Therapeutic agents and uses thereof

Abstract

Provided are human therapeutic compositions including a compound comprising a plurality of fused polycyclic moieties and a linker moiety. In certain embodiments, the compound is a reaction product of an aldehyde and a harmine component. The compositions exhibit anti-cancer properties, particularly against lymphoma, leukemia, pancreatic, endometrial, ovarian, gastric, breast, renal, cervical, head and neck, and myeloma cell lines.

Description

BACKGROUND TECHNICAL FIELD

[0001] The present invention relates to chemotherapeutic agents for use in the treatment of humans, and in particular for use in the treatment of human cancers, and corresponding methods for treating humans with cancer or other diseases. The present invention further provides dosage forms and regimens for administration to human patients, and methods of formulating and administering such dosage forms to produce improved therapeutic outcomes. More particularly, the present invention relates to the administration of specific chemotherapeutic dosage forms (e.g., liquid mixtures, capsules, pellets, or tablets) that include compounds or agents having multiple fused polycyclic moieties connected or tethered by an appropriate linker. In certain embodiments, compounds having multiple beta-carboline component moieties and a single linker moiety are provided.

[0002] Description of the Related Art

[0003] Cancer is a general term for a large group of diseases that can affect any part of the body. Other terms used are malignant tumors and neoplasm. One defining feature of cancer is the rapid production of abnormal cells that grow beyond their usual boundaries, and that can then invade nearby parts of the body and spread to other organs. This process is called metastasis. Metastasis is the main cause of death from cancer.

[0004] The transformation of normal cells into tumor cells is a multistage process that typically requires years, often beginning with a precursor lesion that can eventually become malignant. These changes are the result of the interaction between a person's genetic makeup and three types of external factors:

[0005] • physical carcinogens, such as ultraviolet light and ionizing radiation

[0006] • chemical carcinogens, such as asbestos, components of tobacco smoke, aflatoxins (a food contaminant), and arsenic (a drinking water contaminant)

[0007] • biological carcinogens, such as infections by certain viruses, bacteria, or parasites.

[0008] Examples of some infections associated with certain cancers:

[0009] • Viruses: hepatitis B and liver cancer, human papillomavirus (HPV) and cervical cancer, and human immunodeficiency virus (HIV) and Kaposi sarcoma.

[0010] • Bacteria: Helicobacter pylori and stomach cancer.

[0011] • Parasites: schistosomiasis and bladder cancer.

[0012] Aging is another fundamental factor in the development of cancer. The incidence of cancer rises dramatically with age, most likely due to the accumulation of risk for specific cancers as one ages. As people age, the total risk accumulation combines with a tendency for cellular repair mechanisms to be less effective.

[0013] In low- and middle-income countries, smoking, drinking alcohol, low intake of fruits and vegetables, and chronic infection with hepatitis B virus (HBV), hepatitis C virus (HCV), and some types of human papillomavirus (HPV) are the main risk factors for cancer. Cervical cancer, caused by HPV, is the leading cause of cancer death among women in low-income countries. In high-income countries, smoking, drinking alcohol, and being overweight or obese are the main risk factors for cancer.

[0014] The most common ways of treating cancer are surgery, chemotherapy, and radiation therapy. All of these techniques have significant drawbacks in terms of side effects and patient discomfort. For example, chemotherapy can cause a significant drop in white blood cell count (neutropenia), a significant drop in red blood cell count (anemia), and a significant drop in platelet count (thrombocytopenia). This can result in pain, diarrhea, constipation, mouth sores, hair loss, nausea, and vomiting.

[0015] Biological therapy (sometimes called immunotherapy, biotherapy, or biologic response modifier therapy) is a relatively new addition to the family of cancer treatments. Biological therapy harnesses the body's immune system, either directly or indirectly, to fight cancer or to alleviate some of the side effects that can result from some cancer treatments.

[0016] Drug adverse events are common during chemotherapy involving multiple drug treatments, and in fact, drug-drug interaction related toxicities are one of the leading causes of hospitalization in the United States. Obach, R. S. “Drug-Drug Interactions: An Important Negative Attribute in Drugs.” Drugs Today 39.5 (2003): 308-338. In fact, one in five of all surveyed adults in the United States report a drug adverse reaction in any one month time period. Hakkarainen, K. M. et al. “Prevalence and Perceived Preventability of Self-Reported Adverse Drug Events - A Population-Based Survey of 7,099 Adults.” PLoS One 8.9 (2013): e73166. A large scale study of adults aged 57-85 found that 29% were taking more than five prescription drugs and nearly 5% were at risk for serious adverse drug-drug interactions. In the field of oncology, a review of over 400 cancer patients determined that 77% of patients taking drugs were considered to have a moderate risk of adverse drug-drug interactions and 9% had a serious risk of adverse drug-drug interactions. Ghalib, M. S. et al. “Alterations of Chemotherapeutic Pharmocokinetic Profiles by Drug-Drug Interactions.” Expert Opin. Drug Metabl. Toxicol 5.2 (2009): 109-130.

[0017] Such interactions are a global health problem and the WHO has identified adverse drug interactions as a major cause of morbidity and mortality worldwide, with up to 7% of all hospitalizations in the United States due to adverse drug interactions. A recent survey of one hospital showed that 83% of inpatients were taking a combination of prescription drugs that could cause adverse reactions. Patel, P. S. et al. "A Study of Potential Adverse Drug-Drug Interactions Among Prescribed Drugs in a Medicine Outpatient Department of a Tertiary Care Teaching Hospital." J. Basic Clin. Pharm. 5.2 (2014): 44-48.

[0018] Examples of well-known adverse drug interactions include the development of rhabdomyolysis (a serious muscle disease) when taking simvastatin and amiodarone. As a result, the FDA introduced a warning on the drug labels about the interaction. The calcium channel blocker Mibefradil was removed from the market due to harmful interactions with drugs that affect heart electrical activity.

[0019] U.S. Patent No. 8,039.025 describes cancer treatment in the form of Arum palaestinum Boiss extract supplemented with separate amounts of beta-sitosterol, iso vanillin, and linoleic acid, and is incorporated by reference in its entirety herein.

[0020] U.S. Patent No. 9,402,834, issued August 2, 2016, describes anticancer compositions comprising various components in mixtures, such as a mixture of curcumin, peganum harmala, and isovanillin components, or component mixtures comprising curcumin / peganum harmala, curcumin / isovanillin, and peganum harmala / isovanillin components.

[0021] Despite the large amount of research and efforts being conducted worldwide to curb the trend of cancer and its side effects, many of the clinical manifestations of the disease remain a huge problem. Therefore, any new cancer treatment that has the ability to affect the course of the disease and / or improve the symptoms of cancer and improve the patient's lifestyle is highly significant and important. SUMMARY

[0022] The present invention provides compositions useful as improved chemotherapeutic agents for the treatment of humans, and in particular, the treatment of human cancers, as well as corresponding methods for making such compositions and uses thereof. Generally, the chemotherapeutic agents of the present invention comprise (or consist essentially of, or consist of) one or more compounds and related forms thereof. Thus, as used herein in the specification and claims, a defined "therapeutic compound" or "compound" means the defined compound per se, as well as its dimers, isomers, tautomers, derivatives, solvates, metabolites, esters, metal complexes (e.g., Cu, Fe, Zn, Pt, V), prodrugs, and salts. Thus, a "dimer" refers to a molecule or molecular complex consisting of two identical molecules joined together by a bond, which can be strong or weak (e.g., covalent or hydrogen bond); an "isomer" refers to each of two or more compounds having the same molecular formula but different arrangements of atoms, and includes structural isomers and stereoisomers (e.g., geometric isomers and enantiomers); a "tautomer" refers to two or more equilibrating isomers of an isomeric pair, such as keto-enol and imine and enamine tautomers; a "derivative" refers to a compound that can be imagined to arise from the defined parent compound by the substitution of one atom for another atom or group of atoms, or actually synthesized therefrom; a "solvate" refers to the interaction of the defined compound with a solvent to form a stable solvate species; a "metabolite" refers to the defined compound metabolized in the body by digestion or other chemical processes; and a "prodrug" refers to the defined compound produced by metabolic processes. The compounds can be used directly in partially or substantially completely purified form, or can be modified as indicated above. The compounds can be in crystalline or amorphous form, and can be lyophilized.

[0023] The present invention also provides novel methods for treating cancer by administering an appropriate amount of a composition comprising a therapeutic compound as described herein. Thus, these compositions are specifically designed for use in treating cancer, and the compositions are useful in the manufacture of a medicament for anti-cancer therapeutic applications. Further, the present invention provides compositions for treating cancer comprising administering a therapeutically effective amount of the novel compositions, prepared by processes known per se with a pharmaceutically acceptable carrier.

[0024] As used herein, "chemotherapeutic," "chemotherapeutic agent," or simply "therapeutic agent" refers to one or more compounds described herein that are useful in the treatment of human disorders, particularly human cancers. Chemotherapeutics can have cytostatic, selective toxicity, or destructive properties on cancerous tissues and / or cells, including cancer stem cells, but also include non-discriminatory cytotoxic compounds for use in cancer treatment.

[0025] The therapeutic compounds or agents of the present application have been found to be effective in treating a variety of human cancer cells, and in particular, lymphoma, leukemia, pancreatic cancer, endometrial cancer, ovarian cancer, gastric cancer, breast cancer, kidney cancer, cervical cancer, head and neck cancer, and myeloma.

[0026] The compounds or agents of the present application broadly include multiple fused polycyclic moieties linked or tethered by appropriate linkers; preferably, two tricyclic moieties are present. Each polycyclic moiety includes at least one N-containing ring. Beta-carboline moieties are particularly useful in the present application, such as harmaline or similar moieties. In certain embodiments, a pair of beta-carboline moieties are bonded by a linker moiety, and in particular, by a single atom forming at least a portion of the entire linker. Reaction products of beta-carboline compounds and aldehyde compounds yield a number of useful anti-cancer compounds according to the present application. While the compounds of the present application, per se, are not part of an anti-cancer composition, and do not include compounds consisting of the reaction product of two harmaline moieties or two securinine moieties with a p-nitrobenzaldehyde benzaldehyde linker moiety, the anti-cancer compositions of the present application (which generally include at least one other agent, component or compound) and methods of treatment do include such compounds. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a plot of cell number versus amount of dose for the o-vanillin / harmaline compound (GZ523.001) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0028] Figure 2 is a plot of cell number versus amount of dose for the o-vanillin / harmaline compound (GZ523.002) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0029] Figure 3 is a plot of cell number versus amount of dose for the o-vanillin / harmaline compound (GZ523.003) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0030] Figure 4 is a plot of cell number versus amount of dose for the o-vanillin / harmaline compound (GZ523.004) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0031] Figure 5 is a plot of cell number versus amount of dose for the o-vanillin / harmaline compound (GZ523.005) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0032] Figure 6is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.006) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0033] Figure 7 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.007) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0034] Figure 8 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.008) showing its effect in inducing death of lymphoma cells, as described in Example 2:

[0035] Figure 9 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.001) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0036] Figure 10 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.002) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0037] Figure 11 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.003) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0038] Figure 12 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.004) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0039] Figure 13 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.005) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0040] Figure 14 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.006) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0041] Figure 15 is a plot of cell number versus dose number for the o-vanillin / harmine compound (GZ523.007) showing its effect in inducing death of leukemia cells, as described in Example 2:

[0042] Figure 16 is a plot of cell number versus dose quantity for the o-vanillin / harmine compound (GZ523.008) demonstrating its effect in inducing leukemia cell death as described in Example 2:

[0043] Figure 17 is a plot of cell number versus dose quantity for the composition comprising one or more high molecular weight dilignan compounds derived from the o-vanillin / harmine reaction demonstrating its effect in inducing lymphoma cell death as described in Example 3;

[0044] Figure 18 is a plot of cell number versus dose quantity for the composition comprising one or more high molecular weight dilignan compounds derived from the o-vanillin / harmine reaction demonstrating its effect in inducing leukemia cell death as described in Example 3;

[0045] Figure 19 is a plot of cell number versus dose quantity for the vanillin / harmine compound (GZ518.000) demonstrating its effect in inducing lymphoma cell death as described in Example 5;

[0046] Figure 20 is a plot of cell number versus dose quantity for the vanillin / harmine compound (GZ518.001) demonstrating its effect in inducing lymphoma cell death as described in Example 5;

[0047] Figure 21 is a bar graph demonstrating EC 50 values determined by treating a plurality of lymphoma cell lines with GZ523.006 as explained in Example 6;

[0048] Figure 22 is a bar graph depicting results of a caspase 3 / 7 assay comparing a plurality of lymphoma cell lines using GZ523.006, demonstrating the cytotoxic properties of GZ523.006 by inducing apoptosis;

[0049] Figure 23 is a plot demonstrating cell growth as a function of concentration of 518B562 compound in the MIA PaCa-2 cell proliferation assay described in Example 8;

[0050] Figure 24 is a plot demonstrating cell growth as a function of concentration of 518B562 compound in the ASPC-1 cell proliferation assay described in Example 8;

[0051] Figure 25 is a plot demonstrating cell growth as a function of concentration of 518B562 compound in the BxPC-3 cell proliferation assay described in Example 8;

[0052] Figure 26 is a plot showing cell growth as a function of concentration of 518B562 compound in the AN3CA cell proliferation assay described in Example 8;

[0053] Figure 27 is a plot showing cell growth as a function of concentration of 518B562 compound in the HEC- la cell proliferation assay described in Example 8;

