Goring-nevus syndrome, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma, and malignant fibrous histiocytoma, brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, and spinal cord tumors. In one embodiment, the compositions and methods described herein are for treating conditions such as carcinoid tumors, tumors of unknown primary origin, central nervous system tumors, atypical malformations/striated tumors, meningitis, embryonal tumors of the central nervous system, central nervous system lymphomas, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, and cutaneous T-cell lymphomas (including but not limited to, Sezary syndrome, and fungal infections). In one embodiment, the compositions and methods described herein are for treating embryonal tumors of the central nervous system, endometrial cancer, endothelioma, ependymoma, esophageal cancer, Ewing sarcoma family tumors, extracranial germ cell tumors, exosomal cell tumors, extrahepatic bile duct cancer, and ocular cancer. In one embodiment, the compositions and methods described herein are for treating conditions 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 hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's 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, non-Hodgkin's lymphoma, and primary central nervous system lymphoma. In one embodiment, the compositions and methods described herein are for treating diseases such as macroglobulinemia (lymphoplasmacytic lymphoma), bone and malignant osteosarcoma, malignant fibrous histiocytoma, neuroblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, oral cancer, multiple myeloma/plasmacytoma, fungal infections, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, and myeloproliferative disorders. In one embodiment, the compositions and methods described herein are for treating cancer. In one embodiment, the compositions and methods described herein are for treating diseases such as nasal and paranasal sinus cancer, nasopharyngeal carcinoma, and neuroblastoma. In one embodiment, the compositions and methods described herein are for treating diseases such as oral cancer, oral and pharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian germ cell tumors, ovarian epithelial cancer, and low-grade ovarian tumors. In one embodiment, the compositions and methods described herein are for treating cancers such as pancreatic cancer, papilloma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, intermediately differentiated pineal medullary tumors, pineal cell tumors and supratentorial primitive neuroectodermal tumors, pituitary tumors, pleuropulmonary blisteroma, pregnancy and breast cancer, primary central nervous system lymphoma, and prostate cancer. In one embodiment, the compositions and methods described herein are for treating cancers selected from rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract cancer involving chromosome 15 of the NUT gene, retinoblastoma, and rhabdomyosarcoma. In one embodiment, the compositions and methods described herein are for treating advanced prostate cancer. In one embodiment, the compositions and methods described herein are for treating moderate prostate cancer. In one embodiment, the compositions and methods described herein are for treating low-grade prostate cancer. In one embodiment, the compositions and methods described herein are for treating castration-resistant prostate cancer.

The inventors have discovered that ONC201 (compound (1)) exhibits broad-spectrum anticancer activity, low toxicity (if any), minimal adverse reactions, low genotoxicity, and high bioavailability, including oral bioavailability, in in vitro and in vivo models, animal models, and human clinical trials. These characteristics make ONC 201 and its analogues particularly suitable for pediatric patients. These characteristics also make ONC 201 and its analogues particularly suitable for chronic treatment, high-risk patients, and for ensuring a durable response or stabilization of disease or prevention of disease relapse.

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 leukemias, and pediatric lymphomas).

In one embodiment, the compositions and methods described herein are used to treat proliferative skin diseases, such as psoriasis. In one embodiment, the compositions and methods described herein are used to treat cancers selected from salivary gland carcinoma, sarcoma, Sézary syndrome, skin cancer, eye cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, and occult primary and epithelial primitive neuroectodermal tumors. In one embodiment, the compositions and methods described herein are used to treat cancers selected from T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal vessels and ureter, and gestational trophoblastic tumor. In one embodiment, the compositions and methods described herein are used to treat cancers selected from cancers of unknown primary location, cancers of unknown primary origin, unusual cancers in children, transitional cell carcinoma of the renal vessels and ureter, urethral cancer, and uterine sarcoma. In one embodiment, the compositions and methods described herein are used to treat cancers selected from vaginal cancer and Vulvar cancer. In one embodiment, the compositions and methods described herein are used to treat cancers selected from Wilms' tumors and women's cancers.

In some embodiments, the compositions and methods described herein are used as first-line treatment (sometimes referred to as primary treatment). In some embodiments, the compositions and methods described herein are used as second-line therapy. In some embodiments, the compositions and methods described herein are used as third-line treatment. In some embodiments, the compositions and methods described herein are used as salvage therapy. The term "salvage therapy" refers to a therapeutic agent that can be administered by any regimen after a subject's initial treatment regimen has failed or after a subject's condition has not responded to the initial treatment. In some embodiments, the compositions and methods described herein are used as rescue therapy. In one embodiment of rescue therapy, the composition is used as a rescue agent to counteract the effects of the initial treatment. In one embodiment of rescue therapy, the composition is used as a rescue agent and is administered to a subject who has developed resistance to standard or initial treatment. In some embodiments, the compositions and methods described herein are used as neoadjuvant therapy. In one embodiment, neoadjuvant therapy includes administering one or more of the therapeutic agents described herein to a subject prior to primary or first-line treatment. In one embodiment, neoadjuvant therapy reduces the size or extent of the cancer being treated prior to administration of primary or first-line treatment to the treated subject. In some embodiments, the compositions and methods described herein are used as adjuvant therapy. In one embodiment, adjuvant therapy includes administering one or more therapeutic agents described herein to a subject, wherein one or more therapeutic agents modify or are administered simultaneously to the subject or subsequently to the subject.

In some embodiments, the compositions and methods described herein show a reduced chance of drug interactions. In some embodiments, compound (1) or compound (10) or its analogues are eliminated from the patient before interacting with other pharmaceutically active agents.

In some embodiments, the compositions and methods described herein exhibit enhanced toxicity levels when combined with other pharmaceutical formulations.

The methods and compositions described herein are not limited to specific animal species. In one embodiment, the subject treated according to the method and the composition described herein may be a mammal or a non-mammal. In one embodiment, the mammal subject includes, but is not limited to: humans; non-human primates; rodents such as mice, rats, or guinea pigs; domesticated pets such as cats or dogs; horses, cattle, pigs, sheep, goats, or rabbits. In one embodiment, the non-mammal subject includes, but is not limited to, poultry such as ducks, geese, chickens, or turkeys. In one embodiment, the subject is a human, regardless of sex or age. The compositions and methods can also be used for cancer prevention. The compositions and methods can also be used to stimulate the immune system.

The methods and compositions described herein are not limited to the specific age range described herein. In one embodiment, the subject treated according to the method and using the compositions described herein may be 50 years of age or older, 55 years of age or older, 60 years of age or older, or 65 years of age or older. In one embodiment, the subject treated according to the method and using the compositions described herein may be under 50 years of age, under 55 years of age, under 60 years of age, or under 65 years of age.

In one embodiment, the subject treated according to the method described herein and using the composition may be a pediatric patient. In one embodiment, the pediatric patient's CT age is less than 18 years, less than 17 years, less than 16 years, less than 15 years, less than 14 years, less than 13 years, less than 13 years under 12 years, less than 10 years, less than 10 years, less than 9 years, less than 8 years, less than 7 years, less than 6 years, less than 5 years, less than 4 years, less than 3 years, less than 2 years, and less than 1 year. In one embodiment, the pediatric patient's age is less than 12 months, less than 11 months, less than 10 years, less than 9 months, less than 8 months, less than 7 months, less than 6 months, less than 5 months, less than 4 months, less than 3 months, less than 2 months, and less than 1 month. In one embodiment, the pediatric patient is less than 4 weeks old, less than 3 weeks old, less than 2 weeks old, and less than 1 week old. In one implementation, the pediatric patient is 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, or less than 1 year old. In one implementation, the pediatric patient is a newborn. In one implementation, the pediatric patient is prematurely born. In one implementation, the pediatric patient is a newborn.

In one implementation scheme, the patient's weight is less than 45 kg, less than 40 kg, less than 35 kg, less than 30 kg, less than 25 kg, less than 20 kg, less than 15 kg, less than 14 kg, less than 10 kg, less than 5 kg, less than 4 kg, less than 3 kg, less than 2 kg, or less than 1 kg.

In one implementation, the subject has received at least one prior treatment. In another implementation, the subject has received at least two, three, or four prior treatments. In one implementation, the prior treatment is ibrutinib, bortezomib, carfilzomib, temozolomide, bevacizumab, cyclophosphamide, hydroxychloroquine, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, or lenalidomide.

In one implementation, the subject has been treated with radiation. In one implementation, the subject has been treated with surgery. In one implementation, the subject has received adoptive T-cell therapy.

In some implementations, cancer is no longer affected by ibuprofen, bortezomib, carfromycin, temozolomide, bevacizumab, cyclophosphamide, hydroxychloroquine, vincristine, prednisone, cytarabine, cisplatin, rituximab, 5-fluorouracil, oxaliplatin, leucovorin, lenalidomide, radiation, surgery, or a combination thereof.

In some embodiments, the compositions and methods described herein have dose-response relationships in cancer cells that differ from those in normal cells with the same composition and methods. Figure 1 illustrates, for example, the dose-response relationships of compound (1) to proliferation and cell death in normal and tumor cells. Figure 1 shows cell viability after treatment with a specified concentration of compound (1) for 72 hours. Tumors tested included human colon cancer cell line (HCT116), breast tumor cell line (MDA-MB-231), and human primary glioblastoma cell line (U87). Normal cells tested included human foreskin fibroblasts (HFF), human fetal lung fibroblasts (MRC-5), and human lung fibroblast cell line (WI-38). Doxorubicin was used as a positive control at 1 μg/mL in normal fibroblasts. As shown in Figure 1, cell viability of normal cells was at least about 75% at concentrations of compound (1) of about 1–5 mg/mL, while the viability of tumor cells was significantly reduced (e.g., equal to or less than 50%) at the same concentration of compound (1). Furthermore, as the concentration of compound (1) increased to more than about 5 mg/mL, the survival rate of tumor cells dropped to below 25%, while the viability of normal cells remained at about 75%.

Figure 2 illustrates the cell viability assay in human fetal lung fibroblast (MRC-5) cells after treatment with compound (1) (5 μM) or DMSO for 72 hours, indicating the recovery period in completely drug-free culture medium after treatment. The time point is the time after removal of compound (1) 72 hours after treatment. As shown in Figure 2, cell recovery was observed with compound (1) but not with DMSO.

In some embodiments, the compositions and methods described herein have practical value in treating cancer. In one embodiment, the compositions and methods described herein can be used to treat cancer in a human subject. In some embodiments, the treatment method includes, for a subject requiring such treatment, a pharmaceutically effective amount of compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

In one embodiment, the treatment method comprises administering to a subject requiring such treatment: (i) a salt comprising compound (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable first therapeutic agent thereof, and (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially. The second therapeutic agent may be any suitable therapeutic agent, including any pharmaceutically active agent disclosed herein. A pharmaceutically acceptable salt of compound (1) comprises the following dihydrochlorides:

It is understood that a dihydrochloride salt of chemical compound (1) or its analogues (including, but not limited to, compounds of formula (10)) or an alternative dihydrochloride that is obvious from the teachings of this disclosure may replace compound (1) or its analogues in the compositions or dosing regimens described herein.

In some embodiments, the treatment method includes the simultaneous or sequential administration of a synergistic drug combination comprising (i) a first therapeutic agent (1) or compound (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof, comprising a compound; and (ii) a second therapeutic agent. In one embodiment, the treatment method includes the simultaneous or sequential administration of a synergistically effective amount of a first therapeutic agent and a second therapeutic agent to a subject requiring such treatment. In one embodiment, the treatment method includes the administration of a combination of an effective amount of a first therapeutic agent and an effective amount of a second therapeutic agent, the combination providing a synergistic effect in vivo to treat cancer sensitive to the combination, wherein the first and second therapeutic agents are administered simultaneously or sequentially. In one embodiment, the treatment method includes administering to a subject receiving such treatment an effective amount of a combination of a first therapeutic agent and an effective amount of a second therapeutic agent, wherein the combination provides a synergistic effect in vivo to treat minimal residual disease sensitive to the combination, wherein the first and second therapeutic agents are administered simultaneously or sequentially.

In some implementations, the second therapeutic agent may be administered before or prior to the first therapeutic agent.

In one implementation, the treatment method targets cancer selected from solid tumors, liquid tumors, lymphomas, leukemia, or myeloma.

In one implementation, the treatment method targets solid tumors selected from: cervical cancer, endometrial cancer, extracranial germ cell tumors; external germ cell tumors; gestational trophoblastic tumors; ovarian cancer, ovarian germ cell tumors, ovarian epithelial cancer and malignant ovarian tumors; penile cancer, prostate cancer; gestational and breast cancer; advanced 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 carcinoma (liver cancer); kidney (renal cell) cancer. Symptoms; Liver cancer, renal cell carcinoma, renal pelvis and ureter; Basal cell carcinoma; Basal cell nevus syndrome, Gorlin-Nevus syndrome, melanoma, Merkel cell carcinoma, papilloma, multiple endocrine neoplasia syndrome; Pancreatic cancer, parathyroid carcinoma, ocular melanoma; Ocular cancer; Retinoblastoma; Malignant fibrous histiocytoma, Ewing sarcoma family tumors; Defibrotic round cell tumor; Chondrosarcoma, Kaposi's sarcoma, rhabdomyosarcoma; Spinal cord tumors, meningitis, embryonal tumors of the central nervous system, spinal cord tumors ... Corticoma, embryonic tumors of the central nervous system, endothelioma, ependymoma, neuroblastoma; intermediately differentiated pineal solid tumors, pineal cell tumors; adrenocortical carcinoma; bone cancer, osteosarcoma bone and malignant fibrous histiocytoma of osteosarcoma; osteosarcoma and malignant fibrous histiocytoma of bone; carcinoid tumors, unknown primary carcinomas, bronchial tumors, lung cancer, pleural alveolar tumors; respiratory tract cancers involving the NUT gene on chromosome 15, astrocytoma, atypical teratoma/rhabdomyosarcoma; atypical teratomas of the central nervous system. Rhabdomyosarcoma, craniopharyngioma, glioma, brain cancer, medulloblastoma, myeloid tumor, epithelial primitive neuroectodermal tumor; pituitary tumors, gastric cancer (stomach), gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), bladder cancer, anorectal cancer, adnexal cancer, esophageal cancer, hypopharyngeal cancer; laryngeal cancer, lip and oral cancer, metastatic squamous cell carcinoma and occult primary cancer, oral cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity and oral cavity cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer; head and neck cancer and mesothelioma.

Choose one implementation scheme where the treatment method for lymphoma is selected from: diffuse large cell lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, Sezary syndrome, mycosis fungoides (MF); histiocytosis; Burkitt lymphoma and central nervous system lymphoma; non-Hodgkin lymphoma, primary central nervous system lymphoma, Hodgkin lymphoma, macroglobulinemia; fungal primary central nervous system lymphoma; lymphoplasmacytic lymphoma and primary central nervous system lymphoma.

In one implementation, the treatment for a non-Hodgkin lymphoma (NHL) is selected from: mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, lymphocytic lymphoma NHL, Waldenstrom macroglobulinemia, and cutaneous lymphoma.

In one implementation, the treatment method for leukemia is selected from: 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 implementation, the treatment method for acute leukemia is selected from: acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and myeloproliferative disorders.

In one implementation, the treatment method targeting myeloma is selected from: IgA myeloma; IgG myeloma; IgM myeloma; IgD myeloma; IgE myeloma; light chain myeloma; non-secreting myeloma; multiple myeloma/plasmocytoma, multiple myeloma, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, and myeloproliferative disorders.

In one implementation, the treatment method targets cancer selected from: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal or rectal cancer, appendiceal cancer, astrocytoma, and atypical malformations/tumors.

In one implementation, the treatment method targets cancers selected from: basal cell carcinoma, basal cell nevus syndrome, Goring-nevus syndrome, cholangiocarcinoma, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain tumors, bronchial tumors, Burkitt lymphoma and spinal cord tumors.

In one implementation, the treatment method targets cancers selected from: carcinoid tumors, unknown primary cancers, atypical malformations/striated tumors of the central nervous system, embryonal tumors of the central nervous system, lymphomas of the central nervous system, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, and cutaneous T-cell lymphomas (including but not limited to, Sezary syndrome, and fungal infections).

In one implementation, the treatment method targets cancers selected from: embryonal tumors of the central nervous system, endometrial cancer, endothelioma, ependymoma, esophageal cancer, Ewing sarcoma family tumors, defibrotic round cell tumors, chondrosarcoma, extracranial germ cell tumors, germ cell tumors, extrahepatic bile duct cancer, and eye cancers, including intraocular melanoma and retinoblastoma.

Choose a cancer option: gallbladder cancer, stomach cancer (gastric cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic tumor, and glioma.

In one implementation, the treatment method targets cancers selected from: hairy cell leukemia, head and neck cancer, hepatocellular carcinoma (liver cancer), histiocytosis, Hodgkin's lymphoma, and hypopharyngeal cancer.

In one implementation, the treatment targets cancers selected from Kaposi's sarcoma and kidney (renal cell) cancer.

In one implementation, the treatment method targets cancers selected from: Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, including non-small cell lung cancer and small cell lung elimination, non-Hodgkin lymphoma, and primary central nervous system lymphoma.

In one implementation, the treatment method targets cancers selected from: macroglobulinemia (lymphoplasmacytic lymphoma), malignant fibrous histiocytoma of bone and osteosarcoma, neuroblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous cell carcinoma, multiple endocrine neoplasia syndrome, oral cancer, multiple myeloma/plasmacytoma, fungal diseases, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, and myeloproliferative disorders.

In one implementation, the treatment method can be used to treat cancer selected from: nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, and neuroblastoma.

In one implementation, the treatment method can be used to treat cancers selected from: oral cancer, oral and pharyngeal cancer, osteosarcoma and malignant fibroblastoma of bone, ovarian cancer, ovarian germ cell tumors, ovarian epithelial cancer and low-grade ovarian tumors.

In one implementation, the treatment method can be used to treat cancers selected from: pancreatic cancer, papilloma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, intermediately differentiated pineal parenchymal tumors, pine ligament tumors and epithelial primitive neuroectodermal tumors, pituitary tumors, pleural and pulmonary tumors, pregnancy and breast cancer, primary central nervous system lymphoma and prostate cancer.

In one implementation, the treatment method can be used to treat cancers selected from rectal cancer, renal cell carcinoma, kidney stones and urinary tract cancers involving the NUT gene on chromosome 15, retinoblastoma and rhabdomyosarcoma.

In one implementation, the treatment method can be used to treat cancers selected from: salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, occult primary squamous neck cancer, and supratentorial primitive neuroectodermal tumors.

In one implementation, the treatment method can be used to treat cancers selected from: T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal vessels and ureter, and gestational trophoblastoma.

In one implementation, the treatment methods are useful options for treating cancer: cancer of unknown primary site, cancer of unknown primary site, unusual cancer in children, transitional cell cancer of the renal vessels and ureter, urethral cancer, and uterine sarcoma.

In one implementation, the treatment method may be used to treat cancer selected from: vaginal cancer and vaginal cancer.

In one implementation, the treatment method is selected from: Wilms' tumors and women's cancers.

In some implementations, cancer treatment includes preventing tumor growth in the cancer subject. In some implementations, cancer treatment includes preventing the formation of cancer metastases. In some implementations, cancer treatment includes targeted therapy for minimal residual disease in cancer subjects known to have minimal residual disease, or for subjects at risk of minimal residual disease.

This may be indicated by surgical treatment of the primary tumor and/or after chemotherapy (radiotherapy) has begun or been proven effective. Spreading tumor cells may be dormant and generally cannot be attacked by chemotherapy (radiotherapy). A patient treated in this way appears to be in a healing state and is considered to have “minimal residual disease.” However, if dormant cells, after a longer period of dormancy, become metastatic due to growth stimulation, they have the potential to form metastatic tumors.

As used herein, “minimal residual disease” refers to a small number of cancer cells remaining in a subject during or after treatment when the subject is in remission (showing no symptoms or signs of disease). The methods described herein are preferably applicable to the forms of the diseases listed herein, including both adult and pediatric forms.

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

In one implementation, the treatment method can be used to treat autoimmune and inflammatory diseases of the peripheral nervous system, such as amyotrophic lateral sclerosis (ALS), which are based on various causes, including metabolic disorders such as diabetes, vitamin B12 and folic acid deficiency, chemotherapy drugs and medications used to treat HIV, toxins that cause peripheral nerve damage, cancers that develop peripheral neuropathy, paraneoplastic syndromes, alcohol abuse, chronic kidney disease, damage that causes compression of nerves and other lesions, such as Lyme disease, Guillain-Barré syndrome, connective tissue diseases, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, certain inflammatory diseases such as sarcoidosis, celiac disease, hereditary diseases such as Charcotmarie's dental syndrome, Friedreich's ataxia, and/or idiopathic diseases where no specific cause has been found but inflammatory and/or autoimmune mechanisms are the cause of the disease.

In one implementation, the treatment method can be used to treat autoimmune and inflammatory diseases with ocular manifestations. These ocular manifestations include, but are not limited to, ocular cicatricial pemphigoid, Moren's keratitis, various forms of uveitis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, relapsing polychondritis, Wegener's granulomatosis, scleroderma, Bechtel's disease, Reiter's disease, inflammatory bowel disease (ulcerative colitis and Crohn's disease) and ankylosing spondylitis, retinitis pigmentosa, macular degeneration, keratoconjunctivitis, scleritis, parascleritis, keratitis, peripheral corneal ulcers, and less common solids such as choroiditis, retinal vasculitis, scleral nodules, retinal detachment, and/or macular edema.

In one embodiment, the treatment method can be used to treat acute allogeneic transplant rejection in transplant patients. In one embodiment, the treatment method can be used to treat ischemic stroke. In one embodiment, the treatment method is useful for treating inflammatory diseases, including but not limited to arthritis, psoriasis, asthma, and colitis.

In one embodiment, the therapeutic agent comprises a single salt (1) of a pharmaceutically acceptable compound or an analogue thereof (e.g., a compound of formula (10)). In one embodiment, the therapeutic agent comprises a disalt of a pharmaceutically acceptable compound (1) or an analogue thereof (e.g., a compound of formula (10)). As described herein, some analogues may be trisalts. In one embodiment, the therapeutic agent comprises a single or disalt of compound (1) or an analogue thereof (e.g., a compound of formula (10)) in a pharmaceutically acceptable form, selected from hydrochloride, hydrobromide, hydrogen sulfate, sulfate, phosphate, fumarate, succinate, oxalate and lactate, hydrogen sulfate, hydroxyl, tartrate, nitrate, citrate, tartrate, carbonate, malate, maleate, fumarate sulfonate, methanesulfonate, formate, acetate and carboxyl. In one embodiment, the therapeutic agent comprises a chemically synthesized compound (1) or an analogue thereof in a pharmaceutically acceptable form selected from p-toluenesulfonate, benzenesulfonate, methanesulfonate, oxalate, succinate, tartrate, citrate, fumarate, and maleate. In one embodiment, the therapeutic agent comprises a compound (1) or an analogue thereof in a pharmaceutically acceptable mono- or di-salt having an anti-ion selected from ammonium, sodium, potassium, calcium, magnesium, zinc, lithium, and/or with anti-ions such as methylamino, dimethylamino, diethylamino, triethylamino, and combinations thereof. In one embodiment, the therapeutic agent comprises a di-salt of a compound in the halide form described herein, such as a dihydrochloride or dihydrobromide.

In some embodiments of the treatment method, the second therapeutic agent includes an anticancer agent. In some embodiments of the treatment method, the second therapeutic agent is selected from, but is not limited to, ivermectin, azolibicine, atrazodazole, oxazolidine, aspirin, alpha-interleukin, ivermectin, allopurinol, atrazine, cyclomycin, isoproterenol, amifostine, aminoglutamyl imide, anastrozole, anesthetine, arsenic trioxide, asparaginase, astragalin, azacitidine, azetepa, azotomycin, barmastolomew, benzogluconate, bevacizumab, bicalutamide, bisantrene, bisnafidedimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactusmycin, capecitabine, capecitabine, carboplatin, carmustine, carbimazole, carnestacin, carfiltrates. Celecoxib, chlorambucil, siromoline, cisplatin, cladribine, sinoproxetine mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, donomycin, decitabine, dexamethasone, desoxyguanine, dezagunine mesylate, diazoquine, docetaxel, doxorubicin, droloxifene, dibromomorphone, duzomycin, edaraxamin, etesulin, ifloxacin, cisplatin, etoposide, etopostatin Etiazole, Etoposide, Etoposide, Fadazole, Fazalofibril, Fenivel Aamine, Fluorouracil, Fludarabine, Fluoxetine, Fluoxetine, Fosterlin, Fulvestrant, Gemcitabine, Hydroxyurea, Demethoxydaunorubicin, Ifosfamide, Sarcosine, Interleukin II (IL-2, including recombinant interleukin II or rIL2), Interferon α-2a, Interferon α-2b, Interferon α- α1, Interferon α-α3, Interferon β-1a, Midazolam, Midazolam, Meropenem, Megamol, Megamol, Mercaptopurine, Methotrexate, Metopine, Meturedepa, Mimodimethicone, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mitopine, Mycophenolic acid, Nerapine, Nocodazole, Nocarbazine, Olanzapine, Oxicamurean, Paclitaxel, Pegaspargase, Promycin, Promoxetine, Pipobroman, Piposulfan, Piroxazone Hydrochloride, Glucosamine, Promimetane, Porphyrin, Porphyrin, Porphyrin, Prednimustine, Procarbazine, Puromycin, Pyrifurine, Rhodotorula Letimide, safingol, semustine, simtrazene, sparsosycin, sparsomycin, spirogermanium, spiromustine, serofenur, tebimycin, tamoxifen, tegalan, tegaflu, tetopafen, temopofen, teniposide, teroxirone, testolactone, thiamiprine, thiotepa, thiazofuran, telapasamine, topotecan, toremifene, trestolone, vitamin B1, vitamin B2, velinolide, vitamin B2, vezocillin, vezolorol, zineb, zolbutine, zoledronic acid, zorimycin and combinations thereof.

In some embodiments of the treatment, the second therapeutic agent is selected from, but not limited to, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, growth factor inhibitors, growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors; anti-metabolites; antitumor antibiotics; platinum derivatives; alkylating agents; antimitotic agents; tubuline 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, rapacustrine analogs, amiodarone, anagrelate, clodronate, filgrastim, interferon, interferon-alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate and porphyrinol, 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epomycin B, A 105972, A 204197, abiraterone, interleukin Acitretin, allocitric acid, amoxidipine, amoxidipine, anthraquinone, amoxidipine, amoxidipine, amoxidipine, apoxidipine, apoxidipine, AG-2037, AP-5280, aposide, apoinsulin, vidarabine, agliptin, azoxifene, atacerceptin, ateligiblecan, aspirin PE, AVLB, AZ10992, ABX-EGF, AMG-479 (ganitomab), 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, ARQ68 0, AS-703026 (primasertib), avastin, AZD-2014, azacytidine, azabetazolin B, azonafide, BAY-43-9006, BAY 80-6946, BBR-346 4. BBR-3576, Bevacizumab, BEZ-235, Diploid Gibberellin, BCX-1777, BKM-120, Bucocillin, 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, B I 2536, BI 6727, BI 836845, BI 847325, BI 853520, BUB-022, Bleomycin A, Bleomycin B, Brivanil, Lichenin-1, Bortezomib, Myristyl Peptide, Cholesthiane, BYL-719, CA-4 Prodrug, CA-4, CapCell, Calcitriol, Canertinib, Canfosfamide, Capecitabine, Carboxybiphenyl Platinum, CCl-779, CC-115, CC-223, CEP-701, CEP-751, C BT-1 Cefixime, Cefotaxime, Ceftriaxone, Celecoxib, Celmoleukin, Cemadotin, CH4987655/RO-4987655, Trichlorophenylamine, Silencitracin, Cyclosporine, CDA-II, CKD-602, CKI-27, Clofarabin, Colchicine, Cobustatin A4, COT Inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 Cryptocycin 52, CTP-37, CTLA-4 Monoclonal Antibody CP-461, CV-247, Cyanomorpholine Doxorubicin, Cytarabine, D24851, Decitabine, Deoxyrubicin, Deoxyvitamin, Deoxybectomycin, Deoxycorticosterone B, Dexamethasone, Decicloxacin, Diethylstilbestrol, Diflomotecan, DMD, Dolasatin 10, Dolasatin, DS-7423, E7010, E-6201, Edatrexat, Edotreotide, Efaproxiral, Eflornithine, EGFR inhibitors, EKB-569, EKB-509 Enzastaurin, Enzapromide, Efloxacin, Epomycin B, 86526, Erlotinib, ET-18-0CH3, Ethynylcytidine, Ethynylestradiol, Exatecan Mesylate, Exemestane, Exisulind, Fenivel A, Etomidumab, Fluorouracil, Folic Acid, FOLFOX, FOLFOX4, FOLFIRI, Mesylate, Flutoxine, Camptothecin, Maltol Gallium, Gefinitib, GLC-100, GDC-0623, GDC-0941 (pictrelisib), G DC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 (dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795 GW2016, Granisetron, Herceptin, Hexamethylmelamine, Histamine, Homoserine, Hyaluronic Acid, Hydroxyurea, Hydroxyprogesterone Caprate, Ibandronic Acid, Itramycin, Itratamivir, IDN-5109, IGF-1R Inhibitor, IMC-1C11, IMC-A12 (cixutumumab), Immunotherapy, Hemolysin, Interferon Alpha-2a, Interferon Alpha-2b, Pegylated Interferon Alpha-2b, Interleukin-2, INK-1117, INK-128, INSM-18, Ionafamib, Ipilimumab, Isobutanol Ketones, Isohinson's B, Isoflavones, Isotretinoin, Ixabepilone, JRX-2, JSF-154, J-107088, Conjugated Estrogens, Kahalid F, Ketoconazole, KW-2170, KW-2450, Roprolin, Leflunomide, Rhinostraline, Leuprorelin, Rifampin, Lysaderano, LGD-1550, Linezolid, Safeline, Lomotral, Loosxantrone, LU 223651, Lurtotecan, LY-S6AKT1, LY-2780301, Mafosf amide, malamastamine, mechloroethamine, MEK inhibitor, MEK-162, methyltestosterone, methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minophosphoric acid, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexafin gadolinium, MS-209, MS -275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitroglycerin, norepinephrine, N-acetylcysteine, 6-benzylguanine, omeprazole, oncophage, oncoVEXGM-CSF, orplatin, ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, oxantrazole, estrogen, panitumuma b, patupilone, pegfilgrastim, PCK-3145, pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, albumin-stabilized paclitaxel, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, peri Fosine, perillylalcohol, pertuzumab, PI3K inhibitor, PI3K/mTOR inhibitor, PG-TXL, PG2, PLX-4032/-5185426 (vemurafenib), PLX-3603/RO-5212054, PT-100, PWT-33597, PX-866, pyrplatin, neopentyloxymethylbutyrate, pyranone, phenoxydiol O, PKI166, xplevitrexed, scintillan, polymalonic acid, ranisone, prednisolone.