[0054] Figure 28 is a plot showing cell growth as a function of concentration of 518B562 compound in the MDA-MB-231 cell proliferation assay described in Example 8;

[0055] Figure 29 is a plot showing cell growth as a function of concentration of 518B562 compound in the MDA-MB-468 cell proliferation assay described in Example 8;

[0056] Figure 30 is a plot showing cell growth as a function of concentration of 518B562 compound in the HCC70 cell proliferation assay described in Example 8;

[0057] Figure 31 is a plot showing cell growth as a function of concentration of 518B562 compound in the H1975 cell proliferation assay described in Example 8;

[0058] Figure 32 is a plot showing cell growth as a function of concentration of 518B562 compound in the H1650 cell proliferation assay described in Example 8;

[0059] Figure 33 is a plot showing cell growth as a function of concentration of 518B562 compound in the A2780 cell proliferation assay described in Example 8;

[0060] Figure 34 is a plot showing cell growth as a function of concentration of 518B562 compound in the A2780CP cell proliferation assay described in Example 8;

[0061] Figure 35 is a plot showing cell growth as a function of concentration of 518B562 compound in the RXF-393 cell proliferation assay described in Example 8;

[0062] Figure 36 is a plot showing cell growth as a function of concentration of 518B562 compound in the A498 cell proliferation assay described in Example 8;

[0063] Figure 37 is a graph showing cell growth as a function of concentration of 518B562 compound in the N87 cell proliferation assay described in Example 8;

[0064] Figure 38 is a graph showing cell growth as a function of concentration of 518B562 compound in the SiHA cell proliferation assay described in Example 8;

[0065] Figure 39 is a graph showing cell growth as a function of concentration of 518B562 compound in the FaDu cell proliferation assay described in Example 8;

[0066] Figure 40 is a graph showing cell growth as a function of concentration of 518B562 compound in the DOHH-2 cell proliferation assay described in Example 8;

[0067] Figure 41 is a graph showing cell growth as a function of concentration of 518B562 compound in the SU-DHL-4 cell proliferation assay described in Example 8;

[0068] Figure 42 is a graph showing cell growth as a function of concentration of 518B562 compound in the OCI-LY3 cell proliferation assay described in Example 8;

[0069] Figure 43 is a graph showing cell growth as a function of concentration of 518B562 compound in the JIM1 cell proliferation assay described in Example 8;

[0070] Figure 44 is a graph showing cell growth as a function of concentration of 518B562 compound in the KMM-1 cell proliferation assay described in Example 8;

[0071] Figure 45 is a graph showing cell growth as a function of concentration of 518B562 compound in the KMS-11 cell proliferation assay described in Example 8;

[0072] Figure 46 is a graph showing cell growth as a function of concentration of 518B562 compound in the KMS-27 cell proliferation assay described in Example 8;

[0073] Figure 47 is a graph showing cell growth as a function of concentration of 518B562 compound in the KMS-34 cell proliferation assay described in Example 8;

[0074] Figure 48is a plot showing cell growth as a function of concentration of 518B562 compound in the H929 cell proliferation assay described in Example 8;

[0075] Figure 49 is a plot showing cell growth as a function of concentration of 518B562 compound in the L363 cell proliferation assay described in Example 8;

[0076] Figure 50 is a plot showing cell growth as a function of concentration of 518B562 compound in the MM. Is cell proliferation assay described in Example 8;

[0077] Figure 51 is a plot showing cell growth as a function of concentration of 518B562 compound in the MOLP-8 cell proliferation assay described in Example 8;

[0078] Figure 52 is a plot showing cell growth as a function of concentration of 518B562 compound in the Jeko-1 parental cell proliferation assay described in Example 8;

[0079] Figure 53 is a plot showing cell growth as a function of concentration of 518B562 compound in the Jeko-1 lenalidomine resistant cell proliferation assay described in Example 8;

[0080] Figure 54 is a plot showing cell growth as a function of concentration of 518B562 compound in the Jeko-1 bortezomib resistant cell proliferation assay described in Example 8;

[0081] Figure 55 is a plot showing cell growth as a function of concentration of 560 compound in the MIA PaCa-2 cell proliferation assay described in Example 8;

[0082] Figure 56 is a plot showing cell growth as a function of concentration of 560 compound in the ASPC-1 cell proliferation assay described in Example 8;

[0083] Figure 57 is a plot showing cell growth as a function of concentration of 560 compound in the BxPC-3 cell proliferation assay described in Example 8;

[0084] Figure 58 is a plot showing cell growth as a function of concentration of 560 compound in the AN3CA cell proliferation assay described in Example 8;

[0085] Figure 59is a plot showing cell growth as a function of concentration of 560 compound in the HEC-1a cell proliferation assay described in Example 8;

[0086] Figure 60 is a plot showing cell growth as a function of concentration of 560 compound in the MDA-MB-231 cell proliferation assay described in Example 8;

[0087] Figure 61 is a plot showing cell growth as a function of concentration of 560 compound in the MDA-MB-468 cell proliferation assay described in Example 8;

[0088] Figure 62 is a plot showing cell growth as a function of concentration of 560 compound in the HCC70 cell proliferation assay described in Example 8;

[0089] Figure 63 is a plot showing cell growth as a function of concentration of 560 compound in the H1975 cell proliferation assay described in Example 8;

[0090] Figure 64 is a plot showing cell growth as a function of concentration of 560 compound in the H1650 cell proliferation assay described in Example 8;

[0091] Figure 65 is a plot showing cell growth as a function of concentration of 560 compound in the A2780 cell proliferation assay described in Example 8;

[0092] Figure 66 is a plot showing cell growth as a function of concentration of 560 compound in the A2780CP cell proliferation assay described in Example 8;

[0093] Figure 67 is a plot showing cell growth as a function of concentration of 560 compound in the RXF-393 cell proliferation assay described in Example 8;

[0094] Figure 68 is a plot showing cell growth as a function of concentration of 560 compound in the A498 cell proliferation assay described in Example 8;

[0095] Figure 69 is a plot showing cell growth as a function of concentration of 560 compound in the N87 cell proliferation assay described in Example 8;

[0096] Figure 70 is a plot showing cell growth as a function of concentration of 560 compound in the SiHA cell proliferation assay described in Example 8;

[0097] Figure 71 is a graph showing cell growth as a function of concentration of 560 compound in the FaDu cell proliferation assay described in Example 8;

[0098] Figure 72 is a graph showing cell growth as a function of concentration of 560 compound in the DOHH-2 cell proliferation assay described in Example 8;

[0099] Figure 73 is a graph showing cell growth as a function of concentration of 560 compound in the SU-DHL-4 cell proliferation assay described in Example 8;

[0100] Figure 74 is a graph showing cell growth as a function of concentration of 560 compound in the OCI-LY3 cell proliferation assay described in Example 8;

[0101] Figure 75 is a graph showing cell growth as a function of concentration of 560 compound in the JIM1 cell proliferation assay described in Example 8;

[0102] Figure 76 is a graph showing cell growth as a function of concentration of 560 compound in the KMM-1 cell proliferation assay described in Example 8;

[0103] Figure 77 is a graph showing cell growth as a function of concentration of 560 compound in the KMS-11 cell proliferation assay described in Example 8;

[0104] Figure 78 is a graph showing cell growth as a function of concentration of 560 compound in the KMS-27 cell proliferation assay described in Example 8;

[0105] Figure 79 is a graph showing cell growth as a function of concentration of 560 compound in the KMS-34 cell proliferation assay described in Example 8;

[0106] Figure 80 is a graph showing cell growth as a function of concentration of 560 compound in the H929 cell proliferation assay described in Example 8;

[0107] Figure 81 is a graph showing cell growth as a function of concentration of 560 compound in the L363 cell proliferation assay described in Example 8;

[0108] Figure 82 is a graph showing cell growth as a function of concentration of 560 compound in the MM.1s cell proliferation assay described in Example 8;

[0109] Figure 83 is a plot showing cell growth as a function of concentration of 560 compound in the MOLP-8 cell proliferation assay described in Example 8;

[0110] Figure 84 is a plot showing cell growth as a function of concentration of 560 compound in the Jeko-1 parental cell proliferation assay described in Example 8;

[0111] Figure 85 is a plot showing cell growth as a function of concentration of 560 compound in the Jeko-1 lenalidomide-resistant cell proliferation assay described in Example 8;

[0112] Figure 86 is a plot showing cell growth as a function of concentration of 560 compound in the Jeko-1 bortezomib-resistant cell proliferation assay described in Example 8;

[0113] Figure 87 is a plot showing cell growth as a function of concentration of 561 compound in the MIA PaCa-2 cell proliferation assay described in Example 8;

[0114] Figure 88 is a plot showing cell growth as a function of concentration of 561 compound in the ASPC-1 cell proliferation assay described in Example 8;

[0115] Figure 89 is a plot showing cell growth as a function of concentration of 561 compound in the BxPC-3 cell proliferation assay described in Example 8;

[0116] Figure 90 is a plot showing cell growth as a function of concentration of 561 compound in the AN3CA cell proliferation assay described in Example 8;

[0117] Figure 91 is a plot showing cell growth as a function of concentration of 561 compound in the HEC-1a cell proliferation assay described in Example 8;

[0118] Figure 92 is a plot showing cell growth as a function of concentration of 561 compound in the MDA-MB-231 cell proliferation assay described in Example 8;

[0119] Figure 93 is a plot showing cell growth as a function of concentration of 561 compound in the MDA-MB-468 cell proliferation assay described in Example 8;

[0120] Figure 94is a graph showing cell growth as a function of concentration of 561 compounds in the HCC70 cell proliferation assay described in Example 8;

[0121] Figure 95 is a graph showing cell growth as a function of concentration of 561 compounds in the H1975 cell proliferation assay described in Example 8;

[0122] Figure 96 is a graph showing cell growth as a function of concentration of 561 compounds in the H1650 cell proliferation assay described in Example 8;

[0123] Figure 97 is a graph showing cell growth as a function of concentration of 561 compounds in the A2780 cell proliferation assay described in Example 8;

[0124] Figure 98 is a graph showing cell growth as a function of concentration of 561 compounds in the A2780CP cell proliferation assay described in Example 8;

[0125] Figure 99 is a graph showing cell growth as a function of concentration of 561 compounds in the RXF-393 cell proliferation assay described in Example 8;

[0126] Figure 100 is a graph showing cell growth as a function of concentration of 561 compounds in the A498 cell proliferation assay described in Example 8;

[0127] Figure 101 is a graph showing cell growth as a function of concentration of 561 compounds in the N87 cell proliferation assay described in Example 8;

[0128] Figure 102 is a graph showing cell growth as a function of concentration of 561 compounds in the SiHA cell proliferation assay described in Example 8;

[0129] Figure 103 is a graph showing cell growth as a function of concentration of 561 compounds in the FaDu cell proliferation assay described in Example 8;

[0130] Figure 104 is a graph showing cell growth as a function of concentration of 561 compounds in the DOHH-2 cell proliferation assay described in Example 8;

[0131] Figure 105 is a graph showing cell growth as a function of concentration of 561 compounds in the SU-DHL-4 cell proliferation assay described in Example 8;

[0132] Figure 106is a graph showing cell growth as a function of concentration of 561 compounds in the OCI-LY3 cell proliferation assay described in Example 8;

[0133] Figure 107 is a graph showing cell growth as a function of concentration of 561 compounds in the JIM1 cell proliferation assay described in Example 8;

[0134] Figure 108 is a graph showing cell growth as a function of concentration of 561 compounds in the KMM-1 cell proliferation assay described in Example 8;

[0135] Figure 109 is a graph showing cell growth as a function of concentration of 561 compounds in the KMS-11 cell proliferation assay described in Example 8;

[0136] Figure 110 is a graph showing cell growth as a function of concentration of 561 compounds in the KMS-27 cell proliferation assay described in Example 8;

[0137] Figure 111 is a graph showing cell growth as a function of concentration of 561 compounds in the KMS-34 cell proliferation assay described in Example 8;

[0138] Figure 112 is a graph showing cell growth as a function of concentration of 561 compounds in the H929 cell proliferation assay described in Example 8;

[0139] Figure 113 is a graph showing cell growth as a function of concentration of 561 compounds in the L363 cell proliferation assay described in Example 8;

[0140] Figure 114 is a graph showing cell growth as a function of concentration of 561 compounds in the MM.1s cell proliferation assay described in Example 8;

[0141] Figure 115 is a graph showing cell growth as a function of concentration of 561 compounds in the MOLP-8 cell proliferation assay described in Example 8;

[0142] Figure 116 is a graph showing cell growth as a function of concentration of 561 compounds in the Jeko-1 parental cell proliferation assay described in Example 8;

[0143] Figure 117 is a graph showing cell growth as a function of concentration of 561 compounds in the Jeko-1 lenalidomide-resistant cell proliferation assay described in Example 8;

[0144] Figure 118 is a graph showing cell growth as a function of concentration of 561 compound in the Jeko-1 bortezomib-resistant cell proliferation assay described in Example 8;

[0145] Figure 119 is a graph showing cell growth as a function of concentration of 560 compound identified in the S2-007 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

[0146] Figure 120 is a graph showing cell growth as a function of concentration of 560 compound identified in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

[0147] Figure 121 is a graph showing cell growth as a function of concentration of 562 compound identified in the S2-007 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