In some embodiments of the treatment method, the second therapeutic agent is selected from tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, bicalutamide, aminoglutamylimide, cyproterone acetate, finasteride, busherin acetate, flucolone, fluorometholone, octreotide, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent is selected from, but not limited to, the group consisting of LHRH agonists and LHRH antagonists. In some embodiments, the LHRH agonist is selected from gorelin acetate, flurbiproline acetate, triptorelinpamoate, and combinations thereof. In some embodiments, the second therapeutic agent includes an LHRH antagonist selected from combinations of Degarelix, Cetrorelix, Abarelix, Ozarelix, and Degarelix. In some embodiments of the treatment method, the second therapeutic agent includes an inhibitor of growth factors. In some implementations, growth factor inhibitors are selected, but not limited to, 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, with HER2, HER3, and HER4 selected.

In some embodiments of the treatment method, the second therapeutic agent comprises a tyrosine kinase inhibitor. In some embodiments of the treatment method, the tyrosine kinase inhibitor is selected from, but not limited to, cetuximab, gefitinib, imatinib, lapatinib, trastuzumab, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent comprises an aromatase inhibitor. In some embodiments of the treatment method, the aromatase inhibitor is selected from anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane, and combinations thereof.

In some embodiments of the treatment method, the second therapeutic agent includes an antimetabolite. In some embodiments of the treatment method, the antimetabolite includes an antifolate agent. In some embodiments of the treatment method, the antiseptic is selected from methotrexate, raltitrexed, pyrimidine analogs, and combinations thereof. In some embodiments of the treatment method, the antimetabolite is a pyrimidine analog. In some embodiments of the treatment method, the pyrimidine analog is selected from 5-fluorouracil, capecitabine, gemcitabine, and combinations thereof. In some embodiments of the treatment method, the antimetabolite is a purine analog or an adenosine analog. In some embodiments of the treatment method, the purine analog or adenosine analog is selected from mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine, fludarabine, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent includes an antitumor antibiotic. In some embodiments of the treatment method, the antitumor drug includes doxorubicin, daunomycin, doxorubicin and demethoxydaunorubicin, serine-C, bleomycin, actinomycin D, sclerosomycin, streptozocin, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent comprises a platinum derivative. In some embodiments of the treatment method, the platinum derivative is selected from cisplatin, oxaliplatin, carboplatin, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent comprises an alkylating agent. In some embodiments of the treatment method, the alkylating agent is selected from estramustin, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosourea, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent comprises nitrosourea. In some embodiments of the treatment method, the second therapeutic agent comprises an antimitotic agent. In some embodiments of the treatment method, the second therapeutic agent comprises an antimitotic agent. In some embodiments of the treatment method, the antimitotic agent is selected from vinblastine alkaloids and taxanes. In some embodiments of the treatment method, paclitaxel is selected from paclitaxel, docetaxel, and combinations thereof. In some embodiments of the treatment method, vinblastine alkaloids are selected from vitamins, virinol, virinolone, vincristine, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent comprises a topoisomerase inhibitor. In some embodiments of the treatment method, the topoisomerase inhibitor is epipodophyllotoxin. In some embodiments of the treatment method, the topoisomerase inhibitor, which is epipodophyllotoxin, is selected from etoposide, vanbex, teniposide, amsacrin, topotecan, irinotecan, mitoxantron, and combinations thereof. In some embodiments, the second therapeutic agent includes a serine/threonine kinase inhibitor. In some embodiments of the treatment method, the serine/threonine kinase inhibitor is selected from PDK1 inhibitors, β-Raf inhibitors, mTOR inhibitors, mTORC1 inhibitors, PI3K inhibitors, dual mTOR/PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors, CDK inhibitors, Aurora kinase inhibitors, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent includes a tyrosine kinase inhibitor. In some embodiments of the treatment method, the second therapeutic agent includes a PTK2/FAK inhibitor. In some embodiments of the treatment method, the second therapeutic agent includes a protein-protein interaction inhibitor. In some embodiments of the treatment method, the protein-protein interaction inhibitor is selected from IAP, Mcl-1, MDM2/MDMX, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent includes a rapamycin analogue. In some embodiments, the rapamycin analogue is selected from everolimus, sirolimus, ridaforolimus, sirolimus, and combinations thereof. In some embodiments of the treatment method, the second therapeutic agent is selected from aripipridine, anagrelide, clodronate, filgrastim, interferon, interferon-alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer, and combinations thereof. In some implementations of the treatment method, the second therapeutic agent is selected from 2-chlorodeoxyadenosine, 2-fluorodeoxycytidine, 2-methoxyestradiol, 2C4, 3-alethine, 131-1-TM-601, 3CPA, 7-ethyl-10-hydroxycamptothecin, 16-aza-epomycin B, A 105972, A204197, abiraterone, interleukin, ivermectin, allovocin-7, atrazine, alvocidib, amonafide, anthraquinone, AG-2037, AP-5280, apaziquone, apomine, ivermectin, atratriptan, atracetan, atrastan, aspirin, aspirin, AVL B, AZ10992, ABX-EGF, AMG-479(ganitumab), ARRY162, ARRY 438162, ARRY-300, ARRY-142886/AZD-6244(selumetinib)/AZD-8330, AR-12, AR-42, AS-7 03988, AXL-1717, AZD-8055, AZD-5363, AZD-6244, ARQ-736, ARQ 680, AS-703026 (primasertib), avastin, -2014, azacytidine, azabetalidine B, azonafide BAY-43-9006, BAY 80-6946, BBR-3464, BBR-3576, Bevacizumab, BEZ-235, Disodium dislactate, BCX-1777, BKM-120, BLP-25, BMS-184476, BMS-247550, BMS-188797, BMS-275291, BMS-663513, BMS-754807, BNP-1350, BNP-7787, BIBW2992 (afatinib, tomtoVok), BIBF1120 (vargatef), BI836845, BI 2536, BI 6727, BI 836 845, BI 847325, BI 853520, BUB-022, Bleomycin A, Bleomycin B, Brivanib, Brystone-1, Bortezomib, Brostallicin, Busulphan, BYL-719, CA-4 Prodrug, CA-4, CapCell, Calcitriol, Canertinib, Canfosfamide, Capecitabine, Carboxybiphenylplatin, CC1-779, CC-115, CC-223, CEP-701, CEP-751, CBT-1 Cefixime, Cefotaxime, Ceftriaxone, Celecoxib, Celmoleukin, c Emadotin, CH4987655/RO-4987655, Tribenzozepam, Cyclosporine, CDA-II, CDC-394, CKD-602, CKI-27, Clofarabin, Colchicine, Cobustatin A4, COT Inhibitor, CHS-828, CH-5132799, CLL-Thera, CMT-3 Cryptocycin 52, CTP-37, CTLA-4 Monoclonal Antibody, CP-461, CV-247, Cyanomorpholine Doxorubicin, Cytarabine, D24851, Decitabine, Deoxyrubicin, Deoxyvitamin, Deoxythymidine, Deoxycephalosporin B, Dexamethasone, Dexamethasone Diethylstilbestrol, diflomotecan, didox, DMDC, dolastatin 10, dorastinazole, DS-7423, E7010, E-6201, edatrexat, edotreotide, efaproxiral, eflornithine, EGFR inhibitors, EKB-569, EKB-509, enzastaurin, itramycin B, epremycin B, epremycin B, epremycin, ER-86526, erlotinib, ET-18-0CH3, ethinylcytidine, ethinylestradiol, exatecan, exatecan mesylate, exemestane, exisulin d, Fenivel Aamine, Fenoxaparin, Fenoxaparinone, Folic Acid, FOLFOX, FOLFOX4, FOLFIRI, Gefitinib, Gefitinumab, Gefitinib, Gefitinib, Gefitinib, Gefitinib, GFC-100, GDC-0623, GDC-0941 (pictrelisib), GDC-0980, GDC-0032, GDC-0068, GDC-0349, GDC-0879, G17DT Immunogen, GMK, GPX-100, gp100 peptide vaccine, GSK-5126766, GSK-690693, GSK-1120212 (trametinib), GSK-2118436 ( dabrafenib), GSK-2126458, GSK-2132231A, GSK-2334470, GSK-2110183, GSK-2141795, GW2016, granisetron, Herceptin, hexamethylmelamine, histamine, homoserine protein, hyaluronic acid, hydroxyurea, hydroxyprogesterone acetate, ibandronic acid, ibutatatabine, itrastatin, edetrazol, IDN-5109, IGF-1R inhibitor, IMC-1C11, IMC-A12 (cixutumumab), immunoglobulin, hemolysin, interferon alpha-2a, interferon alpha-2b, pegylated interferon alpha-2b, interleukin-2, INK-1117. INK-128, INSM-18, Ionafarnib, Isoproterenol, Isomalpine B, Isoflavones, Isotretinoin, Ixabepilone, JRX-2, JSF-154, J-107088, Conjugated Estrogen, Kahalid F, Ketoconazole, KW-2170, KW-2450, Lopaplatin, Leflunomide, Leuporelin, Lexidronam, LGD-1550, Linezolid, Lutex, Lomotriol, Loosxantrone, LU 223651, Lurtotecan, LY-S6AKT1, LY-2780301, Mafosfamide, Mar imastat, mechloroethylamine, MEK inhibitor, MEK-162, methyltestosterone, methylprednisolone, MEDI-573, MEN-10755, MDX-H210, MDX-447, MDX-1379, MGV, midostaurin, minodronate, mitomycin, mivobulin, MK-2206, MK-0646 (dalotuzumab), MLN518, motexaf in gadolinium, MS-209, MS-275, MX6, neridronate, neratinib, Nexavar, neovastat, nilotinib, nimesulide, nitrate Glycerin, Nortraxe, Norreline, N-acetylcysteine, O6-benzylguanine, Oblimersen, Omeprazole, Oncophage, OncoVEXGM-CSF, Orplatin, Ortataxel, OX44 antibody, OSI-027, OSI-906 (linsitinib), 4-1BB antibody, Oxantrazole, Estrogen, Panitumumab, Pegfilgrastim, PCK-3145, Pegfilgrastim, PBI-1402, PBI-05204, PDO325901, PD-1 antibody, PEG-paclitaxel, Albumin-stabilized violet... Taxol, PEP-005, PF-05197281, PF-05212384, PF-04691502, PHT-427, P-04, PKC412, P54, PI-88, pelitinib, pemetrexed, pentrix, perifosine, perillylalol, pertuzumab, PI3K inhibitors, PI3K/mTOR inhibitors, PG-TXL, PG2, PLX-4032/RO-5185426 (vemurafenib), PLX-3603/RO-5212054, PT-100, PWT-33597, PX-866, pyridine, neopentyl Oxymethylbutyrate, Vibivolinone, Phenoxydiol O, PKI166, Plevitrexed, Glomerulone, Polyepinephrine, Prednisolone, Quinine, Quinupristin, R115777, RAF-265, Ramosetron, Rapensetron, Rapastetarine, RDEA-119/BAY 869766, RDEA-436, Rabemycin analogue, Receptor tyrosine kinase (RTK) inhibitor, Revimid, RG-7167, RG-7304, RG-7421, RG-7321, Rojietinib, Ritudronate, Rituximab, Rofecoxib, RO-31-7453, RO-5126766, RO-5068760 RPR109881A, Naringin, Erythromycin, R-Flurbiprofen, RX-0201, S-9788, SAHA, Sargramostim, Satraplatin, SB408075, 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, Spirocycline, Veratrum Nectaride, Simvastatin Hydroxime Acid, Cisplatin, T 900607, T 138067, TAK-733, TAS- 103, tacedinaline, talaporfin, Tarceva, tariquitar, tasisulam, Taxoprexin, testosterone, temofifine, testosterone, testosterone propionate, tesmetifine, testosterone, tetracycline, tezazolium, thalidomide, citric acid, enalapril, thymine, thymosin, thiazosin, tivavani, tilabadamine, tolvaprine, TransMID-107, retinoid, trastuzumab, tremelimumab, retinoic acid, triaceturidine, triapine, triciribine, trimetrexate, TLK-286TXD 258, tyker b/tyverb, urocidin, valrubicin, vtalanib, vincristine, vitamins, virulizin, WX-UK1, WX-554, vectibix, xeloda, XELOX, XL-147, XL-228, XL-281, XL-518/R-7420/GDC-0973, XL-765, YM-511, YM-598, ZD-4190, ZD-6474, ZD-4054, ZD-0473, ZD-6126, ZD-9331, ZD1839, ZSTK-474, zoledronat, zosuquidar and combinations thereof.

In some embodiments, other therapeutic agents include steroids. Steroids include, but are not limited to, dexamethasone, prednisolone, methylprednisolone, prednisone, hydrocortisone, triamcinolone, betamethasone, and corticosteroids. In some embodiments, other therapeutic agents include antiemetics. Dopamine agonists (such as domperidone, olanzapine, haloperidol, chlorpromazine, prochlorperazine, alisapride, atorvastatin, docetaxel, prochlorpromazine, and metoclopramide), NK1 receptor antagonists (such as aspirin and placebo), antihistamines (such as cimetidine, diphenylamine, xyleneamine, doxycycline, meadil, promethazine, and hydroxyzine), cannabinoids (such as cannabis, droscarpine, and sativex), benzodiazepines (such as midazolam and lorazepam), anticholinergics (such as hyoscine), trimethobenzamide, ginger, emetrol, propofol, menthol, thymol, and ferulic acid.

The pharmaceutical composition can be administered to the subject via any suitable route of administration. In one embodiment, the pharmaceutical composition is administered orally, parenterally, transdermally, or transmucosally. In one embodiment, the pharmaceutical composition is administered parenterally. In one embodiment, the pharmaceutical composition is administered to the subject via intravenous (IV), subcutaneous (SC), and intramuscular (IM) parenterial routes of administration. In one embodiment, the pharmaceutical composition is administered to the subject via a route of administration selected from rectal and transdermal. In one embodiment, the pharmaceutical composition is given to the subject in a dosage form selected from sterile solutions, suspensions, suppositories, tablets, and capsules. In one embodiment, the pharmaceutical composition is administered to the subject in an oral dosage form selected from tablets, pouches, capsules, lozenges, syrups, liquids, suspensions, and elixirs. In one embodiment, the pharmaceutical composition is given to the subject in an oral dosage form selected from tablets, hard-shell capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solids, and semi-solids.

In some implementations, the pharmaceutical composition is administered to the subject as a dosage form, choosing a sustained-release form, a controlled-release form, a delayed-release form, or a responsive-release form.

In some embodiments, the pharmaceutical composition is administered to the subject once daily. In some embodiments, the pharmaceutical composition is administered according to an infrequent dosing regimen (e.g., once weekly or less frequently). In some embodiments, the pharmaceutical composition is administered to the subject according to a frequent dosing regimen (e.g., more than once weekly). In some embodiments, the pharmaceutical composition is administered to the subject once weekly. In some embodiments, the pharmaceutical composition is administered once every four weeks. In some embodiments, the pharmaceutical composition is administered twice weekly. In some embodiments, the pharmaceutical composition is administered once every two weeks. In some embodiments, the pharmaceutical composition is administered once every three weeks. In some embodiments, the pharmaceutical composition is administered to the subject in repeated cycles of once weekly, once every two weeks, once every three weeks, once every four weeks, or a combination thereof.

In one embodiment, the treatment method comprises administering to a subject in need of such treatment: (i) a first therapeutic agent comprising compound (1), compound (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, and (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially; and further comprises testing the expression of endoplasmic reticulum (ER) stress response genes in a biological sample. In some embodiments, the ER stress response genes are selected from the group comprising, but not limited to, C/EBP homolog (CHOP), activated transcription factor 3 (ATF3), and CHOP and ATF3. ATF3, activated transcription factor 4 (ATF4), CHOP, IRE1, binding immunoglobulin (BiP), eukaryotic translation initiation factor 2A (eIF2a), ER stress response genes, X-box binding protein 1 (XBP1). The biological sample may be a tumor, peripheral blood mononuclear cells, or skin biopsy. The biological sample may be obtained before, during, or after administration. In some embodiments, the treatment method also includes adjusting the dose of the first therapeutic agent to achieve approximately 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, 500%, 525%, 550%, 575%, 600%, or more of the ER stress gene. In some embodiments, the treatment method further includes adjusting the dose of the first therapeutic agent to induce about 50% to about 100%, about 100% to about 150%, about 150% to about 200%, about 200%, about 250% to about 300%, about 300% to about 350%, about 350% to about 400%, about 400% to about 450%, about 450% to about 500%, about 500% to about 550%, about 550% to about 600%, or greater than 600% of the ER-stressing gene. In some embodiments, the treatment method further includes adjusting the dose of the first therapeutic agent to induce about 50% to about 100%, about 100% to about 200%, about 200% to about 300%, about 300%, about 400%, about 400% to about 500%, about 500% to about 600%, or greater than 600% of the ER-stressing gene.

In one embodiment, the treatment method comprises administering to a subject requiring such treatment: (i) a pharmaceutically acceptable salt of a first therapeutic agent comprising compound (1) or compound (10), or an analogue thereof, or a compound therein, and (ii) a second therapeutic agent, wherein the first and second therapeutic agents are administered simultaneously or sequentially; and further comprises testing for the expression of proteasome activity in a biological sample. In some embodiments, the proteasome activity may be trypsin-like, trypsin-like, and/or cysteine-like activity. In some embodiments, the biological sample may be a tumor, peripheral blood mononuclear cells, or skin cells. The biological sample may be obtained before, during, or after drug administration. In some embodiments, the treatment method further comprises adjusting the dose to achieve about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the proteasome activity. In some embodiments, the treatment method further includes adjusting the dose to achieve at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% proteasome activity. In some embodiments, the treatment method further includes adjusting the dose to achieve about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or greater than 90% proteasome activity.

On the one hand, this article provides a treatment method comprising administering to a subject requiring such treatment a first therapeutic agent comprising a compound of formula (1) or formula (10) or an analogue thereof, or a pharmaceutically acceptable salt thereof (e.g., a di- or tri-salt) and a second therapeutic agent, the method comprising:

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

(ii) wait until the scheduled waiting time has elapsed since the first treatment administration time has arrived at the subject; and/or until the adverse event is resolved or resolved;

(iii) Administering a second therapeutic agent to the subject, wherein a predetermined waiting time is selected to obtain a delayed therapeutic effect of the first therapeutic agent without increasing the risk of potential combined toxicity of the first and second therapeutic agents. In some embodiments of the treatment method, the predetermined waiting time is determined based on the clearance rate of the first therapeutic agent or its metabolite compound. In some embodiments of the treatment method, the predetermined waiting time is determined by a quantitative assessment of renal function and renal parameters. In some embodiments of the treatment method, the predetermined waiting time is determined by measuring renal function, wherein the test is selected from blood levels of the first therapeutic agent or its metabolite compound; clearance rate of the first therapeutic agent or its metabolite compound; and 24-hour urinary clearance of the first therapeutic agent or its metabolite compound.

In one embodiment of the treatment method, the predetermined waiting time is substantially equal to the time required for complete clearance of the first therapeutic agent or its metabolite compound from the subject's body. In one embodiment of the treatment method, the predetermined waiting time is substantially equal to the time required for clearance of the first therapeutic agent or its metabolite chemical from the subject. In one embodiment of the treatment method, the predetermined waiting time is substantially equal to the time required for clearance of the first therapeutic agent or its metabolite compound from the subject's liver. In one embodiment of the treatment method, the predetermined waiting time is substantially equal to the time required for complete clearance of the first therapeutic agent or its metabolite compound from the subject. In one embodiment of the treatment method, the predetermined waiting time is about 4 hours. In other embodiments, the waiting time is 1 day. In some embodiments, the waiting time continues until the _Cmax of the first therapeutic agent compound has passed. In other embodiments, the waiting time is after most adverse events have been resolved or resolved. In one embodiment of the treatment method, the predetermined 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 of the treatment method, the predetermined waiting time is about 1-7 days, about 1-6 days, about 1-5 days, about 1-4 days, about 1-3 days, or about 1 to 2 days. In one implementation, the waiting time is up to three weeks. This was previously considered the "treatment period".

When the order of administration of the first therapeutic agent is reversed, it can be administered after the Cmax of the second therapeutic agent (i.e., the drug administered the first time) has passed. In one embodiment, administration of the first therapeutic agent may be based on the fact that most or substantially all of the drug administered the first time has been eliminated from the body, or that the toxicity of the first administration has been resolved or is being resolved.

In some embodiments, the treatment method further includes monitoring the levels of a first therapeutic agent or its metabolite compound in the subject using pharmacokinetic analysis. In some such embodiments, monitoring the levels of a first therapeutic agent or its metabolite compound in the subject using pharmacokinetic analysis includes using concentrations to construct a pharmacokinetic profile of the first therapeutic agent or its metabolite compound in samples obtained from the subject at at least two time points suitable for constructing a pharmacokinetic profile. In some embodiments of the method, this includes monitoring the levels of a first therapeutic agent or its metabolite compound in the subject using pharmacokinetic analysis, with samples taken from the subject at a point of care or point of use via a point-to-point care device or point-of-use device or a matrix suitable for storing samples prior to laboratory quantification. In some embodiments of the treatment method, each point-to-point care device or point-of-use device is capable of quantifying the first therapeutic agent or its metabolite compound. In some embodiments of the method, this includes monitoring the levels of a first therapeutic agent or its metabolite chemical substance in the subject, with one or more samples collected from the subject at a point-to-point or point-of-use site via a biopsy site prior to analysis at a laboratory site, or at a point-of-care or point-of-use device. In some embodiments of the method, a biopsy is performed at a time interval of 3-8 hours after administration of the first therapeutic agent. In some embodiments of the method, a biopsy is performed at a time interval of hours after administration of the first therapeutic agent. In some embodiments of the method, a biopsy is performed 8-24 hours after administration of the first therapeutic agent to the subject. In some embodiments of the method, a biopsy is performed 2 days after administration of the first therapeutic agent to the subject. In some embodiments of the method, a biopsy is performed 3 days after administration of the first therapeutic agent. In some embodiments of the method, a biopsy is performed 4 days after administration of the first therapeutic agent. In some embodiments of the method, a biopsy is performed at a time interval of 1-7 days after administration of the first therapeutic agent.

In some embodiments of the treatment method, pharmacokinetic characteristics include pharmacokinetic parameters of the first therapeutic agent suitable for guiding the treatment of the subject. In one embodiment of the treatment method, the maximum concentration of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) (“ _Cmax ”) ranges from about 1000 ng/dL to 1500 ng/dL during the treatment period. In some embodiments, _Cmax is less than 1500 ng/dL and greater than 85 ng/dL during the treatment period. In one embodiment of the treatment method, the maximum concentration of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) (“ _Cmax ”) ranges from about 1000 ng/mL to 1500 ng/mL during the treatment period, _Cmax is less than 1500 ng/mL and greater than 85 ng/mL.

In some implementations, the maximum concentration of the drug administered to the subject in the blood (whole blood, plasma, or serum) (“ _Cmax ”) is approximately 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310. 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng/dL to about 1500 ng/dL; about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113... 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL to about 150 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.

In some implementations, the maximum concentration of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) (“ _Cmax ”) after treatment is approximately 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310. 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480 or 1490 ng/mL to about 1500 ng/mL; about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134... 135, 136, 137, 138, 139, 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.

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

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

In one implementation, the first therapeutic agent in the final concentration of blood (whole blood, plasma, or serum) (“ _Cmax ”) is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505. 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485, 1495 or 1500 ng/dL. In some implementations, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma, or serum) (“ _Cmax ”) of the treated subject is selected from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/dL. In some embodiments, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma, or serum) (“ _Cmax ”) of the treated subject is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/dL.

In one implementation, the first therapeutic agent in the final concentration of blood (whole blood, plasma, or serum) (“ _Cmax ”) is selected from about 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, 475, 485, 495, 505. 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895, 905, 915, 925 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485, 1495 or 1500 ng/mL. In some implementations, the maximum concentration of the first therapeutic agent in the blood (whole blood, plasma, or serum) (“ _Cmax ”) of the treated subject is selected from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or 149 ng/mL. In some embodiments, the most recent maximum concentration in blood (whole blood, plasma, or serum) (“ _Cmax ”) is selected from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5 ng/mL.

In one implementation of the treatment method, the maximum concentration of the first therapeutic agent in the subject's blood (whole blood, plasma, or serum) ("C _max ") is in the range of about 85 ng/dL to 1500 ng/dL; from about 8.5 ng/dL to 150 ng/dL; or from about 0.85 ng/dL to 15 ng/dL. In some implementations, the final concentration of the first therapeutic agent in blood (whole blood, plasma, or serum) (“ _Cmax ”) is primarily selected from approximately 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465. 475, 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885 895, 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255, 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485 or from about 1495 ng/dL to about 1500 ng/dL; from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or from about 149 ng/dL to about 150 ng/dL; or from about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or from about 14.5 ng/dL to about 15ng/dL.

In one embodiment of the treatment method, the administration range is from about 85 ng/mL to 1500 ng/mL of the maximum concentration of the first therapeutic agent in blood (whole blood, plasma or serum) (“C _max ”); from about 8.5 ng/mL to 150 ng/mL; or from about 0.85 ng/mL to 15 ng/mL. In some implementations, the final concentration of the first therapeutic agent in blood (whole blood, plasma, or serum) (“ _Cmax ”) is selected to be approximately 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, 395, 405, 415, 425, 435, 445, 455, 465, or 475. 485, 495, 505, 515, 525, 535, 545, 555, 565, 575, 585, 595, 605, 615, 625, 635, 645, 655, 665, 675, 685, 695, 705, 715, 725, 735, 745, 755, 765, 775, 785, 795, 805, 815, 825, 835, 845, 855, 865, 875, 885, 895 905, 915, 925, 935, 945, 955, 965, 975, 985, 995, 1005, 1015, 1025, 1035, 1045, 1055, 1065, 1075, 1085, 1095, 1105, 1115, 1125, 1135, 1145, 1155, 1165, 1175, 1185, 1195, 1205, 1215, 1225, 1235, 1245, 1255 1265, 1275, 1285, 1295, 1305, 1315, 1325, 1335, 1345, 1355, 1365, 1375, 1385, 1395, 1405, 1415, 1425, 1435, 1445, 1455, 1465, 1475, 1485, or from about 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, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, or from about 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.

In some embodiments of this method, the total drug exposure over time, measured as the area under the curve (“AUC”) of the drug concentration in the subject’s blood (whole blood, plasma, or serum), ranges from about 150 ng/mL to about 8000 ng/mL; from about 15 ng/mL to about 800 ng/mL; or from about 1.5 ng/mL to about 80 ng hr/mL. In some embodiments, the AUC is less than 8000 ng hr/mL and greater than or equal to 150 ng hr/mL. In some embodiments, the AUC is less than 800 ng hr/mL and greater than or equal to 15 ng hr/mL. In some embodiments, the AUC is less than 80 ng hr/mL and greater than or equal to 1.5 ng hr/mL.

In some embodiments of this method, the total drug exposure over time is about 100 ng/mL to about 8000 ng/mL AUC; about 10 ng hr/mL to about 800 ng hr/mL; or about 1 ng hr/mL to about 80 ng hr/mL. In some implementations, the total drug exposure over time is an AUC ranging from approximately 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 from approximately 7800 ng hr/mL to approximately 8000 ng hr/mL. In some implementations, the total drug exposure over time is an AUC of approximately 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 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 approximately 800 ng hr/mL. In some implementations, the total drug exposure over time is approximately from about 1.5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 76, or 78 ng hr/mL to about 80 ng hr/mL.

In some embodiments of the method, the total drug exposure over time is an AUC of about 100 ng/mL to about 8000 ng/mL, from about 10 ng hr/mL to about 800 ng hr/mL; or about 1 ng hr/mL to about 80 ng hr/mL. In some implementations, the total drug exposure over time is approximately 150 ng/mL to approximately 7800, 7600, 700, 7200, 7000, 6800, 6600, 6400, 6200, 6000 AUC, 5800, 5600, 5400, 525000, 5400, 4000, 4600, 4200, 4200, 4000, 3200, 3600, 3400, 3200, 3000, 2800, 2600, 3400, 2200, 2000, 1800, 2400, 14200, 1200, 1000, 8 00, 600, 400 or 200 ng/mL, and in some embodiments, the total drug exposure over time is about 15 ng/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, 3300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 80, 60, 40 or 20 ng hr/mL AUC. In some embodiments, the total drug exposure over time ranges from about 1.5 ng/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 some embodiments, the total drug exposure over time is an AUC of about 100 ng/mL to about 200 ng/mL; from about 10 ng/mL to about 20 ng hr/mL; or from about 1 ng hr/mL to about 2 ng hr/mL.