[0148] Figure 122 is a graph showing cell growth as a function of concentration of 562 compound identified in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell proliferation assay described in Example 15;

[0149] Figure 123 is a series of photographs depicting colony formation as a function of concentration of 560 compound identified in the S2-007 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

[0150] Figure 124 is a series of photographs depicting colony formation as a function of concentration of 560 compound identified in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

[0151] Figure 125 is a series of photographs depicting colony formation as a function of concentration of 562 compound identified in the S2-007 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

[0152] Figure 126 is a series of photographs depicting colony formation as a function of concentration of 562 compound identified in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

[0153] Figure 127 is another series of photographs depicting colony formation as a function of concentration of 562 compound identified in the MiaPaCa-2 pancreatic ductal adenocarcinoma cell colony formation assay described in Example 16;

[0154] Figure 128 is a set of bar graphs showing the results of cell cycle assays using the identified 560 compounds with S2-007 cells over 24 and 48 hours, as described in Example 17, with Sub G0;

[0155] Figure 128A is a set of bar graphs showing the results of cell cycle assays using the identified 560 compounds with S2-007 cells over 24 and 48 hours, as described in Example 17, without Sub G0;

[0156] Figure 129 is a set of bar graphs showing the results of cell cycle assays using the identified 560 compounds with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0;

[0157] Figure 129A is a set of bar graphs showing the results of cell cycle assays using the identified 560 compounds with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0;

[0158] Figure 130 is a set of bar graphs showing the results of cell cycle assays using the identified 562 compounds with S2-007 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0;

[0159] Figure 130A is a set of bar graphs showing the results of cell cycle assays using the identified 562 compounds with S2-007 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0;

[0160] Figure 131 is a set of bar graphs showing the results of cell cycle assays using the identified 562 compounds with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, with Sub G0; and

[0161] Figure 131A is a set of bar graphs showing the results of cell cycle assays using the identified 562 compounds with MiaPaCa-2 cells over 24, 48, and 72 hours, as described in Example 17, without Sub G0. DETAILED DESCRIPTION

[0162] The therapeutic agents of the present application are used in therapeutically effective amounts, i.e., amounts that will elicit a biological or medical response of the tissue, system, or subject sought to be affected, and in particular, for a variety of human diseases, and in particular cancer, elicit some desired therapeutic effect; in the case of cancer, these agents work by preventing and / or inhibiting the proliferation and / or survival of cancer cells (including cancer stem cells) and / or by slowing the progression of the cancer. Those skilled in the art recognize that an amount can be considered therapeutically effective even if the condition is not completely eradicated or prevented, but is ameliorated or alleviated in part in the subject, or its symptoms and / or effects. Of course, the appropriate composition of the agents herein and the dosing regimen in which such agents are used will depend on the particular cancer being treated, the extent of the disease, and other factors relating to the patient as determined by one skilled in the art. Thus, the terms "therapeutic" or "treatment" as used herein refer to a product or process according to the present application that is intended to result in a beneficial alteration of an existing condition (e.g., cancerous tissue, tumor size, metastasis, etc.) in a subject, such as by reducing the severity of clinical symptoms and / or the impact of the condition, and / or reducing the duration of symptoms / impact in the subject.

[0163] The chemotherapeutic agents of the present application can include additional ingredients for administration to a subject. Such additional ingredients include other active agents, preservatives, buffers, salts, carriers, excipients, diluents, or other pharmaceutically acceptable ingredients. Active agents that can be included in the compositions include antiviral, antibiotic, or other anticancer compounds; the latter can include the compounds described in PCT Application Serial No. PCT / US2015 / 055968, such as curcumin, hashishol, and isovanillin, as well as metabolites, dimers, derivatives, isomers, enantiomers (both D and L), tautomers, esters, complexes, and salts of any of the foregoing.

[0164] The therapeutic agents of the present application give rise to significant and unexpected therapeutic results, particularly in the context of anticancer results. In use, a therapeutically effective amount of an agent or composition according to the present application is administered to a subject in need thereof. This can include a single unit dose, or more typically, lower doses administered over a period of time (e.g., daily).

[0165] The dosage can be administered in any convenient way, such as by oral, rectal, nasal, ocular, parenteral (including intraperitoneal, intragastric, intrathecal, intravenous, dermal (e.g., transdermal patch), subcutaneous (e.g., injection or implantation), or intramuscular) administration. The dosage forms of the present application can be in the form of a liquid, gel, suspension, solution, or solid (e.g., tablet, pill, or capsule). Further, a therapeutically effective amount of an agent of the present application can be co-administered with another chemotherapeutic agent or agents, wherein the two products are administered substantially simultaneously or in any order.

[0166] The dosage level of the compositions of the present application is very variable with factors such as the age, body weight, physical condition, type of disease(s) being treated (e.g., one or more particular cancers) and severity of the disease(s) in the patient. However, generally speaking, regardless of the dosage form or route of administration employed, such as liquid solutions or suspensions, capsules, pills, or tablets, via oral, parenteral, or injection, the compositions should be administered at about 5 to 2000 mg per day, and more usually about 100-800 mg per day. Such dosages can be given on a once-a-day basis, but more usually are given in divided doses.

[0167] Additional advantages of various embodiments of the application will be apparent from a review of the disclosure and the working examples that follow. It should be understood that the various embodiments described herein are not necessarily mutually exclusive. For example, features described or depicted in one embodiment can also be included in other embodiments, but are not necessarily included. Accordingly, the present application encompasses various combinations and / or permutations of the specific embodiments described herein.

[0168] As used herein, the phrase "and / or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or not containing components A, B, and / or C, the composition can contain or not contain: only A; only B; only C; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0169] This specification also uses numerical ranges to quantify certain parameters associated with the various embodiments of the application. It should be understood that when a numerical range is provided, such range is to be interpreted as providing literal support for every number within the range. For example, the disclosure of a range of 1 to 10 should be interpreted to provide literal support for the range from 1 to 10 inclusive of each integer in the range. In other words, the disclosure of a range of 1 to 10 provides literal support for the range from 1 to 10 inclusive of every possible integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10). The same applies to any range that is provided in this specification.

[0170] As used herein, a pharmaceutically acceptable salt of a therapeutic compound of the present application means a salt of a compound that is acceptable for use in pharmaceutical compositions, which is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and that is acceptable for use in humans and in a desired degree of pharmacological activity. Such pharmaceutically acceptable salts include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-l-carboxylic acid), 4-methyldicyclo[2.2.2]oct-2-ene-l-carboxylic acid, acetic acid, aliphatic mono- and di-carboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucametacin acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, Mandela acid, methanesulfonic acid, mucic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, t-butylacetic acid, trimethylacetic acid and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It will be recognized that the particular anion or cation that is part of the salt of any compound of the present application is not critical, so long as the salt as a whole is pharmacologically acceptable, and that the pharmaceutically acceptable salts of the compounds of the present application are meant to also include the zwitterionic forms in which the acidic proton has been replaced by a cation such as Na+, K+, or Ca2+. Additional examples of pharmaceutically acceptable salts and their preparation methods and uses are presented in Handbook of Pharmaceutical Salts Properties, and Use, P. H. Stahl & C. G. Wermuth eds., ISBN 978-3-90639-058-1 (2008).

[0171] In preparing compounds of the present application, it can be necessary to protect reactive functional groups, such as carboxylic acid groups, to prevent side reactions. Conventional protecting groups and the methods for protection and deprotection can be found in Protective Groups in Organic Synthesis, 3rdEd., Greene and Wuts, John Wiley & Sons, 1999. The protecting groups can be removed at the appropriate time, usually using methods known in the art.

[0172] Accordingly, preferred starting compounds or components of the present application are either synthetically derived or derived from one or more naturally occurring products that have been significantly modified to comprise at least about 90% by weight (more preferably at least about 98% by weight) of the desired component. As used herein, "synthetically derived" means that the component in question is synthesized using specific starting ingredients and one or more chemical and / or biological reactions to obtain a substantially pure compound. Modification of naturally occurring products can include extraction or any other physical or chemical step to obtain the desired end product.

[0173] As used herein, the terms "alkyl," "alkenyl," "alkynyl" mean and are intended to encompass straight chain, branched chain, and cyclic groups. "Amine" means and is intended to encompass primary, secondary, and tertiary amines. "Sulfur groups" means and is intended to encompass thiols, sulfides, disulfides, and sulfoxides. "Derivative" means and is intended to encompass compounds, moieties, and / or groups that are substituted with atoms, groups, or side chains that do not substantially degrade the performance of the compound, moiety, or group as compared to its unsubstituted version (e.g., degradation of no more than about 20%, preferably no more than about 10%).

[0174] As indicated, certain preferred compounds or agents of the present application include a pair of fused polycyclic moieties, each comprising an N-containing ring, wherein the fused polycyclic moieties are bound or linked by a single tether or linker moiety, which is schematically illustrated as

[0175] PCM1— L— PCM2

[0176] wherein PCM1 and PCM2 are fused polycyclic moieties (which can be the same or different), wherein L is a tether or linker. As indicated by this schematic, the linker L can be attached to PCM1 and PCM2 at any position on any of its rings, and the bonding sites for both PCM1 and PCM2 need not be the same. Fused polycyclic and linker moieties are described below.

[0177] Fused polycyclic compounds or moieties

[0178] The fused polycyclic moieties of the present application are derived or synthesized from starting ingredients, which result in compounds or moieties having the following general structure:

[0179]

[0180] where one of the terminal rings is a 6-membered ring that includes at least one N heteroatom located at any valence-allowed position around the 6-membered ring (the single N atom shown in Structure I is exemplary only in terms of the location of the N atom and the number of N atoms). The 6-membered ring can be aryl in nature (e.g., a pyridino ring) or non- aryl (e.g., a piperidino ring), or include multiple N atoms (e.g., a piperazino ring). Further, with respect to Structure I, RG1 is fused to the terminal 6-membered N-containing ring and has 5-8 atoms (as used herein, "fused" refers to the fact that the fused rings share 2 adjacent atoms or in other words, 1 covalent bond). Ring RG2 can be absent (i.e., the moiety is bicyclic), and if present, ring RG2 is fused to ring RG1 and the terminal six-membered ring, and has 5-8 atoms. In RG1, RG2 (if present), and the six-membered N-containing ring, the majority of the ring atoms in each case are carbon atoms. However, these rings can also include one or more heteroatoms, such as S, O, or N.

[0181] The internal dashed lines shown in RG1 and RG2 represent the fact that the individual rings can have one or more double bonds and can be aryl or non- aryl in nature. As shown, in the six-membered N-containing ring, one or more N atoms can be located at any allowed position around the ring, and the dashed lines represent that the six-membered ring can have 1, 2, or 3 double bonds. The n subscript on each Y1 represents the fact that there can be a single or multiple substituents at any allowed position around the six-membered N-containing ring RG1 and / or RG2; preferably, each n is independently 1, 2, or 3. Y1 and Y2 are each independently selected from the group consisting of absent, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and carboxaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, where any of the above mentioned can be substituted with N, S, O, B, or a halogen atom.

[0182] Exemplary bicyclic compounds corresponding to moieties according to Structure I can include quinoline and its derivatives, purine and its derivatives, and quinoline-2-amine, 6-bromo-2-methylquinoline, 2-hydroxy-4-methylquinoline, 4-chloro-7-methoxyquinoline, 8-quinolineboronic acid, quinoxaline, 8-aminoisoquinoline, 5-chloro-3-methylbenzothiophene, 4-nitroquinoline-N-oxide, 1-methylisoquinoline, 7-methylquinoline, 6,7-dimethoxyquinazoline-2,4-dione, 6-chloroquinoline, 1-chloroisoquinoline, 4-chloroquinoline, 8-chloroquinoline, isoquinolone, 8-hydroxyquinoline-5-sulfonic acid, isoquinoline N-oxide, 6-fluoroquinaldine, 2-chloroquinoline-3-carboxaldehyde, 5-nitroisoquinoline, 2,6-dimethylquinoline, 3-hydroxyquinoline, 2-methyl-6-quinolinecarboxylic acid, 6-bromoisoquinoline, 8-mercaptoquinoline hydrochloride, quinoline-4-carboxylic acid, 6-bromoquinoline, 7-bromoquinoline, 6-nitroquinoline, decahydroquinoline, 4-hydroxyquinoline, 8-methylquinoline, 3-hydroxy-2-methyl-4-quinolinecarboxylic acid, 6-quinolinecarboxylic acid, 3-quinolinecarboxylic acid, 2-hydroxy-4-quinolinecarboxylic acid, 2,4-dimethylquinoline, 1-isoquinolinecarbonitrile, 7-chloro-2-methylquinoline, 1-methyl-3,4-dihydroisoquinoline, 4-methyl-6H,7H-thieno[3,2-c]pyridine, 7-methyl-4H,5H-thieno[2,3-c]pyridine, 1-ethyl-3,4-dihydroisoquinoline, 5-methyl-7,8-dihydro-1,6-naphthyridine, 1,4-dimethyl-3,4-dihydro-2,7-naphthyridine, 5-methyl-7,8-dihydro-1,6-naphthyridine, and 4-methyl-6,7-dihydrothieno[3,2-c]pyridine.

[0183] A variety of fused tricyclic compounds corresponding to moieties according to Structure I can also be used in the present application. One class of fused tricyclic compounds is the β-carboline compounds and derivatives thereof, having a tricyclic system, such as a bicyclic ring composed of a six-membered benzene ring and a fused five-membered pyrrole ring, wherein the terminal N-containing ring is fused to the intermediate pyrrole ring. Exemplary β-carbolines include tryptoline, pinoline, harmane, harmine, harmaline, tetrahydroharmaline, and 9-methyl-β-carboline. In some cases, harmaline is preferred for use in the present application.

[0184] Harmaline (7-methoxy-1-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole) is a fluorescent psychoactive alkaloid from the group of secale alkaloids and β-carbolines, and is found in various plants, such as Peganum harmala. Harmaline is identified as CAS# 304-21-2, and exists in two tautomeric forms:

[0185]

[0186] As used herein, "harmaline" refers to one or both tautomers. Other harmaline components are described below.

[0187] Some harmaline components have the structure

[0188]

[0189] where the numbered 6-membered fused ring is an N-heterocycle having a single N atom in any of positions 2-5, and the R6' substituent can be located in any ring position; R5' is H, OH, C1-C12 (preferably C1-C4) alkoxy, aryloxy (e.g., benzyloxy or phenoxy), carboxyl, biphenyl, nitro, carboxylate; and R6' is H, OH, C1-C12 (preferably C1-C4) alkyl, or C1-C12 (preferably C1-C4) carboxylic acid.

[0190] Such representative compounds include harmaline and the following:

[0191]

[0192] In the above representative compounds, any methoxy substituent can be replaced by a C2-C4 alkoxy group or a phenoxy group.

[0193] Linker compounds and moieties

[0194] Each linker L provides two bonded branches from a single atom that forms at least a portion of the linker moiety. Thus, the linker moiety can assume an effective "V" or "Y" configuration, with the single atom at the lower apex (as depicted in Structure II below), with the two bonded branches bonded to the fused polycyclic moiety, respectively. Thus, the linker can be a single methylene group (CH2), with the fused polycyclic moiety bonded to the carbon atom of the methylene group to assume an effective "V" configuration. In a similar manner, the linker can include a pair of alkyl groups with an intervening carbon atom, such as CH3-C-CH3, such that the fused polycyclic moiety is bonded to the intervening carbon atom. Thus, preferred linkers include multiple atoms, one of which is a bonded atom of the fused polycyclic moiety. The single atom of the linker can be selected from non-metals, and in particular, atoms of carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, chlorine, bromine, and iodine. Metal atoms, such as Pt, are generally less preferred. Moreover, the bond between the single atom and the bonded branches can be a classical covalent bond, meaning that each atom involved in the bond contributes at least one electron as part of a molecular orbital. However, typical metal bonds, such as coordination or dative bonds, are generally less favored.

[0195] It will be recognized that the linker compound or moiety serves to separate fused polycyclic moieties forming part of the compounds of the application, and can also contribute to the conformation and / or stereochemical features of the complete compound. As used herein, and consistent with conventional linker nomenclature, the entirety of any multi-atom linker moiety between fused polycyclic moieties is considered to be a "linker," without any artificial separation of such multi-atom linker moieties, wherein one atom of the linker moiety is considered to be a "linker," and the remainder of the linker moiety is not considered to be part of the "linker." For example, if a propyl moiety is used as a linker moiety, with two fused polycyclic moieties bonded to the terminal carbons of the propyl moiety, it would be inappropriate and inconsistent with the present application to consider one of the terminal CH2of the methylene group as a linker while ignoring the presence of the remaining CH2-CH2group as part of the linker.

[0196] In certain preferred compounds of the application, the fused tricyclic moiety is bonded to the linker by a single atom, and wherein this single atom is a carbon atom of a methine group. The "methine group" is defined by the Illustrated Glossary of Organic Chemistry as a part of a molecular structure equivalent to a methane minus three hydrogen atoms, i.e., a CH group. The methine group is contrasted with the "methylene group" or CH2group, which is defined as a part of a molecular structure equivalent to a methane minus two hydrogen atoms.

[0197] Some linker moieties can be derived from an aldehyde, wherein both of the fused polycyclic moieties are bonded to the carbonyl carbon of the aldehyde functional group, thereby assuming an effective "Y" configuration. In certain embodiments, a suitable aldehyde linker moiety is characterized by a six-membered ring with an attached aldehyde functional group, which has the following structure.

[0198]

[0199] wherein the substituents can be located anywhere around the ring. R1' is a C1-C12 (preferably C1-C4) aldehyde, R2'-R5' are independently and optionally selected from the group consisting of H, OH, C1-C12 (preferably C1-C4) alkyl groups, C2-C12 (preferably C2-C5) alkenyl groups, C1-C12 (preferably C1-C4) alkoxy groups, C1-C12 (preferably C1-C4) aldehyde groups, acetate groups, isobutyrate groups, phenyl groups, phenoxy groups, benzyloxy groups, C2-C12 (preferably C2-C6) alkyl esters, halogens (e.g., F, Br, I, Cl), primary and secondary amines, nitro groups, and mixtures thereof. The dashed bond line in the six-membered ring indicates that the six-membered ring can be a cyclohexane, or have one, two, or three carbon-carbon double bonds (e.g., a cyclohexene, a cyclohexadiene, a benzene, or a derivative thereof).

[0200] Representative compounds of this class include vanillin, benzaldehyde, cinnamaldehyde, cuminic aldehyde, o-vanillin, perillic aldehyde, cyclohexanecarboxaldehyde, and the following:

[0201]

[0202]

[0203] Still other phenyl aldehydes useful as linkers in the present application include moieties of 2-methoxybenzaldehyde, 3-ethoxy-4-hydroxybenzaldehyde, 4- formyl-2-methoxyphenyl isobutyrate, 3,4-dimethoxybenzaldehyde, 4-hydroxy-3- methoxy-5-nitrobenzaldehyde, 4-formyl-2-methoxyphenyl acetate, 3-hydroxy-5- methoxybenzaldehyde, 2-hydroxy-4-methoxybenzaldehyde, 3-chloro-4-hydroxy-5- methoxybenzaldehyde, 4-(benzyloxy)-3-methoxybenzaldehyde, 3-hydroxy-4,5- dimethoxybenzaldehyde, 3-bromo-4-hydroxy-5-methoxybenzaldehyde, 2-bromo-3- hydroxy-4-methoxybenzaldehyde, 3-hydroxy-2-iodo-4-methoxybenzaldehyde, 3- methoxybenzaldehyde, 3-phenoxybenzaldehyde, 4-phenoxybenzaldehyde, [1,1'- biphenyl]-3-carboxaldehyde, 4-fluoro-3-phenoxybenzaldehyde, 3-fluorobenzaldehyde, 4-fluorobenzaldehyde, 3,5-difluorobenzaldehyde, 2,4,5-trifluorobenzaldehyde, 2,3,4,5,6-pentafluorobenzaldehyde, 4-methylbenzaldehyde, terephthaldehyde, 4- chlorobenzaldehyde, 4-(prop-1-en-2-yl)cyclohex-1-en-1-carboxaldehyde, 4- isopropylbenzaldehyde, and cyclohexanecarboxaldehyde.

[0204] In other embodiments, aliphatic or alkenyl aldehydes can be used as linkers. Typically, such linkers are moieties of aldehydes, such as C1-C12alkyl or C2-C12alkenyl aldehydes, and include representative compounds such as (E)-hex-2-enal (C6H10O, exact mass: 98.07), 3-methylbutanal (isovaleraldehyde) (C5H10O, exact mass: 86.07), 3,7-dimethyloct-6-enal (citronellal) (C10H18O, exact mass: 154.14), 7-hydroxy-3,7-dimethyloctanal (hydroxycitronellal) (C10H20O2, exact mass: 172.15), and lauric aldehyde (C12H24O, exact mass: 184.18).

[0205] Complete compounds of the present application

[0206] As previously mentioned, one general form of the compounds is listed in the schematic

[0207] PCM1— L — PCM2.

[0208] Preferred species of this representation are listed in Structure II below

[0209]

[0210] As can be seen, the intermediate tether or linker L is bonded to a six-membered N-containing ring of the respective fused polycyclic moiety of Structure I, and in particular at the single atom forming the lower vertex and at least a portion of the linker L. The bonding site of the linker L to the fused polycyclic moiety can be at any allowed position around the six-membered ring, including at the N-heteroatom (in which case Y2would not be present), and such bonding sites need not be identical for the respective polycyclic moieties. The six-membered terminal N-containing ring, the RG1, RG2, Y1, and Y2substituents, and the value of n are those defined previously with respect to Structure I.

[0211] One class of compounds (Structure III below) has a central linker bonded to the B ring containing N at the ortho carbon atom relative to the nitrogen atom at the respective position, where each beta-carboline group can be independently substituted or unsubstituted. With respect to the first moiety ring and the beta-carboline groups, “substituted” means that they can be substituted at any position (and independently in the case of the respective beta-carboline groups) with any substituent that does not materially degrade (e.g., degrades by no more than about 20%, preferably no more than about 10%) the performance of the compound as compared to its unsubstituted version.

[0212] More particularly, certain other such compounds have the general structure III:

[0213]

[0214] wherein each of X1, X2, X3, and X9 is independently selected from the group consisting of absent, OH, C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and formaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, wherein any of the above recited can be substituted with N, S, O, B, or a halogen atom, Z comprises a single bonding atom as described above, and is selected from the group consisting of C1-C12 alkyl, alkenyl, and alkynyl groups, C1-C12 alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde and formaldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, wherein any of the above recited can be substituted with N, S, O, B, or a halogen atom. Each X3 can be attached at any position around the respective terminal phenyl moiety of the beta-carboline group. Each Y is independently absent (e.g., a direct bond exists between the two B rings, or Z can be directly coupled to one or both of the B rings), H, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, wherein any of the above recited can be substituted with N, S, O, B, or a halogen atom. Preferably, Y is a C1-C12 (preferably C1-C4) group composed of C, CH, and / or CH2 atoms or groups, and Z is C, CH, or CH2. X9 is preferably selected from the group consisting of absent (e.g., M can be bonded to Z), C1-C12 (preferably C1-C4) alkyl groups, and C2-C12 (preferably C2-C5) alkenyl groups. M is selected from the group consisting of Structure IIIA, absent, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, wherein any of the above recited can be substituted with N, S, O, B, or a halogen atom. Each X4, X5, X6, X7, and X8 of Structure IIIA is attached at any position around the A ring and is independently selected from the group consisting of absent, OH, C1-C12 (preferably C1-C4) alkyl, alkenyl, and alkynyl groups, C1-C12 (preferably C1-C4) alkoxy and alkoxyphenyl groups, aryl and aryloxy groups, aldehyde groups, amines, nitro groups, nitrile groups, C2-C6 carboxylic acid groups, boronic acid groups, sulfur groups, and amino acids, wherein any of the above recited can be substituted with N, S, O, B, or a halogen atom. The labels in the A ring refers to the fact that there can optionally be 0, 1, 2, or 3 double bonds (e.g., the A ring can be cyclohexane, cyclohexene, cyclohexadiene, benzene, or a derivative thereof), and wherein the label refers to the fact that there can optionally be 1) one or two non-fused double bonds, in one or both of the B rings at any valence-allowed position, such as in Figures a-d below; 2) a double bond between either or both of the B rings and Y or Z, with or without an additional non-fused double bond at any valence-allowed position around the corresponding N-containing ring, such as in Figures e-g below. In the case of 2), when there is a double bond between either nitrogen atom of the N-containing rings and Y or Z, X1can be absent, such as in Figures a-c and g. However, if there is no such nitrogen double bond, then the corresponding X1is as defined above, and is preferably selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as in Figures d-f; or 3) either or both of the B rings contain no non-fused double bonds, and each X1is as described above, and is preferably selected from the group consisting of H, OH, and C1-C12 (more preferably C1-C4) alkyl groups.

[0215] In preferred cases where Z is a methine CH group, X9is not absent or H.

[0216] Listed below are Figures depicting certain exemplary double bond configurations for either or both of the B rings of Structure III above.

[0217]

[0218] Advantageously, each X1is absent, each X2is H, and each X3is methoxy.

[0219] In certain embodiments of Structure III, M is a 1A ring, both X1are absent, both X3are methoxy, 2 of X4, X5, X6, X7, and X8are H, at least one of X4, X5, X6, X7, and X8is selected from the group consisting of H, -OH, methoxy, ethoxy, phenoxy, a C2-C5 alkenyl group, F, and Cl, with the provisos that: 1) when one or more of X4, X5, X6, X7, and X8is F or Cl, the remainder of X4, X5, X6, X7, and X8are all H; 2) only one of X4, X5, X6, X7, and X8can be phenoxy, and in such a case the remainder of X4, X5, X6, X7, and X8are all H.

[0220] In other embodiments, certain compounds are provided comprising two harmine moieties and a single phenyl moiety derived from a phenyl aldehyde compound, having the general structure IV:

[0221]

[0222] where X10is -CH=CH-, each of X11, X12, X13, and X14is independently selected from the group consisting of H, -OH, methoxy, ethoxy, and phenoxy, F, and Cl, with the provisos that: 1) at least one of X12, X13, or X14is H; 2) when one or more of X11, X12, X13, or X14is F or Cl, the remainder of X11, X12, X13, and X14are all H; 3) the phenoxy group is only present at X12, and X11, X13, and X14are all H, and 4) if a methoxy or ethoxy group is present, at least one such methoxy or ethoxy group must be at the 2 or 3 position, wherein the label refers to the fact that 1) one or both non-conjugated double bonds, at one or both of the N-containing rings, in one or both of the valence-allowed positions, such as in Figures a-c; 2) a double bond between either of the N-containing rings and the adjacent carbon atom, with or without an additional non-conjugated double bond at any valence-allowed position around the corresponding N-containing ring, such as in Figure g; or 3) either of the N-containing rings does not contain a non-conjugated double bond, and each X1is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, can optionally be present.