In some embodiments of this method, the total drug exposure over time is selected from approximately 100, 150, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2002, 2400, 2600, 3200, 3000, 3200, 3400, 3600, 3800, 4000, AUC at 4200, 4600, 400, 4200, 4200, 4200, 4200, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 720000, 7400, 7600, 7800 and 8000 ng/mL. In some implementations, the total drug exposure over time ranges from approximately 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/mL. In some embodiments of this method, the total drug exposure over time is selected from about 1, 15, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80 ng/mL.

On the other hand, this document provides a method for treating or using a composition to treat a disease state, comprising administering a combination of a first therapeutic agent and a second therapeutic agent to a subject requiring such treatment, the method comprising:

(i) administering to the subject a first therapeutic agent comprising a compound of formula (1) or formula (10), or an analogue thereof, or a pharmaceutically acceptable salt thereof;

(ii) Use pharmacokinetic analysis to monitor the levels of the first therapeutic agent or its metabolite in subjects;

(iii) Managing a second therapeutic agent based on the level of the first therapeutic agent. In some embodiments of the method, the monitoring step includes constructing a pharmacokinetic profile of the first therapeutic agent or its metabolite using the concentration of a compound of the first therapeutic agent or its metabolite at multiple samples obtained from the subject at time points suitable for constructing the pharmacokinetic profile. In some embodiments of the method, at least two samples are collected at the point of care or point of use, either sampled at a point of care device or point of use device or self-sampled, or suitable for storing samples for laboratory quantification of the compound or its metabolite. In some embodiments of the method, each point of care device or point of use device is capable of quantifying the compound or its metabolite. In some embodiments of the method, the pharmacokinetic profile includes pharmacokinetic parameters suitable for guiding the administration of the compound or its salt to the subject. In some embodiments of the method, the samples include 2-12 samples. In some embodiments of the method, the sample collection time is up to 8 hours, up to 24 hours, up to 48 hours, or up to 72 hours. In some embodiments of the method, the pharmacokinetic parameters include at least one parameter selected from AUC, AUC _inf , T _max , C _max , time above a threshold, steady-state concentration, absorption rate, clearance rate, distribution rate, terminal T⁻¹/², or parameters extracted from non-compartmental pharmacokinetic (PK) or inter-compartmental PK analysis, including inter-compartmental PK analysis based on physiological models. In some embodiments of the method, the treatment method also includes generating a report including the pharmacokinetic characteristics of the subject. In some embodiments of the method, the report includes recommendations for dosing based on the subject's pharmacokinetic characteristics. In some embodiments of the method, a dose reduction of compound (1), its analogues, or a pharmaceutically acceptable salt thereof is indicated as a reduction in toxicity risk based on one or more pharmacokinetic parameters. In some embodiments of this method, dose reduction of the compound or its salt is expressed based on time above a threshold, where the threshold is above the drug concentration where toxicity occurs, or AUC, AUC _inf , mean residence time (MRT), an index defining pharmacokinetic characteristics, steady-state volume of distribution (Vss), terminal volume of distribution (Vz), or a combination of pharmacokinetic variables to adequately describe the pharmacokinetic characteristics. In some embodiments of this method, dose adjustment of the compound or its salt is indicated based on one or more pharmacokinetic parameters to increase efficacy. In some embodiments of this method, dose increase of the compound or its salt at terminal volume of distribution (Vz) or a combination of pharmacokinetic variables is indicated based on AUC, AUC _inf , MRT, an index defining pharmacokinetic characteristics, steady-state volume of distribution (Vss), or volume of distribution to adequately describe the pharmacokinetic characteristics. In some embodiments of this method, the dose of the compound or its salt is adjusted to within 5% to 25% of the desired target value. In some embodiments of this method, each sample is applied to a care device or a point-of-use device for determining the concentration of a compound or its metabolites, wherein the care device or point-of-use device includes a lateral flow strip having a construction and composition such that applying one or more samples to the lateral flow strip causes a portion of the drug in the sample to bind with a component of the lateral flow strip, resulting in a detectable signal proportional to the drug concentration in the applied sample. In some embodiments of this method, the sample is suitable for storage prior to laboratory quantification. In some embodiments of this method, the sample is stored as a dried blood spot. In some embodiments of this method, the drug concentration is measured by ELISA, LC-MS, LC-UV, or LC-MS. In some embodiments of this method, pharmacokinetic parameters include at least one of steady-state concentration, absorption, and terminal T1/2. In some embodiments of this method, at least one sample is whole blood.

IX. Multimodal treatment methods

In one aspect, this article provides a multimodal treatment method in which a compound of formula (1) or formula (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, is administered to a patient in conjunction with the administration of other treatment modalities. In one embodiment, the multimodal treatment method administers to a subject a pharmaceutical composition comprising a compound of formula (1) or formula (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, in conjunction with radiation therapy or radiation therapy that is determined to be ineffective after radiation. In one embodiment, the multimodal treatment method administers to a subject a pharmaceutical composition comprising a compound of formula (1) or formula (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, in conjunction with radiation therapy, wherein the pharmaceutical composition comprises a compound of formula (1) or formula (10), or an analogue thereof, or a pharmaceutically acceptable salt thereof, and radiation therapy is administered simultaneously or sequentially in any order. In one embodiment, the multimodal treatment method administers to a subject a pharmaceutical composition comprising a compound of formula (1) or formula (10), or an analogue thereof or a pharmaceutically acceptable salt thereof, in conjunction with radiation therapy arranged sequentially. In one embodiment, the multimodal treatment method includes administering a salt of a compound or analogue of formula (1) or formula (10) or a pharmaceutically acceptable composition thereof to a subject requiring such treatment concurrently with radiotherapy. In one embodiment, the multimodal treatment method is used to treat cancer. In one embodiment, the multimodal treatment method includes administering a salt of a compound or analogue of formula (1) or formula (10) or a pharmaceutically acceptable composition thereof to a subject with cancer requiring such treatment and irradiating the cancer cells with a radiation beam. In one embodiment, the multimodal treatment method uses conformal radiotherapy (CRT) to provide a dose-volume histogram (DVH) prescribed to the cancer subject. In one embodiment, the multimodal treatment method uses intensity-modulated radiotherapy (IMRT) to deliver radiation to the cancer cells. In one embodiment, the multimodal treatment method uses techniques to compensate for tumor movement during treatment (e.g., the radiation dose must be applied to a chest tumor that moves during the patient's breathing). For example, the multimodal treatment method uses four-dimensional computed tomography (4D CT) scanning to adjust the delivered radiation field to compensate for tumor movement during the respiratory cycle.

Multimodal treatment methods can utilize any suitable type of radiation, including graded gamma radiation, IMRT (Intensity Modulated Radiotherapy), gamma knife, proton therapy, and brachytherapy. Radiation therapy and the application of compounds of formula (1) or formula (10), or their analogues or pharmaceutically acceptable salts thereof, can be used to treat brain tumors, such as glioblastoma, or diseases that have metastasized to the brain (lung cancer). Multimodal treatment methods can be used to treat lung cancer, pancreatic cancer, rectal cancer, breast cancer, sarcoma, prostate cancer, gynecological malignancies, and lymphoma. Gamma knife is frequently used to treat brain metastases. In one implementation, multimodal treatment methods include the use of proton therapy to treat cancers, including brain tumors, prostate cancer, and any tumors near vital organs. Minimizing toxicity to nearby normal tissues is of paramount importance.

In one implementation, the multimodal treatment method includes administering a pharmaceutical composition comprising a compound of formula (1) or formula (10) or an analogue thereof or a pharmaceutical composition thereof to a subject with cancer who requires such treatment, in concomitant or in combination with 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)).

In one embodiment, the multimodal treatment method eliminates minimal residual disease without increasing the toxicity resulting from treatment with compounds of formula (1) or formula (10) or their analogues or pharmaceutically acceptable salts thereof. In one embodiment, the multimodal treatment method improves prognosis and/or reduces adverse side effects or symptoms in the treated subject associated with the disease state.

X. Compound (1) and related derivatives and chemical substances

In one respect, this document provides analogues and related salts of the compound (1) and the same procedures for their preparation. Those skilled in the art will understand that the same general principles and concepts, including those relating to methods and pharmaceutical compositions, are applied to the compounds (1), (10) and their salts above, to analogues and salts of the compound (1) and their salts that have equivalent potency to the derivatives.

In one embodiment, the analogue has the structure of compound (25):

Wherein Y represents NR ₄ or O, and wherein R ₁ , R ₂ , R ₃ , and R ₄ independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, thioaralkyl, alkanoyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic radicals. In some embodiments, R ₁ , R ₂ , R ₃ , and R ₄ may optionally be substituted. In some embodiments, some or all of the hydrogen atoms in R ₁ , R ₂ , R ₃ , and R ₄ may be replaced by deuterium. In other embodiments, the analogue has the structure of compound (25), wherein _R1 , _R2 , _R3 and _R4 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine and _C1-4 alkylthiophene, wherein _C1-4 alkyl, _C1-4 alkylphenyl, C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine are optionally substituted with _{C1-4 alkyl} , hydroxyl or halogen. In even other embodiments, the analogue has the structure of compound (25), wherein _R1 , _R2 , _R3 and _R4 are independently selected from 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, the analogue has the structure of compound (26):

_R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkylacyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radicals. In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, and _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, and _C1-4 benzyl-piperazine are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, _R1 is selected from 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 some embodiments, _R2 is selected from 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 some embodiments, _R1 is a benzyl group, optionally substituted alone or in combination with one or more of the following substituents 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 , _{-CpX2p+1 , -OCX3} , or _{-OCpX2p +1} , where _p is an integer from 2 to 20 and where X represents a halogen, including fluorine, chlorine, bromine _, or iodine atoms, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine. In some embodiments, _R2 is a substituent of the benzyl ring, one or more of the substituents, or a combination of ortho, meta, and/or para substitutions of the benzyl ring: _-CH3 , _-NO2 , -OCH3, _-CXH2 , _-CX2H , _-CX3 , -CH2(CX3 ₎ , _-CH ( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1 , -OCX3} , or -OCpX2p ₊₁ , where _p is an integer from 2 to 20 and where X represents a halogen.

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In some embodiments, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In some embodiments, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In some embodiments, _R2 is a substituted or substituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridinylmethyl group. In some embodiments, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

In one embodiment, the analogue has the structure of compound (27):

Wherein _R1 represents hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkanoyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic radical. In some embodiments, _R1 is selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, and _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, and _{C1-4 benzyl} -piperazine are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, perhalogenated _C1-4 alkyl, or halogen. In some embodiments, _R1 is selected from H, _CH3 , _CH2Ph , _CH2 -((2-Cl)-Ph), _CH2- (2-thiophene), _CH2CH2Ph , _CH2CH2 (4- _N -benzyl-piperazine), _CH2- ( _2,4 -diF-Ph), _CH2 -((2- _CH3 )-Ph), _CH2CHOHPh , and ( _CH2 ) _3CO -4F-Ph.

In some embodiments, _R1 is benzyl, optionally substituted alone or in combination with one or more of the following substituents 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 , -CpX2p + 1 , -OCX3 ,} or -OCpX2p ₊₁ , where _p is an integer from 2 to 20 and where X represents a halogen, including fluorine, chlorine, bromine _, or iodine atoms, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine. In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In one embodiment, the analogue has the structure of compound (28): wherein _R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkanoyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocyclic radical. In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, and _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, and _C1-4 benzyl-piperazine are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, _R1 is selected from H, _CH3 , _CH2Ph , _CH2 -((2-Cl)-Ph), _CH2- (2-thiophene), _CH2CH2Ph , CH2-(2,4-diF-Ph), _CH2 -(( ₂ - _CH3 )-Ph), _CH2CHOHPh , _CH2CH2 ( ₄ -N-benzyl-piperazine), _{( CH2} ) _3CO -4F-Ph. In some embodiments, _R2 is selected from 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 , ( _CH2 ) _3CO -4F-Ph. In some embodiments, when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph.

In some embodiments, _R1 is any substituent of the benzyl ring, one or more of the substituents, or substituted 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 , -CpX2p _{+1 , -OCX3} , or _{-OCpX2p +1} , _{where p} is an integer from 2 to 20 and where X represents a halogen, including fluorine, chlorine, bromine _, or iodine atoms, preferably fluorine, chlorine, or bromine, more preferably fluorine or chlorine. In some embodiments, _R2 is a substituent of the benzyl ring, one or more of the substituents, or a combination of substituents 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 , _{-CpX2p +1 , -OCX3} , or -OCpX2p ₊₁ , where _p is an integer from 2 to 20 and X represents a halogen.

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In some embodiments, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In some embodiments, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In some embodiments, _R2 is a substituted or substituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridinylmethyl group. In some embodiments, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

In one embodiment, the analogue has the structure of compound (29):

Wherein _R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkylacyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radicals. In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, and _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, and _C1-4 benzyl-piperazine are substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, _R1 is selected from 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 some embodiments, _R2 is selected from 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 , ( _CH2 ) _3CO -4F-Ph. In some embodiments, when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph.

In some embodiments, _R1 is benzyl, optionally substituted alone or in combination with one or more of the following substituents 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 , _{-CpX2p +1} , _-OCX3 or -OCpX2p ₊₁ , _{where p} is an integer from 2 to 20 and where X represents a halogen, including fluorine, chlorine, bromine or _iodine atoms, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine. In some embodiments, _R2 is a substituent of the benzyl ring, one or more of the substituents, or a combination of ortho, meta, and/or para substitutions of the benzyl ring: _-CH3 , _-NO2 , _-OCH3 , _-CXH2 , -CX2H, _-CX3 , -CH2(CX3), _-CH ( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1 , -OCX3 ,} or _{-OCpX2p +1} , where p is an integer from 2 to 20 and X represents a halogen.

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In some embodiments, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In some embodiments, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In some embodiments, _R2 is a substituted or substituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridinylmethyl group. In some embodiments, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

In one embodiment, the analogue has the structure of compound (30):

Wherein _R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkylacyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radicals. In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, and _C1-4 benzyl-piperazine are optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, perhalogenated _C1-4 alkyl, or halogen. In some embodiments, _R1 is selected from 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 some embodiments, _R2 is selected from 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 some embodiments, when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph.

In some embodiments, _R1 is benzyl, optionally substituted alone or in combination with one or more of the following substituents 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 , _{-CpX2p +1} , _-OCX3 or -OCpX2p ₊₁ , _{where p} is an integer from 2 to 20, and where X represents a halogen including fluorine, chlorine, bromine or _iodine atoms, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine. In some embodiments, _R2 is a substituent of the benzyl ring, one or more of the substituents, or a combination of ortho, meta, and/or para substitutions of the benzyl ring: _-CH3 , _-NO2 , _-OCH3 , _-CXH2 , -CX2H, _-CX3 , -CH2(CX3), _-CH ( _CX3 ) ₂ , -C( _CX3 ) ₃ , _{-CpX2p +1 , -OCX3 ,} or _{-OCpX2p +1} , where p is an integer from 2 to 20 and X represents a halogen.

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In some embodiments, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In some embodiments, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In some embodiments, _R2 is a substituted or substituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridinylmethyl group. In some embodiments, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

In one embodiment, the analogue has the structure of compound (31):

_R1 and _R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, thioaralkyl, alkylacyl, mercapto, thioalkyl, thioaryl, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic free radicals. In some embodiments, _R1 and _R2 are independently selected from H, _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine, _C1-4 alkylthiophene, wherein the _C1-4 alkyl, _C1-4 alkylphenyl, _C1-4 alkylphenyl ketone, _C1-4 benzyl-piperazine is optionally substituted with _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some implementation schemes, _R1 is selected from H, _CH3 , _CH2Ph , _CH2 -((2-Cl)-Ph), _CH2- (2-thiophene), _CH2CH2Ph , _CH2CH2 (4-N-benzyl-piperazine), _CH2- ( _{2,4 -} diF-Ph), _CH2 -((2- _CH3 )-Ph), _CH2CHOHPh , ( _CH2 ) _3CO -4F-Ph. In some embodiments, _R2 is selected from 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 some embodiments, when _R1 represents _CH2Ph , _R2 does not represent _CH2 -((2- _CH3 )-Ph.

In some embodiments, _R1 is benzyl, optionally substituted alone or in combination with one or more of the following substituents 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 , _{-CpX2p +1} , _-OCX3 or -OCpX2p ₊₁ , _{where p} is an integer from 2 to 20, and where X represents a halogen including fluorine, chlorine, bromine or _iodine atoms, preferably fluorine, chlorine or bromine, more preferably fluorine or chlorine. In some embodiments, _R2 is a substituent of the benzyl ring, one or more of the substituents, or a combination of ortho, meta, and/or para substitutions of the benzyl ring: _-CH3 , _-NO2 , _-OCH3 , -CXH2, -CX2H, _-CX3 , -CH2 _{(CX3), -CH(CX3)2, -C(CX3)3 , -CpX2p + 1 , -OCX3 , or -OCpX2p + 1} , where _p is an integer from 2 to 20, and where X represents a halogen.

In some embodiments, _R1 is hydrogen. In some embodiments, _R1 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl group, a _C1-4 alkoxy group, a hydroxyl group, a fully halogenated _C1-4 alkyl group, or a halogen.

In some embodiments, _R2 is a substituted or unsubstituted aralkyl group, such as benzyl or phenethyl. In some embodiments, the aralkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the aralkyl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 . In some embodiments, _R2 is a substituted or unsubstituted heterocyclic alkyl group, such as morpholinoalkyl or piperazineylalkyl group. In some embodiments, _R2 is a substituted or substituted heteroaryl alkyl group, such as isoxazolylmethyl or pyridinylmethyl group. In some embodiments, the heterocyclic alkyl or heteroaryl alkyl group is substituted with a _C1-4 alkyl, _C1-4 alkoxy, hydroxyl, fully halogenated _C1-4 alkyl, or halogen. In some embodiments, the heterocyclic alkyl or heteroaryl group is substituted with one or more substituents selected from the following: halogenated, _-CH3 , _-CF3 , and _-OCH3 .

XI. Examples

It should be understood that the descriptions and specific examples provided below are for illustrative purposes only 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 them. Other compounds besides those described below can be prepared using the above-described reaction schemes or suitable variations or modifications thereof.

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

2-Chlorobenzylamine (141 mg, 1.0 mmol) was added to a solution of 2-methylthio-2-imidazoline hydrochloride (244 mg, 1.00 mmol) in anhydrous dioxane (2.0 mL). The reaction mixture was stirred at 70 °C for 90 min under an argon atmosphere. The solution was cooled to room temperature, filtered through a sintered funnel, washed with cold dioxane (2 mL), and dried under vacuum. A white solid compound 4.HI (R2 = 2-chlorobenzyl) (242 mg, 72%) was given, which could be used without further purification.

Example 2.2 Synthesis of 2-chlorobenzylamino-2-imidazoline

1.0 N sodium hydroxide (2 mL) was added to a 3 mL aqueous solution of 2-chlorobenzylamino-2-imidazoline hydrochloride (242 mg, 0.72 mmol) at 7 °C. The reaction mixture was stirred at 7 °C under argon for 30 min. Then, methyl olefin chloride (5 mL) was added, and the mixture was stirred for another 5 min. The reaction mixture was extracted with methyl chloride (2 × 2.5 mL). The organic layer was dried over anhydrous _Na₂SO₄ , filtered, and evaporated. _The resulting free base (150 mg, 100%) was a viscous liquid and could be used for the next reaction without further purification. MS (ESI)₂¹⁰(M+H).

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

Triethylamine (6 mL) was added to a stirred solution of methyl-1-benzyl-4-oxo-3-piperidine carboxyl hydrochloride (5.7 g, 20 mmol) in ethyl acetate (50 mL) at 7 °C. The reaction mixture was stirred at 7 °C under an argon atmosphere for 30 min. 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), a viscous oil, was ready for use on MS (ESI) 248 (M+H) without further purification.

Example 4. Synthesis of ONC902 (compound (14))

PPTS (10 mg) was added to a methanol solution of 2-chlorobenzylamino-2-imidazoline (150 mg, 0.72 mmol) in 1-benzyl-4-oxo-3-piperidinecarboxylate (5,1R=benzyl) (195 mg, 0.79 mmol) in 1-butanol (2 mL), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed from 125 °C to 130 °C for 2 hours. The solvent was removed under vacuum, and the mixture was 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 RP HPLC (10%–40% acetonitrile/water) to give ONC902TFA salt (228 mg, 50% yield) as a white solid (MS(ESI) 407(M+H)).

Various analogues, such as ONC903, 904, 905, 906, 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.

Example 5. Synthesis of ONC907 (compound (19))

Dimethyl carbonate (4.32 g, 48.0 mmol) was added dropwise over 0.5 hours to a suspension of sodium hydride (3.5 g, 88 mmol) in anhydrous toluene (50 mL) at 60 °C under a nitrogen atmosphere and at room temperature. After adding a few drops of methanol, a solution of 1-tert-butoxycarbonyl-4-piperidinone (4.8 g, 24 mmol) dissolved in anhydrous toluene (20 mL) was added dropwise to the reaction mixture while stirring at 80 °C for more than 1 hour. The reaction mixture was stirred at the same temperature for 3 hours, then cooled to 0 °C (ice bath) and adjusted to pH 6–6.5 with acetic acid. The resulting cold mixture was diluted with water (10 mL) and adjusted to pH 8 with 5% sodium hydroxide solution. The toluene layer was separated, and the aqueous layer was extracted with toluene (20 mL). The combined organic layers were dried over _{anhydrous Na₂SO₄} and concentrated under reduced pressure. The compound was dried under vacuum to give methyl-1-tert-butoxycarbonyl-4-oxo-3-piperidinecarboxylate (5.0 g, 80%). The obtained compound was then used in the next reaction without further purification.

2-Methylbenzylamino-2-imidazoline (190 mg, 1 mmol) and methyl 1-tert-butoxycarbonyl-4-oxo-3-piperidinecarboxylate (315 mg, 1.1 mmol) were added to 2 mL of 1-butanol with 10.0 mg of PPTS, and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed from 125 °C to 130 °C for 2 hours. The solvent was removed under vacuum, and the mixture was 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 in dichloromethane with 10% trifluoroacetic acid and purified by RPHPLC (10%–40% acetonitrile/water) to give a white solid MS(ESI)₂97(M+H).

Example 6. Synthesis of ONC909 (compound (21))

A mixture of ONC907 (100 mg, 0.2 mmol), phenyl 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, and the mixture was 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 RPHPLC (10%–40% acetonitrile/water) to give a white solid MS(ESI)₄O₁(M+H) of ONC909 (62 mg, 50%) TFA salt.

ONC910 and 214 were obtained from different halides using the same method. Compounds 227, 228, 229, and 230 (along with the compounds listed in Table X in the preparation) of different benzylamines were prepared using similar methods to those in Examples 1 and 5.

Compound ONC911 was prepared from ONC910 by treatment with TFA.

Compound (72) was prepared by reacting a precursor NH compound, similar to that prepared in Example 5, with styrene oxide.

Example 7. Synthesis of ONC908 (compound (20))

PPTS (10.0 mg) was added to 2.0 mL of methyl 1-methyl-4-oxo-3-piperidinecarboxylate (185.0 mg, 1.0 mmol) in 1-butanol (2.0 mL), and the mixture was stirred at room temperature for 48 hours. The reaction mixture was then refluxed from 125 °C to 130 °C for 2 hours. The solvent was removed under vacuum, and the mixture was extracted with ethyl acetate (10 mL) and washed with saturated sodium bicarbonate solution (2 × 10 mL) and water (10 mL). The organic layer was dried over _{anhydrous Na₂SO₄} , filtered, and evaporated. The crude free base was purified by HPLC with 10%–40% acetonitrile and water to give a white solid MS(ESI)₃11(M+H) of ONC908 (270.0 mg, 50%) TFA salt.

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

Compound (3) (239.7 g, 0.845 mol, 1.6 equivalence) was added in portions to 800 mL of saturated NaHCO3 in _a stirred 2 L round-bottom flask. 500 mL of n-butanol 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 2 L three-necked round-bottom flask equipped with a mechanical stirrer, _N2 inlet, thermocouple, condenser, and Dean-Stark water separator. Compound (5) (100 g, 0.528 mol, 1 equivalence) and pyridinium p-toluenesulfonate (PPTS) (6.63 g/mL, 0.026 mol, 5 mol%) were added to the flask. The resulting mixture was refluxed for 6 hours. Water in the reaction mixture was separated using a Dean-Stark water separator as needed. The reflux temperature was increased from 93 °C to 118 °C. The reaction progress was monitored by HPLC. The reaction was stopped when the peak area of compound (1) on HPLC remained constant over time.

Example 9. Synthesis of the disalt of ONC201 (compound (1)·2HCl)

Without separating compound (1), the reaction mixture from Example 8 was washed with 500 mL of water and diluted with methyl tert-butyl ether (MTBE) (800 mL). The organic phase was washed with water (500 mL × 2) and transferred to a 3 L three-necked round-bottom flask equipped with a mechanical stirrer, _N₂Cl , thermocouple, condenser, and Dean-Stark separator. While stirring the reaction mixture, 1N HCl in a 1N dioxane-METBE solution (4N HCl in dioxane solution: 300 mL, 1.2 mol, 2.27 equivalents; MTBE: 1200 mL) was added dropwise until no solid HCl precipitated from the reaction mixture upon addition. The reaction mixture was refluxed at 60–65 °C for 2 hours. The water was separated into Dean-Stark traps as needed. After cooling 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 overnight (16 hours) at 65°C in a vacuum oven to obtain 200g of yellow solid.

The above solid (200 g) was added to a 2 L three-necked round-bottom flask equipped with a mechanical stirrer, _N2 inlet, thermocouple, and condenser, followed by ethanol (1000 mL). The mixture was heated under reflux at 78 °C for 2 hours. After cooling to room temperature, the solid was filtered through a sintered glass funnel and washed with ethanol (200 mL × 3). The wet solid was dried in a vacuum oven at 85 °C for 3 days until the residual solvent reached the specified concentration. 120 g of compound (2) was obtained as a white solid with a yield of 49% and an HPLC purity of 99.7%.

Example 10. Activity of analogues of compound (1)

Analogs of several exemplary compounds (1) were prepared based on the synthesis described herein. For each of these compounds, the viability of human cancer cells was measured 72 hours after treatment with the compound. The changes in potency (relative to ONC201) were determined, as shown in the table below.

Relative potency of analogues of compound (1)

No. Identification code R1 R2 Relative effectiveness* 1 ONC201 CH2Ph CH2-((2-CH3)-Ph) N/A 14 ONC902 CH2Ph CH2(2-Cl-Ph) B 15 ONC903 CH2Ph CH2-(2-Thienyl) C 16 ONC904 CH2Ph CH2CH2Ph B 17 ONC905 CH2Ph CH2CH2(4-N-Benzyl-piperazine) C 18 ONC906 CH2Ph CH2-(2,4-diF-Ph) A 19 ONC907 H CH2-((2-CH3)-Ph) C 20 ONC908 CH3 CH2-((2-CH3)-Ph) B twenty one ONC909 CH2CH2Ph CH2-((2-CH3)-Ph) B 32 ONC910 (CH2)3-NH-BOC CH2-((2-CH3)-Ph) B 33 ONC911 (CH2)3-NH2 CH2-((2-CH3)-Ph) B 41 ONC210 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(3,5-DiF-Ph)]]> A 51 ONC211 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(3,4-DiCl-Ph)]]> A 52 ONC212 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(4-CF₃-Ph)]]> A 53 ONC213 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(3,4-DiF-Ph)]]> A 54 ONC214 <![CDATA[CD₂C₆D₅]]> <![CDATA[CH₂-((2-CH₃)-Ph)]]> B 43 ONC217 <![CDATA[CH₂Ph]]> <![CDATA[CH₂(2-F-Ph)]]> B 55 ONC218 <![CDATA[CH₂Ph]]> <![CDATA[CH₂(2-CH₃, 4-F-Ph)]]> A 56 ONC219 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(2,4-DiCl-Ph)]]> A 57 ONC220 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-((4-OCH₃)-Ph)]]> A 34 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(3-pyridyl)]]> 35 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(3-isooxazolidinyl)]]> B 36 <![CDATA[CH₂Ph]]> <![CDATA[CH₂CH₂-(4-morpholinyl)]]> A 38 H <![CDATA[CH₂-(4-CF₃-Ph)]]> A 72 <![CDATA[CH₂Ph]]> <![CDATA[CH₂-(2-F, 4-CF₃-Ph)]]> A

*Effectiveness relative to ONC201; A indicates an increase of more than 2 times in the effectiveness of ONC201; B indicates an effectiveness within 2 times that of ONC201; and C indicates a decrease of more than 2 times in the effectiveness of ONC201.

In addition, a single dose of compound (52) administered orally or intraperitoneally to human colon cancer xenograft mice resulted in a significant reduction in tumor volume compared to the vector-treated control group. Compound (52) has a broad therapeutic window and is well tolerated in mice at doses of at least 225 mg/kg.

Example 11. Dosing regimen

Compound (1) was administered to tumor-bearing mice in a 7-day cycle according to the following administration regimen.

1) Day 1: 200 mg/kg orally;

2) Day 1/Day 4: 100mg/kg orally per dose;

3) Day 1/Day 2: 100mg/kg orally per dose;

4) Day 1: Two doses of 100 mg/kg orally, each dose taken 6 hours apart.