[0223]

[0224] When X10is absent, and X11, X12, X13, and X14are all H, the resulting structure is the 560 compound described in Example 12; when X10is absent, X11is H, X12is methoxy, X13is -OH, and X14is H, the resulting structure is the 562 compound described in Example 14; when X10is absent, X11is -OH, X12is methoxy, and X13and X14are both H, the resulting structure is the primary 523 compound described below; when X10is absent, X11, X13, and X14are all H, and X12is phenoxy, the resulting structure is the 594 compound described in Example 24; and when X10is -CH=CH-, and X11, X12, X13, and X14are all H, the resulting structure is the 561 diharmal compound listed below, as a primary compound of harmaline and cinnamaldehyde.

[0225] In other embodiments, certain compounds comprising two harmaline moieties and a single linker moiety within the scope of structure IV are provided. These compounds are selected from the group consisting of:

[0226]

[0227] Synthesis of complete compounds of the present invention

[0228] In preparing the compounds of the present application, it is preferred that the starting ingredients be of relatively high purity, typically at least about 90% pure by weight, and more preferably at least about 98% pure by weight. The use of naturally occurring sources of ingredients is generally not suitable or desirable, as these naturally occurring products can contain relatively small amounts of the desired component and / or have potentially interfering compounds therein. Moreover, the use of low purity ingredients generally results in little or no compound according to the present application.

[0229] Accordingly, preferred starting compounds or components of the present application are either synthetically derived or derived from one or more naturally occurring products that have been significantly modified to contain at least about 90% by weight (more preferably at least about 98% by weight) of the desired component. As used herein, "synthetically derived" means that the component in question is synthesized using a particular starting ingredient and one or more chemical and / or biological reactions to obtain a substantially pure compound. The modification of naturally occurring products can include extraction or any other physical or chemical step to obtain the desired end product.

[0230] One method of preparing the compounds of the present application, particularly where the linker moiety is derived from an aldehyde, involves a direct reaction between the aldehyde and the fused polycyclic compound of interest. Thus, the product produced by this method is the reaction product of the aldehyde and the fused polycyclic compound.

[0231] In carrying out the aldehyde reaction between any type of aldehyde and one or more fused polycyclic compounds, the weight ratio of the one or more aldehyde components to the one or more fused polycyclic compounds in the reaction mixture should range from about 0.5:1 to 25:1, more preferably from about 0.7:1 to 6:1, and most preferably from about 1.5:1 to 4:1. In terms of weight amounts, the amount of the one or more aldehyde components should range from about 25-95% by weight, and the weight amount of the one or more fused polycyclic compounds should range from about 5-75% by weight, the total weight of these reactants being 100% by weight. In most cases, it is preferred that the weight amount of the one or more aldehyde components be present in excess weight relative to the amount of the one or more fused polycyclic compounds.

[0232] These components are typically mixed with an organic solvent such as a C1-C4 lower alcohol (e.g., methanol, ethanol, or propanol) and / or dimethyl sulfoxide (DMSO), and allowed to stand at ambient pressure at a temperature ranging from about 20-60 °C for a period of time (typically from about 12 hours to 4 weeks). Alternatively, the mixture can be refluxed (e.g., refluxed in ethanol at 50-85 °C for 30 minutes to 2 hours, or in methanol at 55 °C for 30 minutes). The reaction product can then be recovered in either liquid or solid form. Depending on the solvent chosen, the reaction product can exhibit different colors, but this does not affect the anti-cancer properties of the reaction product. Moreover, the particular reaction conditions are generally not critical.

[0233] The production of effective esters, metal complexes, and pharmaceutically acceptable salts of the compounds is quite simple and within the skill in the art. For example, salts can be formed by reacting the product with an inorganic or organic acid.

[0234] The above reactants, reaction ratios, amounts of reactants, and reaction conditions apply to all aldehyde reactions according to the present application, and the skilled artisan can readily determine the optimal conditions through routine experimentation.

[0235] In some cases using aldehyde reactions, it can be difficult to determine the precise structure(s) of the reaction product. However, the molecular weight of the active reaction product can be determined, and this is an important criterion for the active product. Thus, an important reaction product of benzaldehyde and harmine has a molecular weight of approximately 516, while such a reaction product of vanillin and harmine has a molecular weight of approximately 562. By "approximately" in connection with the molecular weights referred to herein is meant the listed molecular weight plus or minus 5 weight units. Moreover, the molecular weight of a reaction product derivative (e.g., a reduced product produced by hydrogenation, ester, or salt) will be somewhat different; however, such weights are readily calculated depending on the nature of the derivative. Thus, the preferred molecular weights described herein are for the non-derivative version of the reaction product.

[0236] A second synthetic method can be used when it is desired to produce a fused tricyclic compound such as a β-carboline and its derivatives. In general, this method involves reacting an indole alkylamine with a diacid to produce an intermediate, followed by a ring closure reaction to produce the final compound of interest.

[0237] A third reaction method is shown below, particularly for producing compounds having a fused bicyclic moiety bonded to a linker moiety.

[0238]

[0239] Benzaldehyde / harmine 560 compound

[0240] Benzaldehyde is a benzene ring with an aldehyde substituent, and is a major component of bitter almond oil. It is identified by CAS # 100-52-7.

[0241]

[0242] The aldehyde reaction between benzaldehyde and harmine is preferably carried out by mixing the two components together in a weight ratio of about 2: 1 (benzaldehyde : harmine). Ethanol is then added to obtain a final reaction mixture concentration of about 10: 1000 mg / mL, more preferably about 700: 1000 mg / mL to form a dispersion. The vial is then capped and the mixture within the vial is allowed to stand in a warm water bath at about 50 °C (more broadly, about 40-60 °C) for about 3 days (more broadly, about 1-10 days). The solid compound is then washed with water and methanol to obtain a final product of about 90-95% purity by weight.

[0243] Certain compounds are formed from one harmine moiety and one benzaldehyde moiety, with one having a molecular weight of about 320. Other products also have one harmine moiety and one benzaldehyde, but with a molecular weight of about 302 due to water loss accompanying the reaction. These products are listed below.

[0244]

[0245] Additionally, useful compounds are formed from two harmine moieties and a single linker moiety derived from benzaldehyde, with a molecular weight of about 516, as follows.

[0246]

[0247] Other compounds comprising one benzaldehyde moiety and two harmine moieties include the following.

[0248]

[0249] As explained in Example 12, the confirmed compound is

[0250]

[0251]

[0252] Analogues of the above compound have a molecular weight of 520.68, and are reduced versions in which the nitrogen atoms of the two harmine moieties are hydrogenated, eliminating the double bonds therein, as listed below:

[0253]

[0254] More broadly, however, suitable benzaldehyde / harmine compounds include one or more of the following structures:

[0255]

[0256] and dimers, isomers, and tautomers thereof, wherein the -O-R3groups can independently be located anywhere on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of absent, H, OH, a C1-C12 (preferably C1-C4) alkyl group, and a halogen (such as I and Br), each R2is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, and a halogen (such as I and Br), each R3group is independently selected from the group consisting of a C1-C12 (preferably C1-C4) alkyl group, and a substituted or unsubstituted phenyl group, and wherein the notation refers to the fact that 1) one or both non-conjugated double bonds can optionally be present in one or both of the valence-allowable positions around either of the six-membered N-containing rings, such as in Figures a-d; 2) a double bond between either of the N-containing rings and the adjacent carbon of the central moiety, with or without an additional non-conjugated double bond at any valence-allowable position around the corresponding N-containing ring, such as in Figures e-g. In the case of a double bond between the nitrogen atom of either of the N-containing rings and its adjacent carbon atom, R1is absent, such as in Figures a-c and g. However, if there is no such nitrogen double bond, then the corresponding R1is selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, such as in Figures d-f; or 3) either or both of the B rings do not contain a non-conjugated double bond, and each R1is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group.

[0257] Listed below are diagrams depicting certain exemplary double bond configurations of either of the N-containing rings of Structure V.

[0258]

[0259] In other embodiments, the following compounds are useful

[0260]

[0261] wherein R7and R8are connected anywhere around the phenyl ring, and are independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkoxy group, and wherein preferably R7is OH and R8is a C1-C12 (preferably C1-C4) alkoxy group.

[0262] Cinnamaldehyde / harmalene 561 compounds

[0263] Cinnamaldehyde is found in the bark of the cinnamon tree and exists as cis and trans isomers. It is identified by CAS# 104-55-2.

[0264]

[0265] These compounds are produced using the aldehyde reaction in the same way as the benzaldehyde / harmaline product using the aldehyde reaction, and have molecular weights of approximately 346, 328, and 542, as shown below. The MW 542 compound includes a first cinnamaldehyde moiety, with two harmaline moieties bonded to the first moiety. The MW 346 compound is composed of a single cinnamaldehyde moiety and a single harmaline moiety, while the MW 328 product is a dehydrated version of the MW 346 product. The primary compound is the MW 542 product.

[0266]

[0267]

[0268] Primary:

[0269]

[0270] Likewise, analogs of the above primary structure are hydrogenated versions in which the N atoms of the two harmaline moieties are hydrogenated, eliminating the double bonds therein.

[0271] However, more broadly, suitable cinnamaldehyde / harmaline compounds include one or more of the following structures:

[0272]

[0273] and dimers, isomers, and tautomers thereof, wherein the -O-R3 groups can independently be located at any position on the terminal phenyl groups, wherein each R1 is independently selected from the group consisting of absent, H, OH, and a C1-C12 (preferably C1-C4) alkyl group, each R2 is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, each R3 group is independently selected from the group consisting of a C1-C12 (preferably C1-C4) alkyl group and a substituted or unsubstituted phenyl group, and wherein the labels refers to the fact that 1) one or both non-conjugated double bonds, located at one or both valence-allowed positions around either of the six-membered N-containing rings, such as Figures a-d below; 2) a double bond between either of the N-containing rings and the adjacent carbon of the central moiety, with or without an additional non-conjugated double bond at any valence-allowed position around the corresponding N-containing ring, such as Figures e-g. In the case of a double bond between the nitrogen atom of either N-containing ring and its adjacent carbon atom, R1is absent, such as Figures a-c and g. However, if there is no such nitrogen double bond, then the corresponding R1is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as Figures d-f; or 3) either or both of the N-containing rings do not contain a non-conjugated double bond, and each R1is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

[0274] Listed below are diagrams depicting certain exemplary double bond configurations of either or both of the N-containing rings of Structure VI.

[0275]

[0276] Vanillin / Harmine 562 Compounds

[0277] The aldehyde reaction between vanillin and harmine components proceeds in the same manner as the benzaldehyde / harmine reaction, resulting in the products listed below.

[0278]

[0279] As explained in Example 14, the confirmed vanillin / harmine compounds are

[0280]

[0281] Analogues of the above structures involving hydrogenation of the N-containing ring nitrogen atom and are listed below:

[0282]

[0283] However, more broadly, suitable vanillin / harmine compounds include one or more of the following structures:

[0284]

[0285] and dimers, isomers, and tautomers thereof, wherein each R4is independently selected from the group consisting of absent, H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R5is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, each R6group is independently located anywhere around the corresponding terminal phenyl group, or at either of the two open positions of the two N-containing rings, and is selected from the group consisting of C1-C12 (preferably C1-C4) alkoxy groups, H, OH, and substituted or unsubstituted phenyl groups, R7and R8are connected anywhere around the phenyl ring, and are independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkoxy groups, with the proviso that R7and R8are not both H, and wherein, preferably, R7is OH, R8is a C1-C12 (preferably C1-C4) alkoxy group, and each R9is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, and wherein the label refers to the fact that 1) zero, one, or two non-conjugated double bonds can optionally be present, in one or both of the valence-allowable positions around either or both of the six-membered N-containing rings, such as in Figures a'-d'; 2) a double bond between either or both of the N-containing rings and the adjacent carbon of the central moiety, with or without an additional non-conjugated double bond at any valence-allowable position around the corresponding N-containing ring, such as in Figures e'-g'. In the case of either of 2) where a double bond exists between the nitrogen atom of either N-containing ring and its adjacent carbon atom, R4is absent, such as in Figures a'-c' and g'. However, if no such nitrogen double bond is present, then the corresponding R4is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as in Figures d'-f'; or 3) either or both of the N-containing rings do not contain a non-conjugated double bond, and each R4is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

[0286] Listed below are Figures depicting certain exemplary double-bond configurations of either or both of the N-containing rings of Structure VII.

[0287]

[0288]

[0289] An exemplary compound consistent with 3) above is the hydrogenated form of the preferred 562 compound having the structure

[0290]

[0291] Phenoxybenzaldehyde / harmaline 594 compound

[0292] The primary 594 compound has the following structure as described in Example 24:

[0293]

[0294] O-vanillin / harmine 523 compound

[0295] The aldehyde reaction between o-vanillin and harmine is very diverse and the resulting products are equally variable. Four reaction schemes have been identified as potential candidates, as listed below.