The effects of the dosing regimens were evaluated and compared.

Example 12. Identification of the target of compound (1)

The target spectrum of compound (1) in human colon cancer cell line HCT116 was determined.

In short, compound (33) (ONC911) was immobilized onto agarose beads at varying immobilization densities. For analysis by quantitative mass spectrometry, HCT116 human colon cancer cells were grown in a medium containing amino acids labeled with different forms of isotopes (SILAC = stable isotopes labeled with amino acids in cell cultures). The corresponding proteomes could be distinguished by the introduced mass differences. The binding experiments were repeated with partial label switching to eliminate labeling artifacts. The bound proteins were completely eluted from the affinity matrix, separated by SDS-PAGE, and triedpsinized. The recovered peptides were analyzed by LC-MS/MS on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher). The raw data generated by LC-MS/MS were processed by MaxQuant to obtain quantitative protein abundance data.

By incubating with compound (1), the enrichment of proteins and competition for binding proteins compared to the control matrix were analyzed. This specific target protein binding and substitution pattern is expected.

result

First, HCT116 cells were cultured and metabolically labeled using SILAC. High efficiency in SILAC encoding was achieved, with incorporation rates of arginine and lysine isotope variants exceeding 95%. Sufficient cells were prepared for subsequent experiments. Cell extracts were generated via detergent-mediated cell lysis. Additionally, the remaining nuclei were extracted by lysis in the presence of 400 mM NaCl to include nucleoproteins. The cytosol and nuclear extracts were then combined.

Connector compound (33) (ONC911) was immobilized on agarose beads via its amino group. Beads with four different immobilization densities of 6 mM, 3 mM, 1 mM, and 0.3 mM were prepared. These matrices were used to enrich proteins in HCT116 extract and to investigate the substitution of proteins bound by 50 μM compound (1).

A total of 10 proteins were identified. For all coupling densities and repetitions, specific enrichment of proteins with immobilized chemical substance (33) (ONC911) was observed.

The number of target candidates increased with increasing fixed density. Table 3 summarizes the target candidates for compound (1). The highest coupling density (6 mM) enriched by affinity groups and consistent substitution by two repetitions of compound (1) were observed in 14 proteins. At a coupling density of 3 mM, two potential target candidates were identified, both sharing a high coupling density. At lower coupling densities (1 and 0.3 mM), two and one protein consistently acted as targets.

In addition, several proteins showed enrichment by affinity matrix and shift of compound (1), but only one shift was observed in two replicates for each coupling density. These proteins are referred to as “OK (with outliers)” in Table 3.

In summary, the immobilized compound (33) (ONC911) appears to be functional and capable of specifically enriching proteins from cell lysates. Furthermore, it exhibits distinct competition with compound (1) at 50 μM.

Table 3: Overview of identified target candidates

Uniprot ID: Best Uniprot identifier; Protein name: Protein name based on Uniprot.

Target classification: Proteins were evaluated using the indicated coupling density of compound ONC216. “OK” indicates that the corresponding protein was consistently enriched and competed in more than two independent replicates. “OK (with outliers)” indicates enrichment by affinity matrix and displacement of compound (1), but displacement was observed only once in two replicates.

Example 13. GPCR antagonism of compound (1)

ONC201 was evaluated throughout the cell line as a functional assay of β-arrestin G protein-coupled receptor (GPCR) activity, directly measuring dopamine receptor activity by detecting the interaction of β-arrestin with activating GPCRs that can act as reporters. For each dopamine receptor (DRD1, DRD2S, DRD2L, DRD3, DRD4, and DRD5), cell lines overexpressing the reporter construct were expanded from a cryogenic library. Cells were seeded in white-walled 384-well microplates at a total volume of 20 μL and incubated at 37 °C before testing. The cells were excited with an antagonist and then an agonist at an EC80 concentration. Intermediate dilutions of the sample library were performed to generate 5× samples in test buffer. 3.5 μL of 5× sample was added to the cells and incubated at 37 °C or room temperature for 30 min. The vehicle concentration was 1%. Add 5 μL of 6×EC80 agonist to the test buffer and incubate at 37°C or room temperature for 90 or 180 minutes, then take measurements. % antagonist is calculated using the following formula: % antagonism = 100% × (1 - (mean RLU of test sample / mean RLU of vector control) / (mean RLU of EC80 control - mean RLU of vector control).

Example 14. Evaluation of the interaction between compound (1) and ion and transport protein.

The ability of ONC201 to interfere with the activity of transporter proteins was evaluated to determine the co-dosing regimen of ONC201 with transporter protein substrates. The timing or dose level of ONC201 in combination with other therapeutic agents can be modified based on these test results. Transporters include OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, and MATE2-f solute carriers (SLCs).

The ability of ONC201 to interfere with efflux proteins was assessed to determine whether ONC201 could inhibit the ability of these proteins to expel small molecule substrates. Inhibition of these efflux proteins could increase the efficacy of efflux protein substrates by increasing their intracellular concentration or altering their biodistribution through binding to ONC201. Efflux proteins included MDR1 and BCRP.

The interaction between ONC201 and human MDR1 and BCRP transporters was investigated in vitro using Madin-Darby canine kidneys (MDCKII-MDR1 and MDCKII-BCRP) and parental cells (MDCKII). Bidirectional permeability of each probe substrate in MDCKII-MDR1 and MDCKII monolayers was studied to investigate whether ONC201 is an inhibitor of MDR1 and BCRP. Digoxin and prazosin were used as probe substrates for MDR1 and BCRP, respectively.

The inhibition test results are summarized in Table 4 below. ONC201 is an inhibitor of 200 μmol MDR1 and BCRP. The combination of ONC201 with MDR1 or BCRP substrates can increase the efficacy of the substrates by increasing their intracellular concentration or altering their biodistribution.

Table 4:

A-B: From the apex to the outer side of the base; B-A: From the outer side of the base to the apex; PC: Positive control

Example 15. Evaluation of the inhibitory potential of compound (1) against P450 enzymes

To assess the potential of ONC201 to induce human cytochrome P450 (CYP) enzymes, attention was paid to the use of cryopreserved plateable human hepatocytes for the three main drug-metabolizing enzymes, namely CYP 1A2, 2B6, and 3A4.

The experimental CYP induction results of ONC201 are summarized in Table 5 below. ONC201 did not induce P450 in the positive control (which had a p450 level greater than 20%) in this test. Therefore, it can be used in combination with other drugs without altering CYP enzyme activity.

Table 5: Induction of CYP mRNA in artificial hepatocytes under various treatments

The fold change in ^aCYP mRNA induction was calculated using the standard ΔΔCT method, with the 18S gene used as a reference gene and the vector control used as a baseline to treat hepatocyte target (CYP) gene expression.

The _ΔΔCT method was used with the 18S gene as a reference gene and the vector control as a baseline to treat the expression of hepatocyte target (CYP) genes.

^b NC: Negative control - Flumazetin (25 μM) was used as a negative control treatment.

^c PC: Positive controls - Omeprazole (50 μM), phenobarbital (750 μM) and rifampin (25 μM) were used as positive control treatments for CYP1A2, 2B6 and 3A4, respectively.

The data is calculated from three copies.

Inhibitors of ONC201 of seven (7) human cytochrome P450 (CYP), namely CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, were detected in vitro in concomitant human liver microsomes (HLM) using eight (8)) CYP isomer-specific marker substrate reactions. CYP2C8-mediated N-deethylation of amodiaquine, CYP2C9-mediated 4′-hydroxylation of diclofenac, CYP2C19-mediated 4′-hydroxylation of s-mexazol, CYP2D6-mediated 1′-hydroxylation of furan, CYP3A4-mediated 1′-hydroxylation of midazolam, and 6β-hydroxylation of testosterone.

ONC201 inhibits CYP isoenzymes (CYP 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4) with _IC50 values ranging from 34.9 to 428.6 μM (4 to 48 times higher than 9 μM, and 40 to 480 times higher than the mean plasma concentration of 0.9 μM at 24 hours). The inhibitory effect is not significantly time-dependent (see Table 6). These results indicate that ONC201 can be taken with most other drugs without any safety concerns related to drug-drug interactions.

Table 6:

^{The α-} _IC50 value of ONC201 was determined by fitting normalized data to a nonlinear regression model of the sigmoidal inhibitor γ using the GRAPHPAD PRISM method.

Within the measured concentration range (1.5-500 μM), ^b > 500 - no inhibition > 50%.

Example 16: Clinical trial of oral ONC201 in patients with fire-resistant solid tumors.

ONC201 induces apoptosis in tumor cells, but not in normal cells, at doses that induce high levels of cell death in human cancer cells. The safety profile of ONC201 in GLP-1 safety studies in rats and dogs is consistent with its preferential cytotoxicity in tumors compared to normal cells in vitro. Therefore, the in vitro and in vivo characterization of ONC201 suggests a broad therapeutic window that is highly desirable for cancer therapeutics. Clinical studies were conducted based on a 21-day schedule chosen from preclinical results, which indicated persistent disease progression (PD) in tumors, and preliminary experiments showed that more frequent dosing did not appear to significantly increase in vivo efficacy.

Based on the efficacy and safety of ONC201, and its novel involvement in many important signaling pathways in cancer, a clinical introduction of ONC201 into patients with advanced cancer was initiated. The primary objective of this first-in-human, phase 1 dose-escalation study was to determine the recommended Phase II dose (RP2D) of ONC201 administered orally in patients with advanced cancer, and to assess the safety and tolerability of the drug. Secondary objectives included pharmacokinetic, pharmacodynamic, and preliminary antitumor activity assessments of ONC201.

In summary, this Phase I open-label study treated 10 patients with histologically confirmed advanced solid tumors with dose escalation. An additional 10 patients were accumulated in the ongoing expansion phase to increase safety experience. Patients received ONC201 orally every 3 weeks at doses ranging from 125 to 625 mg using an accelerated titration design.

RP2D was defined as 625 mg, achieving a _Cmax of 1.5–7.5 mg/mL. A saturation plasma concentration of 375 mg indicated that dose escalation above 625 mg was unnecessary. No grade >1 drug-related adverse events occurred. During the dose escalation phase, the mean cycle number (21 days) was 3.1. The safety of ONC201 on RP2D was confirmed in 10 patients during the expansion phase. PK analysis showed a half-life of 9.6 hours and an AUC of 25 h/μg/L. Prolonged induction of serum caspase cleavage of keratin 18 and induction of TRAIL were observed. Eight of the 10 patients had stable disease, and one patient with prostate adenocarcinoma experienced long-term stable disease and continued the study for 27 weeks. One additional patient with endometrial cancer received a mixed response.

ONC201 is well tolerated, with a p-value curve showing absorbable micromolar plasma concentrations, and clinical activity was observed when administered orally at 625 mg every 3 weeks.

Patients and methods

ethics

This study was conducted at Robert Wood Johnson University Hospital/Rutgers Cancer Institute (CINJ) in accordance with the Declaration of Helsinki and the International Conference on Harmonized Clinical Practice Guidelines, and was approved by the relevant regulatory committees and institutional review committees of CINJ. Patients provided written informed consent and participated in the learning process.

patient population

Patients aged 18 years and older with advanced solid tumors lacking or resistant to standard treatment, an ECOG performance status ≥1, and disease assessed according to RECIST 1.1 criteria are eligible. If the patient has received radiation therapy, there must be a measurable lesion outside the irradiated area. The patient must have completed all prior cytotoxic chemotherapy for at least 4 weeks, alkyl therapy for at least 6 weeks, molecularly targeted therapy for at least 28 days, and radiation therapy for at least 14 days prior to the first dose. All prior treatment-related adverse events, except for alopecia areata and neuropathy, of grade ≤2, must be resolved. Patients must have normal bone marrow and organ function as defined by the following parameters: absolute neutrophil count ≥1,500/mcL; platelet count ≥1 million/mcL; hemoglobin ≥9.0 mg/dL, without transfusion for the first 2 weeks; total bilirubin within the normal range (for patients with liver metastases, serum bilirubin ≤1.5×ULN); AST (SGOT)/ALT (SGPT) ≤2.5×upper limit of normal; and a measured or estimated creatinine clearance ≥40 mL/min/1.73 m2, with creatinine levels higher than normal. Exclusion criteria include symptomatic or asymptomatic brain metastases treated with steroids, prior bevacizumab treatment, allergic reactions to compounds similar to ONC201, uncontrolled comorbidities, HIV combined with retroviral therapy, cardiac dysfunction, stroke or seizures that may alter ONC201 uptake, pregnancy, and impairment of GI function treated with hematopoietic colony-stimulating growth factor therapy within 2 weeks prior to starting treatment.

Study Design and Toxicity Assessment

This design is an open-label, dose-escalation phase I trial of a single dose of ONC201 in patients with advanced refractory tumors who have exhausted or rejected standard treatment options for each adaptation. ONC201 capsules (125 mg) are supplied by Oncoceutics Inc. (Philadelphia, PA). ONC201 is administered orally once every 21 days using an accelerated dose-escalation design. The starting oral dose is 125 mg (10% of the observed adverse event level in rats and dogs). This study was conducted using a single-patient accelerated dose-escalation design, which was designed to be discontinued if any patient experienced at least a grade 2 adverse event likely associated with ONC201. In this case, a conventional 3+3 dose escalation design would be used. After a treatment cycle has been completed in the previously administered cohort, dose escalation can be performed, meeting the criteria for the next dose level. Enrollment at each subsequent dose level requires all patients enrolled at the previous dose level to complete cycle 1 of doses and be evaluated for safety after 21 days. Dosage levels range from 125 mg to 250 mg, 375 mg, 500 mg, and finally to 625 mg.

After identifying RP2D, an expansion phase of 22 patients was initiated to recruit additional patients on RP2D to improve the robustness of the safety data generated by the trial.

All toxicities were assessed according to the Common Terminology Criteria for Adverse Events, Version 4. DLT was defined as a drug-related adverse event or abnormal laboratory outcome occurring during the first cycle of treatment that met any of the following criteria: ≥ Grade 3 non-hematologic toxicity; ≥ Grade 3 nausea, vomiting, or diarrhea lasting >72 hours despite optimal antiemetic or antidiarrheal treatment; Grade 3-4 AST/ALT combined with Grade 2 bilirubin elevation; Grade 4 neutropenia lasting = 7 days; Grade 4 neutropenia with fever >38.5°C; Grade 3 neutropenia > Grade 3 infection; any grade of thrombocytopenia associated with clinically significant bleeding; Grade 4 thrombocytopenia; or Grade 4 anemia assessed as unrelated to disease, disease progression, epidemic disease, or concomitant medications; and determined by the investigator to be “probably related,” “possibly related,” or “definitely related” to the administration of ONC201.

Safety assessment

Safety assessments, including complete blood cell counts, serum chemistry, and toxicity, were performed at baseline, then during the first two cycles, followed by every three weeks. Electrocardiographic monitoring was performed prior to ONC201 administration, and then at 15 minutes, 1 hour, and 2 hours post-administration. Adverse events were graded using CTCAE version 4.0. Tumor response was assessed using RECIST every two cycles.

Pharmacokinetic analysis

Plasma samples for PK were collected at baseline, 30 minutes, 2 hours, 4 hours, 6 hours, 24 hours, 48 hours, and 168 hours after the first dose of ONC201 and before the first dose during the 2–6 week treatment period. PK was analyzed in human plasma by LC-MS/MS using a validated GLP method to detect ONC201. PK analysis was performed using version 6.3 (St. Louis, Missouri).

Statistical analysis

Descriptive statistics are used to analyze safety and tumor response data.

Pharmacodynamic analysis

PD blood samples were collected 6 hours after ONC201 treatment cycle 1, and on days 2, 3, 8, and 15, with dosage administered on the day of drug dosing in cycles 2 and 3. Lysed cytokeratin 18 (cCK18) was assessed using the M30 test, and serum levels of all cytokeratin 18 (CK18) were assessed using the M65 test (Perviva AB, Sweden). Other tumor-specific markers were also evaluated according to standards of care.

In vitro sensitivity of tumor type

The in vitro activity of ONC201 was evaluated in 1020 gene-annotated cell lines from the Cancer Drug Sensitivity Genome (http://www.cancerrxgene.org) collection. As previously described, IC50 values were determined at 72 hours post-treatment by cell viability assay. For each tumor type, the estimated IC50 values from multiple cell lines were averaged. Tumor types were divided into three distinct groups, representing the ternary rank of the average IC50 value. Based on their ternary rank in the ONC201 sensitivity spectrum, these groups are designated as “high,” “low,” and “intermediate” in Table 11.

result

Patient characteristics

During the dose escalation phase, 10 evaluable patients participated in this study. Patient characteristics are listed in Table 7. After the dose escalation phase was completed, another 10 patients participated in the ongoing expansion phase (Table 8).

Table 7. Demographics and safety experience of patients with ONC201, conducted every 3 weeks during dose escalation.

Table 8. Demographics and safety experience of patients during the expansion phase of ONC201 RP2D (625 mg every 3 weeks)

* indicates that the patient is still learning.

Dosage escalation method, determination of RP2D and safety

The dosage cohorts are listed in Table 9. 625 mg was the highest dose achieved and was identified as RP2D. The only adverse event possibly attributable to the dose escalation phase of ONC201 was a patient with hypothermia. One patient enrolled in the highest dose cohort was replaced due to rapid disease progression in cycle 1.

The only adverse event among the 10 patients in the dilation phase who may have experienced an episode attributable to ONC201 was vomiting in one patient. Both adverse events were Grade 1 and reversible rapidly. Laboratory and physical examinations revealed no drug-related abnormalities. Similarly, cardiovascular assessments showed no drug-related effects.

Table 9: Cohort of ONC201 Dosage Upgrades and Expansions Every 3 Weeks

Pharmacokinetics

Plasma collected at consecutive time points was used to analyze systemic exposure levels of ONC201 in patients (Figures 5A and 5B). PK parameters for all patients were determined and summarized for the top-dose cohort (Table 10). Although dose escalation was involved in individual patient cohorts, systemic exposure to ONC201, as determined by AUC and _Cmax, appeared to saturate at a dose of 375 mg (Figures 6A and 6B). For the top-dose cohort, the mean _Cmax was 3312 (SD 2133) ng/mL, occurring at a mean of 1.8 hours post-administration. The mean Vz was 381 (SD 164) L, consistent with a large volume of distribution. The mean AUC was 26.3 (SD 10.8) hg/mL, and the mean CL/F was 27.19 (SD 10.95) L/h. The mean t _1/2 was 9.62 (SD 1.76) hours.

Table 10. Mean pharmacokinetic parameters of ONC201 determined in patients receiving 625 mg ONC201 every three weeks.

Generally, CL/F was observed to be variable but consistent across all dose groups. There was no significant relationship between drug CL/F and patient sex and age. A slight trend was observed with patient weight and BSA. An overall increase in CL/F was observed due to increases in weight and BSA. Although a slight upward trend was observed, there was no strong correlation between CL/F and CL _CR .

A stronger correlation was observed between the volume of distribution (VZ) estimate and patient weight and BSA. A significant increase in VZ was observed with increasing patient weight. This trend predicted a VZ greater than 2-fold increase in weight from 50 kg to 100 kg. A similar trend was observed between VZ and BSA. The effect of dose-normalized exposure parameters on patient weight was further investigated. A trend of decreasing exposure with increasing weight was observed in the _Cmax /dose and AUC/dose versus patient weight plots. Weight-normalized CL/F versus dose plots showed a similar trend to the unnormalized CL/F, but significantly lower variability in the 625 mg dose group.

Patient response

Table 11 lists the patient outcomes of 10 evaluable patients enrolled in the dose escalation phase. Of the 10 evaluable patients, at least 2 cycles were completed, 4 completed at least 4 cycles, and 1 patient received 8 cycles and remained on treatment. On average, patients received 3.1 doses of ONC201. Of the 10 patients in the expansion phase, 6 are still on treatment.

Table 11. Pharmacodynamics of clinical response and dose escalation phases.

*MR-mixed response, SD stable disease, PD progressive disease. Tumor type in vitro sensitivity classification is described in the Methods section.

Clinical and laboratory results indicate that the drug is bioactive in treating patients. Patient #3, a 72-year-old patient with advanced clear cell endometrial cancer, showed a mixed response with multiple nodules decreasing by >30% as new nodules developed. Patient #4, a 62-year-old male with renal cell carcinoma and bone metastases, experienced weakness and pain in the clavicle, which was relieved. A 69-year-old patient with prostate adenocarcinoma experienced long-term stable disease and was studied for 27 weeks. Patient #8, a 71-year-old colon cancer patient with stable disease for 12 weeks, received 4 doses of ONC201.

Pharmacodynamics

Given the heterogeneity of tumor types among enrolled patients, no widely used biomarkers are available for uniformly measuring samples from all patients. In particular, serum M30 assays can detect cytokeratin-18 in a caspase-cleaved form during apoptosis, which is useful in studies of solid tumor heterogeneity, as most solid tumors express cytokeratin-18. M30 sandwich ELISAs have been widely used in clinical trials as biomarkers of cell death induced by different cancer chemotherapeutic agents in various solid tumor profiles. In addition to serum M30 assays, M65 sandwich ELISA assays are also used in clinical studies to differentiate tumor cell apoptosis from necrosis by detecting an increase in total cytokeratin-18 that may indicate tumor necrosis and disease progression.

As expected, patients with rapid clinical progression who were undergoing one cycle of the study showed an increase in M65, but not in the M30 assay. In contrast, patients who maintained the study through eight cycles showed an increase in M30, but not in the M65 assay. Four patients participating in the dose-escalation phase of the study were induced in the M30 assay after a single dose of ONC201, typically on day 21 post-treatment (Figures 7A and 7B). To further understand the correlation of the observed heterogeneous M30 induction, the in vitro sensitivity of tumor types identified from a large number of cell lines from the Cancer Protocols for Drug Sensitivity (GDSC) was compared with the in vitro sensitivity of patients in this study (Figure 8). Interestingly, these patients who experienced induction in M30 were also three patients with tumor types that had high in vitro sensitivity to ONC201 (Table 11).

Given the downstream induction of TRAIL by ONC201 in preclinical models, serum TRAIL levels were also quantified using an ELISA assay. Half of the patients experienced a moderate increase in serum TRAIL, which mostly peaked within the first 24 hours of drug administration.

discuss

This example is the first human study investigating the treatment of cancer with ONC201. The primary objective of this study was to determine the RP2D (recommended dose) of ONC201 administered orally every 3 weeks to patients with solid tumors who had exhausted all treatment options. As expected from the benign preclinical safety profile of ONC201, no drug-related grade >1 toxicity was observed at micromolar concentrations in any patient preclinical model. Due to the drug's excellent safety profile, the study allowed progression to the next dose level without requiring additional patient enrollment and completing a discrete design without accelerated titration. This study determined that 625 mg, every 3 weeks, as the RP2D, was non-toxic and achieved therapeutic plasma concentrations at this dose. This RP2D exceeded the saturation threshold observed at 375 mg, therefore no adjustment of body surface area was required to achieve consistently achievable target blood levels. The RP2D has been validated in the expansion phase, and an additional 10 patients can be safely evaluated.

Pharmacokinetic characteristics of ONC201 indicate that the orally administered drug is significantly and rapidly absorbed, as shown by the 1.8-hour average. Importantly, in the highest-dose group treated with RP2D, PK parameters such as Cmax and AUC exceeded those associated with NOAEL in the GLP toxicology studies. The observation of systemic exposure to ONC201 saturating at two dose levels below RP2D suggests absorption saturation. Since absorption saturation occurs at doses that produce well-tolerated therapeutic plasma concentrations, this may serve as a safety feature. These observations support the decision to discontinue further dose increases of ONC201 beyond RP2D, while providing a safety margin around the target dose.

Given that the primary endpoint of the study was based on clinical safety in a highly heterogeneous cohort of patients with aggressive cancer, it is noteworthy that some patients showed evidence of clinical benefit. These included a patient with treatment-resistant clear cell endometrial cancer exhibiting a mixed response, two patients who experienced relief of symptoms related to tumor presentation sites, and two patients with stable disease for >2 months (prostate and colon adenocarcinoma). In this clinical trial, treatment was discontinued after disease progression using RECIST criteria, which define it as a 20% increase in tumor size. The indications of antitumor activity and the absence of any meaningful side effects in this trial suggest that ONC201 can provide clinical benefit without inflicting the typical toxicities of anticancer therapy on patients.

Similar to preclinical findings, PD measurements using the M30 assay showed that the effect of ONC201 persisted in several patients. Serum TRAIL was induced in 2 patients; however, this assay was limited to detecting serum soluble TRAIL because biopsy was unavailable. The ONC201 PK profile and its sustained PD effect provide opportunities for staggered dosing in combination regimens, minimizing the risk of drug-drug interactions while maintaining synergistic biological activity. Synergistic effects between ONC201 and approved cancer therapies have been established using taxanes, bevacizumab, bortezomib, and sorafenib.

In summary, we have demonstrated that ONC201 is well tolerated in 625 mg of RP2D and shows signs of bioactivity in patients with advanced solid tumors.

Those skilled in the art will understand that changes can be made to the exemplary embodiments shown and described above without departing from their broad inventive concept. Therefore, it should be understood that the invention is not limited to the exemplary embodiments shown and illustrated, but is intended to cover modifications within the spirit and scope of the invention as defined by the claims. For example, specific features of the exemplary embodiments may or may not be part of the claimed invention, and features of the disclosed embodiments may be combined. Unless specifically stated herein, the terms "a," "an," and "the" are not limited to one element but should be considered as "at least one."

It should be understood that at least some of the drawings and descriptions of this invention have been simplified to focus on and clearly understand the elements relevant to this invention. Meanwhile, for clarity, other elements that a person skilled in the art will understand may also include a part of this invention. However, because these elements are well known in the art, and because they do not necessarily contribute to a better understanding of this invention, a description of these elements is not provided herein.

Furthermore, the specific order of steps should not be construed as a limitation on the claims, provided that the method does not depend on the order of the specific steps set forth herein. The claims relating to the method of the invention should not be limited to performing the steps in the written order, and those skilled in the art will readily understand that the steps may be varied while still remaining within the spirit and scope of the invention.

All references cited in this article, including publications, patent applications and patents, are incorporated herein by reference to the same extent that each reference is individually and specifically indicated to be incorporated by reference and elaborated throughout this article.

Claims (17)

1. Disalts of compounds having the following structures 2. The compound of claim 1, wherein the disalt is selected from: hydrochloride, hydrobromide, hydrogen sulfate, sulfate, phosphate, fumarate, succinate, oxalate, lactate, tartrate, nitrate, citrate, hydrogen tartrate, carbonate, malate, maleate, sulfonate, methanesulfonate, and carboxylates. 3. The compound of claim 1, wherein the disalt is a formate or an acetate. 4. The compound according to claim 1, wherein the disalt is selected from: p-toluenesulfonate, benzenesulfonate, citrate, methanesulfonate, oxalate, succinate, tartrate, fumarate, and maleate. 5. The compound according to claim 2, wherein the disalt is a dihydrochloride or a dihydrobromate. 6. The compound according to claim 2, wherein the disalt is a dihydrochloride. 7. A pharmaceutical composition comprising a compound according to any one of claims 1-6 and a pharmaceutically acceptable carrier. 8. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating cancer. 9. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating a disease selected from the group consisting of: desmoplastic round cell tumors, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, rectal cancer, appendiceal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, malignant fibrous histiocytoma, breast cancer, bronchial tumors, Burkitt lymphoma, spinal cord tumors, carcinoid tumors, embryonal tumors of the central nervous system, central nervous system lymphoma, cervical cancer, chordoma, chronic lymphocytic leukemia, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, Cezari syndrome, endometrial cancer, endothelioma, ependymoma, esophageal cancer, and Ewing sarcoma family of tumors. Extracranial germ cell tumors, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, head and neck cancer, histiocytosis, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liver cancer, lung cancer, Waldenström macroglobulinemia, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, plasmacytoma, nasal cavity cancer, paranasal sinus cancer, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, intermediately differentiated pineal medullary tumors, pineal cell tumors, supratentorial primitive neuroectodermal tumors, pituitary tumors, pleural pulmonary blastoma, prostate cancer, renal and pelvic cancer, ureteral cancer, cancers involving the NUT gene on chromosome 15, retinoblastoma, and rhabdomyosarcoma. 10. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating diseases selected from: renal cell carcinoma, hepatocellular carcinoma, gastrointestinal carcinoid, respiratory tract cancer, osteosarcoma, acute myeloid leukemia, chronic myeloid leukemia or brain tumor. 11. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating diseases selected from: germ cell tumors, malignant fibrous histiocytoma of bone and osteosarcoma, myelodysplastic syndromes, astrocytomas, basal cell nevus syndromes, extrahepatic cholangiocarcinoma or non-Hodgkin lymphoma. 12. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating a disease selected from the group consisting of chondrosarcoma, multiple myeloma, atypical malformation/striated sarcoma, Goring-nevus syndrome, or occult primary metastatic squamous cell carcinoma. 13. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating malignant fibrous histiocytoma of bone. 14. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating diseases selected from: AIDS-related lymphoma, colorectal cancer, ovarian germ cell tumors, ocular melanoma, myeloproliferative disorders, and oral cancer. 15. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating diseases selected from myeloproliferative neoplasms and low-grade ovarian tumors. 16. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating pancreatic cancer. 17. Use of the compound according to any one of claims 1-6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating primary central nervous system lymphoma, central nervous system atypical malformation/striated tumor, ovarian epithelial cancer, hypopharyngeal carcinoma, nasopharyngeal carcinoma or oropharyngeal carcinoma.