[0296] Scheme 1 monomer:

[0297]

[0298] Scheme 1 dimer:

[0299]

[0300] Scheme 1 trimer:

[0301]

[0302] Scheme 2 monomer:

[0303]

[0304] Scheme 2 dimer:

[0305]

[0306] Scheme 2 dimer via mixed mechanism:

[0307]

[0308] Scheme 2 trimer:

[0309]

[0310] Scheme 3 monomer:

[0311]

[0312] Scheme 3 dimer:

[0313]

[0314] Scheme 3 trimer:

[0315]

[0316] Scheme 4

[0317]

[0318]

[0319] It will be observed that the above compound of Scheme 4 involves coupling between harmine and o-vanillin via the pyrrole nitrogen, i.e. the o-vanillin moiety is bonded to the nitrogen atom of the pyrrole ring forming part of the harmine.

[0320] The initial aldehyde reaction between the o-vanillin and harmine of Scheme 1 can also produce the following compound having the chemical formula C21H20N2O3 and a molecular weight of 348.15. It will be observed that in this case the initial reaction between the o-vanillin and harmine occurs at the cyclohexyl diene nitrogen atom.

[0321]

[0322] The preferred o-vanillin / diharmine compounds are described below:

[0323]

[0324] More generally however, the preferred o-vanillin / diharmine compounds are defined by the following structure VIII:

[0325]

[0326] and dimers, isomers and tautomers thereof, wherein the -O-R3groups can independently be located at any position on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of absent, H, OH and a C1-C12 (preferably C1-C4) alkyl group, each R2is independently selected from the group consisting of H, OH and a C1-C12 (preferably C1-C4) alkyl group, each R3group is independently selected from the group consisting of a C1-C12 (preferably C1-C4) alkyl group and a substituted or unsubstituted phenyl group, and wherein the label refers to the fact that 1) one or both non-conjugated double bonds, located at one or both valence-allowed positions around either of the six-membered N-containing rings, such as Figures a-d below; 2) a double bond between either of the N-containing rings and the adjacent carbon of the central moiety, with or without an additional non-conjugated double bond at any valence-allowed position around the corresponding N-containing ring, such as Figures e-g. In the case of a double bond between the nitrogen atom of either N-containing ring and its adjacent carbon atom, R1is absent, such as Figures a-c and g. However, if there is no such nitrogen double bond, then the corresponding R1is selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups, such as Figures d-f; or 3) either or both of the N-containing rings do not contain a non-conjugated double bond, and each R1is independently selected from the group consisting of H, OH, and C1-C12 (preferably C1-C4) alkyl groups.

[0327] Listed below are figures depicting certain exemplary double bond configurations of either or both of the N-containing rings of Structure VI.

[0328]

[0329] One particular aldehyde reaction for preparing o-vanillin-harmine compounds is to mix together solid particulate o-vanillin and harmine in a weight ratio of about 2: 1, and then add ethanol, DMSO, or a 90% ethanol / 10% DMSO mixture to the particulate. The dispersion is then stirred and allowed to stand at room temperature for 24 hours. The specific steps are: (1) mix together 500 mg of o-vanillin and 250 mg of harmine in a 15 mL bottle; (2) gently shake the bottle until a uniform powder mixture appears; (3) add 10 mL of ethanol and / or DMSO to the dry mixture; (4) stir with a vortex mixer at 1000 rpm for 10 minutes; and (5) allow the dispersion to stand, and the reaction to proceed at room temperature for 24 hours.

[0330] A similar technique involving a reaction between harmine and vanillin includes mixing together particulate harmine and vanillin in a weight ratio of about 2: 1 (vanillin: harmine), and then adding ethanol to a final concentration of about 10-100 mg / mL for the reaction. This mixture is then allowed to stand at 50°C for about 3 days. A blue-colored solid forms, which is filtered and washed with methanol and recovered.

[0331] Phenoxybenzaldehyde / harmine 594 compound

[0332] An aldehyde reaction between the phenoxybenzaldehyde and harmine components is carried out in the same manner as the benzaldehyde / harmine reaction, resulting in the products listed below.

[0333]

[0334] and dimers, isomers, and tautomers thereof, wherein the -O-R3groups can independently be located at any position on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of absent, H, OH, and a C1-C12 (preferably C1-C4) alkyl group, each R2is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, each R3group is independently selected from the group consisting of a C1-C12 (preferably C1-C4) alkyl group and a substituted or unsubstituted phenyl group, and the phenoxy groups can be substituted at any position on the benzyl ring, and wherein the label refers to the fact that 1) one or both non-conjugated double bonds, in one or both of the valence-allowed positions around the six-membered N-containing rings, such as in Figures a-d; or 2) a double bond between either of the N-containing rings and the adjacent carbon of the central moiety, with or without an additional non-conjugated double bond in any valence-allowed position around the corresponding N-containing ring, such as in Figures e-g, can optionally be present. In the case of a double bond between the nitrogen atom of either of the N-containing rings and its adjacent carbon atom, R1is absent, such as in Figures a-c and g. However, if there is no such nitrogen double bond, then the corresponding R1is selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group, such as in Figures d-f; or 3) either of the N-containing rings does not contain a non-conjugated double bond, and each R1is independently selected from the group consisting of H, OH, and a C1-C12 (preferably C1-C4) alkyl group.

[0335] Listed below are diagrams depicting certain exemplary double bond configurations of either of the N-containing rings of Structure VII.

[0336]

[0337] Listed below are particularly preferred compounds of this type, and reference is made to Example 24, in which 3-phenoxybenzaldehyde was used.

[0338]

[0339] Harmaline components

[0340] Some harmaline components are tricyclic compounds of this structure

[0341]

[0342] where the numbered 6-membered fused ring is an N-heterocycle having a single N atom in any of positions 2-5, and the R6substituent can be located in any ring position; R5' is H or C1-C12 (preferably C1-C4) alkoxy; and R6' is H, C1-C12 (preferably C1-C4) alkyl, or C1-C12 (preferably C1-C4) carboxylic acid.

[0343] Representative compounds of this type include harmine and the following:

[0344]

[0345] Pegamine hydrochloride dihydrate

[0346] In the representative compounds described above, any methoxy substituent can be replaced by a C2-C4 alkoxy group or a phenoxy group.

[0347] In addition to those described above and detailed in the examples below, a number of other aldehydes have been reacted with harmine to produce compounds. In each case, the reaction was carried out by mixing 500 mg of the aldehyde and 250 mg of harmine together in a 15 mL vial, then gently shaking the vial until a uniform powder mixture was present. To the dry mixture was then added 10 mL of ethanol, the vial was capped, and it was placed in a warm water bath at about 40 °C for about 24 hours.

[0348] In the table below, the specific aldehyde reacted with harmine is identified along with the resulting compound. For the latter, the composition of each compound is determined from the reactant portion thereof minus any water and / or hydrogen atoms lost due to the reaction, and its approximate molecular weight. For example, a given compound designated "H + A - H2O" refers to a product comprising one harmine moiety and one aldehyde moiety, minus one water molecule, while 2H + A - H2O - 2H refers to a product comprising two harmine moieties, one aldehyde moiety, minus one water atom, and minus two hydrogen atoms.

[0349]

[0350]

[0351]

[0352] Representative structures of some of the compounds listed in the table above, in which two harmine moieties are reacted with the aldehyde listed (in some cases, the aldehyde is identified by a different equivalent name), are as follows:

[0353]

[0354] 1,1'-(2-(2-methoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indol) C34H34N4O3 2-methoxybenzaldehyde, o-anisaldehyde

[0355]

[0356] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- ethoxyphenol C35H36N4O4 3-ethoxy-4-hydroxybenzaldehyde, ethylvanillin

[0357]

[0358] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- methoxyphenyl isobutyrate C38H40N4O5 4-formyl-2-methoxyphenyl isobutyrate, vanillin isobutyrate

[0359]

[0360] 1,1'-(2-(3,4-dimethoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indol) C35H36N4O4 3,4-dimethoxybenzaldehyde, veratraldehyde

[0361]

[0362] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- methoxy-6-nitrophenol C34H33N5O6 4-hydroxy-3-methoxy-5-nitrobenzaldehyde, 5- nitrovanillin

[0363]

[0364] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- methoxyphenyl acetate C36H36N4O5 4-formyl-2-methoxyphenyl acetate, vanillin acetate

[0365]

[0366] 3-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1 -yl)propan-2-yl)-5- methoxyphenol C34H34N4O4 3-hydroxy-5-methoxybenzaldehyde

[0367]

[0368] 2-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1 -yl)propan-2-yl)-5- methoxyphenol C34H34N4O4 2-hydroxy-4-methoxybenzaldehyde

[0369]

[0370] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1 -yl)propan-2-yl)-2-chloro-6- methoxyphenol C34H33CIN4O4 3-chloro-4-hydroxy-5-methoxybenzaldehyde

[0371]

[0372] 1,1 '-(2-(4-(benzyloxy)-3-methoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro- 3H-pyrido[3,4-b]indole) C41 H40N4O4 4-benzyloxy-3-methoxybenzaldehyde

[0373]

[0374] 5-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1 -yl)propan-2-yl)-2,3- dimethoxyphenol C35H36N4O5 3-hydroxy-4,5-dimethoxybenzaldehyde, 3,4-dimethoxy-5- hydroxybenzaldehyde

[0375]

[0376] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1 -yl)propan-2-yl)-2-bromo-6- methoxyphenol C34H33BrN4O4 3-bromo-4-hydroxy-5-methoxybenzaldehyde, 5-bromo- vanillin

[0377]

[0378] 3-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- bromo-6-methoxyphenol C34H33BrN4O4 2-bromo-3-hydroxy-4-methoxybenzaldehyde

[0379]

[0380] 3-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl)propan-2-yl)-2- iodo-6-methoxyphenol C34H33IN4O4 3-hydroxy-2-iodo-4-methoxybenzaldehyde

[0381]

[0382] 1,1'-(2-(3-methoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C34H34N4O3 3-methoxybenzaldehyde, m-anisaldehyde

[0383]

[0384] 1,1'-(2-(3-phenoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C39H36N4O3 3-phenoxybenzaldehyde

[0385]

[0386] 1,1'-(2-(4-phenoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C39H36N4O3 4-phenoxybenzaldehyde

[0387]

[0388] 1,1'-(2-([1,1-biphenyl]-3-yl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C39H36N4O2 [1,1'-biphenyl]-3-carboxaldehyde, biphenyl-3- carboxaldehyde

[0389]

[0390] 1,1'-(2-(4-fluoro-3-phenoxyphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro- 3H-pyrido[3,4-b]indole) C39H35FN4O3 4-fluoro-3-phenoxybenzaldehyde

[0391]

[0392] 1,1'-(2-(3-fluorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C33H31FN4O2 3-fluorobenzaldehyde

[0393]

[0394] 1,1'-(2-(4-fluorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C33H31FN4O2 4-fluorobenzaldehyde

[0395]

[0396] 1,1'-(2-(3,5-difluorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C33H30F2N4O2 3,5-difluorobenzaldehyde

[0397]

[0398] 1,1'-(2-(2,4,5-trifluorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C33H29F3N4O2 2,4,5-trifluorobenzaldehyde

[0399]

[0400] 1,1'-(2-(perfluorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C33H27F5N4O2 2,3,4,5,6-pentafluorobenzaldehyde

[0401]

[0402] 1,1'-(2-(p-tolyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indolizine) C34H34N4O2 4-methylbenzaldehyde

[0403]

[0404] 4-(1,3-bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4-b]indolizol-1-yl)propan-2-yl)benzaldehyde C34H32N4O3 p-xylene dicarboxaldehyde

[0405]

[0406] 1,1'-(2-(4-chlorophenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C33H31CIN4O2 4-chlorobenzaldehyde

[0407]

[0408] 1,1'-(2-(4-(prop-1-en-2-yl)cyclohex-1-en-1-yl)propane-1,3-diyl)bis(7-methoxy- 4,9-dihydro-3H-pyrido[3,4-b]indole) C36H40N4O2 4-(prop-1-en-2-yl)cyclohex-1- en-1-carbaldehyde, perillaldehyde

[0409]

[0410] 1,1'-(2-(4-isopropylphenyl)propane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H- pyrido[3,4-b]indole) C36H38N4O2 4-isopropylbenzaldehyde, cuminic aldehyde

[0411]

[0412] 1,1'-(2-cyclohexylpropane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4- b]indole) C33H38N4O2 cyclohexanecarboxaldehyde

[0413]

[0414] 1,1'-(2-isobutylpropane-1,3-diyl)bis(7-methoxy-4,9-dihydro-3H-pyrido[3,4- b]indole) C31H36N4O2 Exact mass: 496.28

[0415] In addition, many of the harmaline-like compounds were reacted with different aldehydes to produce compounds in addition to those detailed in the following examples. In each case, the reaction was carried out by mixing 500 mg of the selected aldehyde and 250 mg of the harmaline-like compound together in a 15 mL vial, then gently shaking the vial until a uniform powder mixture was present. To the dry mixture was then added 10 mL of ethanol, the vial was capped, and it was placed in a warm water bath at about 40 °C for about 24 hours.

[0416] In the following table, the specific harmalike compound and aldehyde are identified along with the resulting compound. For the latter, the composition of each compound is determined by the reactant moieties therein, minus any water of reaction, and its approximate molecular weight. For example, a given compound referred to as "H+A-H20" refers to a product comprising a harmalike compound moiety and an aldehyde moiety, minus one molecule of water.

[0417]

[0418] 1 = 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (THβC)

[0419] 2 = 6-methoxy-1-methyl-3,4-dihydro-2H-pyrido[3,4-b]indole (6-methoxyharmaline)

[0420] 3 = 4,9-dihydro-3H-β-carboline-1-yl methyl ether

[0421] 4 = 6-methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole (santonine)

[0422] 5 = 2,3,4,5-tetrahydro-8-methoxy-1H-pyrido[4,3-b]indole

[0423] 6 = 4,9-dihydro-1-methyl-3H-pyrido[3,4-b]indol-7-ol hydrochloride (telepathine hydrochloride)

[0424] Examples

[0425] The following examples set forth preferred therapeutic agents and methods in accordance with the application, but it is understood that these examples are given by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the application.

[0426] Example 1

[0427] In this example, a series of 523 compounds were prepared using the aldehyde reaction, including reacting corresponding amounts of solid synthetic o-vanillin (99% purity by weight) and synthetic harmaline (92% purity by weight). In each case, the o-vanillin and harmaline reacted to form one or more compounds. These compositions are referred to as GZ523.001-008, and their compositions and formulations are listed below, along with the amounts and approximate weight percent levels of the two components:

[0428] GZ523.001 - 294 mg o-vanillin (85.5%) + 50 mg harmaline (14.5%), mixed immediately with 5 mL ethanol;

[0429] GZ523.002 - 294 mg o-vanillin (85.5%) + 50 mg harmine (14.5%) mixed with 5 mL DMSO, immediately;

[0430] GZ523.003 - 229.3 mg o-vanillin (66.7%) + 114.7 mg harmine (33.3%) mixed together as dry ingredients and left to stand for 48 hours in a closed container, then 5 mL ethanol was added;

[0431] GZ523.004 - 286.7 mg o-vanillin (83.3%) + 57.3 mg harmine (16.7%) mixed together as dry ingredients and left to stand for 13 days, then 5 mL ethanol was added;

[0432] GZ523.005 - 229.3 mg o-vanillin (66.7%) + 114.7 mg harmine (33.3%) mixed with 5 mL DMSO, immediately, and left to stand for 24 hours;

[0433] GZ523.006 - 229.3 mg o-vanillin (66.7%) + 114.7 mg harmine (33.3%) mixed with 5 mL ethanol, immediately, and left to stand for 24 hours;

[0434] GZ523.007 - 172 mg o-vanillin (50%) + 172 mg harmine (50%) mixed with 5 mL ethanol, immediately, and left to stand for approximately 3 weeks; and

[0435] GZ523.008 - 229.3 mg o-vanillin (66.7%) + 114.7 mg harmine (33.3%) mixed together as dry ingredients and left to stand for 45 minutes in a closed vial, then left to stand for 24 hours in a covered tray, then 5 mL ethanol was added.

[0436] Example 2

[0437] The compound of example 1, 523, was subjected to a series of the same in vitro assays against lymphoma (MO205) and leukemia (jurkat E6-1) cell lines to determine the anti-cancer properties of the composition as determined by cell death. The protocol for the assays is given below.

[0438] Method

[0439] Individual cells were grown in suspension in culture medium (RPMI supplemented with 10% FBS) maintained at approximately 500,000 cells / mL. Cells were plated directly into 96-well plates and each well was exposed to increasing doses of GZ523.001-008 composition for 24 hours (minimum of 4 replicates per dose). PrestoBlue (Life Technologies, Inc) was added to each well after 24 hours of exposure to the selected dose of test composition and fluorescence readings were taken after 4-6 hours using a microplate reader (Enspire Multimode, PerkinElmer) with an excitation wavelength of 485 nm and an emission wavelength of 560 nm. Results were averaged after background subtraction and normalized to untreated cell controls.

[0440] The results of these tests are set out in Figures 1-16 where Figures 1-8 are the lymphoma test results, and Figures 9-16 are the leukemia test results, and in each case the composition exhibits excellent anti-cancer activity at relatively low doses. In general, doses in excess of 10 μg / mL produce very good results, with doses in excess of about 40 μg / mL producing extraordinary results.

[0441] Example 3

[0442] In this example, the 523 compound was prepared using solid synthetic o-vanillin (99% purity) and solid synthetic harmine (92% purity), the compound comprising o-vanillin and harmine in a weight ratio of 2:1. The reactants were dispersed in ethanol to achieve a concentration of 75 mg / mL and allowed to react for a period of 24 hours. Upon completion of the reaction, the compound (designated GZ523F001) was treated by HPLC to recover a high molecular weight fraction consisting primarily (about 70% by weight) of one or more di-oligomers having a molecular weight of approximately 696 and unreacted harmine. These di-oligomers include one or more compounds exemplified by the dimers of Scheme 1.

[0443] The compound was then tested against the same lymphoma and leukemia cells as set out in Example 2. The results of this test are set out in Figure 17 and 18 These results demonstrate that these compounds exhibit very significant anti-cancer activity.

[0444] Example 4

[0445] In this series of tests, the susceptibility of non-Hodgkin's lymphomas to a preferred compound according to the application, GZ523.006, described in Example 1, was tested. The cell lines were grown in suspension according to the supplier's instructions and tested by the method described in Example 2, except that there was no repetition. The following table lists the subtype of each non-Hodgkin's lymphoma tested, the cell line ID number and the median effective dose (EC 50 ). The EC 50 values represent the potency of the GZ523.006 composition against the cell lines and range from 8 to 38 μg / mL, which is considered to be a therapeutically suitable dose range. The magnitude of the effect of the highest concentration of GZ523.006 determines how effective the composition is in killing the corresponding cells directly. For all cell lines tested, at a dose of 25 μg / mL or greater, 100% of the cancer cells died.

[0446] Table 1

[0447]

[0448] Example 5

[0449] In this example, the compound 562 was prepared by mixing 500 mg of vanillin powder and 250 mg of harmine powder in a 15 mL bottle. The powders were gently shaken to form a substantially uniform mixture and 10 mL of dimethyl sulfoxide was added. The mixture was then stirred with a vortex mixer at 1000 rpm for 10 minutes to form a dispersion. In the case of one composition (GZ518.000), one or more of the dispersed compounds were tested immediately against lymphoma cells (MO205) by application to the cells as described in Example 2. A second composition (GZ518.001) was prepared from the dispersion by allowing it to react at room temperature for 24 hours before testing against lymphoma cells by application to the cells. As described in Example 2, the results of the tests are shown in Table 2. Figure 19 and 20 Both compositions exhibited good anti-cancer properties.

[0450] Example 6

[0451] In this example, the EC 50Values. Experiments were performed using two-fold serial dilutions of GZ523.006 between 0.4 μg / mL and 100 μg / mL. Test wells were prepared using media and GZ523.006 controls for background subtraction. Each cell line was plated at 10,000 cells / well with three technical replicates. After 96 hours of exposure, Alamar Blue reagent (Life Technologies) was added to each well and incubated for one hour at 37°C. Fluorescence values were recorded using a 560 nm excitation / 590 nm emission filter set and EC 50 Concentrations. EC 50 Data are listed in Table 2, where: GCB-DLBCL is a germinal center B-cell diffuse large B-cell lymphoma cell line; ABC-DLBCL is an activated B-cell diffuse large B-cell lymphoma cell line; MCL is a mantle cell lymphoma cell line; and FL is a follicular lymphoma cell line. Error bars represent standard error of the mean. Figure 21

[0452] Figure 21 Each cell line was incubated with 5, 10, or 20 μg / mL for 72 hours, followed by incubation with Hoechst 33342 dye (BD Pharmigen) for 60 minutes at 37°C. Cells were washed twice and fluorescence data was collected using an LSRII 4-laser flow cytometer (BD Biosciences). Data were analyzed using Flojo vlO and Modfit v4.05 software to quantify the percentage of dead cells (sub-Gl), senescent cells (Gl peak), and cycling cells (S phase and G2) in each case. Table 2 summarizes the data from this series of tests.

[0453] Table 2

[0454]

[0455]

[0456]

[0457] ​The mechanism of cell death of GZ523.0006 was interrogated using Annexin V and 7-AAD staining using the BD Apoptosis Detection Kit (BD Pharmigen). Four cell lines were selected that exhibited high sensitivity to GZ523.006. Cell lines were incubated with 5, 10, or 20 μg / mL of GZ523.006 for 72 hours, washed, resuspended in IX Annexin V binding buffer, and stained with PE Annexin V and 7-AAD for 15 minutes at room temperature in the dark. Cells were then resuspended in additional binding buffer and analyzed using an LSRII 4-laser flow cytometer (BD Biosciences). Data was analyzed using Flojo v10 by gating on untreated cells. The results of this experiment are summarized in Table 3.

[0458]

[0459] A Caspace 3 / 7 assay was performed by inducing apoptosis using a luminescence-based Caspace cleavage assay. Cells were plated at 10,000 cells per well and exposed to 20 μg / mL of GZ523.006 for 48 hours. Caspace activation was measured using the Caspace Glo 3 / 7 assay (Promega) and compared to vehicle control exposure. Caspace activation was measured using a luminescence plate reader with three technical replicates and two experimental replicates. The results of these tests are summarized in Table 4, where bar 1 is the control and bar 2 is the treated cells. Error bars represent the standard error of the mean. These tests demonstrate that GZ523.006 exhibits cytotoxic properties to induce apoptosis in lymphoma cell lines. Burkitt’s lymphoma cell lines exhibited the highest resistance, while the germinal center B cell-like subtype of diffuse large B cell lymphoma exhibited the greatest sensitivity. Figure 22

[0460] Example 7

[0461] 1 Purpose

[0462] The purpose of this study was to determine the maximum tolerated dose and potential toxicity of GZ523.010 in CD1 mice after 7 days of daily oral dosing. GZ523.010 was prepared by mixing 2433 mg of o-vanillin, 1217 mg of harmaline, and 5 mL of ethanol. The mixture was then sonicated at 35°C for one hour to ensure complete mixing and then allowed to stand at room temperature for 24 hours.

[0463] 2 Study Overview

[0464] ​There were four dose groups consisting of a vehicle control group and 3 dose groups, dosed once daily by oral gavage for 7 days with 10 mice / sex / group. Animals were dosed once daily with GZ523.010 and euthanized on study Day 8. All animals were observed twice daily for any clinical signs following dosing. A gross necropsy was performed on each animal and clinical pathology was performed on all available samples at termination. The first day of dosing was defined as study Day 1. The study design and evaluation variables are presented in Tables 4 and 5.

[0465] Table 4

[0466] Study Design

[0467]

[0468] Table 5

[0469] Evaluation Variables and Intervals

[0470] Parameters Intervals Mortality observations Twice daily Physical examinations Once during acclimation Body weights Daily Food consumption Daily (group mean) Clinical observations Twice daily Clinical pathology Hematology, coagulation, and serum chemistry on three available animals per gender per group Total necropsy All animals retained intact tissue list for future analysis

[0471] 3. Materials and Methods

[0472] 3.1 GZ523.010

[0473] Test / control article name: GZ523.010 Distilled water Lot / batch number: 20160530.1500 S1277 Storage conditions: 4-8℃ Room temperature Supplier: NA (sponsor supplied) Southern Beverage Packers Constituents: 730 mg / mL GZ523.010 in ethanol Water

[0474] 3.2 Test System

[0475] 3.2.1 Animals, Housing, and Environmental Conditions

[0476]

[0477]

[0478] 3.2.2 Diet and Water

[0479]

[0480] 3.3 Dose Procedure

[0481] All animals were dosed once daily by oral gavage for 7 days according to Table 4. Dose volume was calculated based on most recent body weight. Food and water were provided throughout the study period.

[0482] 3.4 Mortality / Moribundity

[0483] Mortality / moribundity was observed twice daily.

[0484] 3.5 Physical Examination

[0485] During acclimation, qualified personnel performed a physical examination on all study animals to determine study eligibility and again prior to termination. The examination included, but was not limited to, examination of the skin and external ears, eyes, abdomen, nervous, behavioral, and general physical condition.

[0486] 3.6 Clinical Observations

[0487] Detailed clinical observations were performed twice daily. Animals were observed for any signs of illness or reaction to treatment. Records of the appearance, changes, or disappearance of clinical signs were maintained on the clinical observation sheet for each individual observation time point.

[0488] 3.7 Body Weights and Food Consumption

[0489] All study animals were weighed daily on Days -1 through termination on Day 8. Group mean food consumption was recorded daily from Day -1 through Day 7.

[0490] 3.8 Termination and Necropsy

[0491] All animals were euthanized with CO2 at termination. A necropsy was performed on each animal and all designated questions / organs were collected for future potential analysis. The following tissues were preserved in 10% neutral buffered formalin, if present, with the exception of testes and eyes. Testes were fixed in modified Davidson’s solution and eyes in Davidson’s solution. Collected tissues were preserved for further evaluation.

[0492]

[0493] The person performing the necropsy can decide at their discretion to collect gross lesions.

[0494] 3.9 Clinical Pathology

[0495] Clinical pathology was performed at termination. Clinical pathology analysis was performed on all designated animals that were euthanized as scheduled. Animals were fasted overnight.

[0496] Serum Chemistry: Blood samples (~0.5 mL) were collected from three study animals of each gender per group, when available, and allowed to clot at room temperature for 15 minutes. No anticoagulant was used. Serum samples were prepared by centrifugation at 3000 RPM for ~15 minutes. Serum chemistry included, preferably ( ). When samples were insufficient for analysis, several samples from the same group were pooled together:

[0497]

[0498] Coagulation: Blood samples (~0.4 mL / animal) were collected from three study animals of each gender per group when available. Sodium citrate (3.2%) was used as anticoagulant. Plasma was prepared by centrifugation at 3000 rpm for approximately 15 minutes at 4°C. Blood clotting analyses included, but were not limited to:

[0499]

[0500] Hematology: Blood samples (~0.4 mL) were collected from three study animals of each gender per group when available. K3-EDTA was used as anticoagulant. Hematology analyses included (preferably (V)):

[0501]

[0502] 4. RESULTS

[0503] 4.1. Dose Administration

[0504] Table 6 summarizes the dose administration. All study animals were successfully dosed with the target amount of the loading vehicle or test article formulation. All dose formulations were prepared prior to dose administration. Prior to preparation of the dose formulation, the stock test article (730 mg / mL) was observed to be a non-uniform consistency. Therefore, due to the large amount of sediment, it was not possible to formulate a dose concentration of 100 mg / mL. The protocol was modified to reduce the dose concentration. The stock formulation was warmed to room temperature and agitated vigorously with sonication to achieve a uniform (muddy) consistency. It was then diluted with ethanol (secondary stock) to 73 mg / mL. The secondary stock was used to prepare each final dose formulation. The final consistency of the dose formulations appeared to be a suspension and was mixed thoroughly prior to dosing.

[0505] Table 6

[0506] Dose Administration - Actual Dose Level Summary

[0507]

[0508] a One mouse (1F17:12-0) was euthanized prior to Day 6 dosing due to tail injury;

[0509] b One mouse was identified as male at termination (suspected to have been misidentified at shipment).

[0510] 4.2. Mortality / Moribundity

[0511] No deaths or severe moribundity were observed during the study period.

[0512] 4.3. Physical Examination

[0513] All study animals were given one physical examination by a veterinarian during acclimation and again prior to termination. All animals were in general good health and were considered suitable for inclusion in the study.

[0514] 4.4 Clinical Observations

[0515] Table 7 lists the clinical observation findings. There were no findings related to the test articles during the exposure period following daily oral gavage dosing.

[0516] Table 7

[0517] Group Clinical Observation Findings Summary

[0518]

[0519] 4.5 Body Weights

[0520] Group Body Weights and Body Weight Change Summary Results are presented in Tables 8 and 9. During the course of the study, most study animals gained weight in general, especially the males. The females maintained or slightly decreased in weight, and there were no significant differences between groups.

[0521] Table 8

[0522] Group Body Weight Results Summary (g)

[0523]

[0524]

[0525] a One mouse (1F17:12-0) was euthanized prior to dosing on Day 6 due to tail injury. b Animals were fasted overnight.

[0526] Table 9

[0527] Group Body Weight Change Results Summary (g / day)

[0528]

[0529]

[0530] a One mouse (1F17:12-0) was euthanized prior to dosing on Day 6 due to tail injury.

[0531] b Animals were fasted overnight.

[0532] 4.6 Food Consumption

[0533] Summary of Group Food Consumption Results are presented in Table 10. Study animals generally had similar food consumption throughout the course of the study. Overall, there were no significant differences between groups.

[0534] Table 10

[0535] Summary of Group Food Consumption (g / day)

[0536]

[0537]

[0538] a Food consumed prior to fasting.

[0539] 4.7 Clinical Pathology

[0540] Blood samples were collected from euthanized mice for hematology and serum chemistry analysis. Some blood samples (serum) did not have sufficient volume to complete all target parameter analysis.

[0541] Hematology and coagulation data are summarized in Table 11. Hematology or coagulation parameters did not appear to be affected in mice treated with different dose levels of the test article when compared to the control group (Group 1).

[0542] Table 12 summarizes serum chemistry data. All serum chemistry results were within the normal range. Serum chemistry parameters did not appear to be affected in mice treated with different dose levels of the test article formulation when compared to the control group (Group 1).

[0543] Table 11

[0544] Group Mean Hematology and Coagulation at Termination

[0545]

[0546]

[0547] Table 12

[0548] Group Mean Serum Chemistry at Termination

[0549]

[0550]

[0551] “.” indicates sample volume was insufficient

[0552] 4.8 Necropsy and Tissue Collection

[0553] A complete necropsy was performed on all study animals. The necropsy included an examination of the external surface, all orifices, and cranial, thoracic, abdominal, and pelvic cavities, including contents. Macroscopic findings are summarized in Table 13. All findings were considered incidental and unrelated to test article administration. All tissues, including all remaining carcasses, were collected and fixed for future potential evaluation.

[0554] Table 13

[0555] Individual Animal Necropsy Findings

[0556]

[0557] 5Summary and Conclusion

[0558] This study was conducted to determine the maximum tolerated dose and potential toxicity of the test article following 7 days of daily oral administration in CD1 mice. There were four dose groups, consisting of a vehicle control group and 3 dose groups. Oral gavage dosing was repeated at 10 mice / sex / group, and the dosing procedure was performed for 7 days. All animals were successfully dosed once daily as recommended, and euthanized on study day 8. All animals were observed for any clinical signs following dosing each day. A gross necropsy was performed on each animal, and clinical pathology was performed on all available samples at termination.

[0559] There were no unscheduled deaths during the study period, and no signs of moribundity were observed. Overall, all animals had normal food consumption and body weight gains throughout the study period as expected. There were no test article-related clinical findings. Clinical pathology analysis and necropsy at termination showed that all study animals were in a normal state.

[0560] In summary, animals tolerated doses of GZ523.010 up to 300 mg / kg / day via 7 days of daily oral administration. The no-observed-adverse-effect level (NOAEL) was determined to be 300 mg / kg / day under the conditions of this study.

[0561] Example 8

[0562] In this example, in vitro cell proliferation assays were performed using (1) a human myeloma tumor cell line; (2) a human lymphoma tumor cell line; (3) a solid human tumor cell line; and (4) parental, lenalidomide-resistant, and bortezomib-resistant Jeko-1 suite of lymphoma tumor cell lines. The compounds tested were three diharmaline compounds, namely the 518B562 (or simply 562), 560, and 561 compounds. In addition, a monoharmaline product designated 518F014 was also tested. This monoharmaline product has the following structure:

[0563]

[0564] 518 The compound 518B562 was prepared by mixing the particulate harmaline with vanillin together in a weight ratio of about 2: 1 (vanillin: harmaline) and then adding ethanol to a final concentration of the reaction of about 10-100 mg / mL. The mixture was then allowed to sit at 50 °C for about 3 days. A blue colored solid formed which was filtered and washed with methanol and recovered. It was found that the addition of an acid such as hydrochloric acid to lower the pH of the product increased its solubility.

[0565] 560 The compound 560 was prepared by mixing 500 mg of benzaldehyde and 250 mg of harmaline together in a 15 mL bottle and then shaking. To the mixture was then added 10 mL of DSMO and then stirred for 10 minutes using a vortex mixer at 1000 rpm. The vortexed mixture was then allowed to sit at room temperature for 24 hours. The resulting product was an orange colored liquid containing 10.6 mM of the compound and was stored at 4 °C until use.

[0566] 561 The compound 561 was prepared by mixing 500 mg of cinnamaldehyde and 250 mg of harmaline together in a 15 mL bottle and then shaking. To the mixture was then added 10 mL of DSMO and then stirred for 10 minutes using a vortex mixer at 1000 rpm. The vortexed mixture was then allowed to sit at room temperature for 24 hours. The resulting product was an orange colored solid containing 10 mM of the compound dispersed in a liquid and was stored at 4 °C until use.

[0567] Each proliferation assay was performed as follows. Test cells were plated in growth media in 384-well microtiter plates in a volume of 50 μL. The cells were incubated in a humidified incubator at 37 °C for 24 hours. After 24 hours of incubation, test compounds were added to the test wells in DSMO solvent at concentrations ranging from 0.0075-100 μM. Control wells received an equal volume of DSMO without compound. Following dosing, the cells were incubated in a humidified incubator at 37 °C for 72 hours. Following this exposure, 100 μL of a 1 : 1 mixture of sterile water and CellTiter-Glo® reagent (Promega) was added to each well. The plate was then incubated at room temperature for 60 minutes and then the luminescence value of each well was recorded using a luminometer as a measure of cell proliferation.

[0568] The following Table 14 lists the cell lines tested using the respective compounds, IC 50 A summary of the results and the identification of the corresponding graphical plot for each assay.

[0569]

[0570]

[0571] ​This data indicates that the preferred diharmaline compounds 560, 561 and 562 have significantly lower IC 50 values compared to the monoharmaline compound 518F014. This phenomenon was found to be consistent throughout the test compounds of the present application, i.e. diharmaline compounds were significantly superior to monoharmaline compounds.

[0572] Example 9

[0573] In this example, a compound mixture was prepared by reacting harmaline and 3- phenoxybenzaldehyde in a 2:1 ratio by weight (3-phenoxybenzaldehyde: harmaline). The reaction mixture had three components, i.e. fractions having molecular weights of 608 (47% by weight), 788 (32% by weight) and 394 (21% by weight). The MW 608 product comprises two harmaline moieties and one 3-phenoxybenzaldehyde with one water molecule removed; the MW 788 product comprises two harmaline moieties and two 3-phenoxybenzaldehyde moieties with two water molecules removed; and the MW 394 product comprises one mole each of harmaline and 3-phenoxybenzaldehyde with one water molecule removed. The following table sets out the results of a series of assays using this compound mixture on 31 different cell lines, where the assays were carried out as set out in Example 2. In each case, two IC 50 values were determined and the results averaged to give the average IC 50 values.

[0574] Table 15

[0575]

[0576] Example 10

[0577] In this example, the aforementioned compound 518B562 was used to treat pancreatic cancer cells (S2-007 and Mia-PaCa2) at different times and concentrations, and then proliferation assays were generated using the techniques described above. The product significantly inhibited the proliferation of the cells in a dose dependent manner at doses of 1-25 μg / mL and in a time dependent manner over 24-72 hours. The IC 50 values of the compound on S2-007 and Mia-PaCa2 cells were determined to be 3 μg / mL and 5 μg / mL, respectively, after 72 hours of treatment.

[0578] Example 11

[0579] In this example, clustergrams / heatmaps of RNA sequences for cancer stem cell (CSC) markers were performed before and after treatment of S2-007 human pancreatic cancer cells with 518B562. The experiment was performed using whole transcriptome shotgun sequencing (WTSS) followed by bioinformatics data analysis for CSC markers. RNA sequence / heatmaps were generated to obtain the whole genome gene expression profile of pancreatic cancer cells.

[0580] One sample of pancreatic cancer cell line was untreated while the same sample was treated with 5 microg / mL of 518B562. The samples were compared for RNA sequencing using an Illumina HISeq 2500 sequencer at 100 bp single read resolution. Sequence reads were mapped to the human genome (component GRCh38.rel77) using STAR software (Dobin et al., 2012). Transcript abundance estimates were generated using Cufflinks software (Trapnell et al., 2010) and differential gene expression estimates were calculated using Cuffdiff software (Trapnell et al., 2013). RNA sequencing yielded approximately 48.6 and 60.1 reads, with between 97.2% and 98.3% of reads mapping to the reference genome.

[0581] The clustergrams or heatmaps indicate that 518B562 significantly inhibited gene clusters on proliferation, anti-apoptosis, and angiogenesis markers, while upregulating anti-proliferation and apoptosis markers. In particular, 518B562 upregulated apoptosis markers [ICAM5, WNK4, ALPP, LTRC26, SHBG, MT1X] and anti-proliferation markers [NRP1, ATF2A, CYP1b1, ALPP, DEPTOR, MT1F]. In addition, 518B562 downregulated angiogenesis markers [OXTR, SYCP2, CRHR1, SPEG], anti-apoptosis markers [TUG1, FABP1, PI3, DOK5], and proliferation signaling markers [FOXj1, SPP1, C3].

[0582] Example 12

[0583] A 250 mL round bottom flask was charged with 6 g of benzaldehyde, 3 g of harmine, and 50 mL of ethanol. This dispersion was refluxed at a temperature of about 78 °C for about 4 hours, after which it was allowed to gradually cool to room temperature. The resulting solid was collected by vacuum filtration, rinsed with about 200 mL of cold water, and dried at room temperature to yield multiple twinned, racemic crystalline chunks. A piece was cut from one of the chunks and gave usable diffraction data, allowing all hydrogen atoms to be located and refined as independent isotropic atoms; both nitrogen atoms also appeared to be protonated. The resulting two-dimensional structure of the 560 compound was determined to be:

[0584]

[0585] The above structure is referred to herein as the "determined 560 compound".

[0586] A three-dimensional representation of the above 560 compound is shown below, where the large circles represent carbon atoms, and the small circles represent hydrogen atoms; the double bonds are not shown in this representation. In addition, the hydrogen bond between nitrogen N1 and N4 is shown in dashed lines. This hydrogen bond is believed to be important to the functionality of the compound. Other numbered atoms are provided for reference.

[0587]

[0588] The above compound can isomerize, particularly during NMR analysis, to give the following two-dimensional isomeric structure:

[0589]

[0590] The reduced form of either of the above isomers can be less prone to additional isomerization. The two-dimensional structure of this reduced compound (resulting from hydrogenation of the above compound) is listed below.

[0591]

[0592] Example 13

[0593] In this example, several 560 compounds were prepared by mixing 6 g of benzaldehyde and 3 g of harmine together in a closed 40 mL vial, then shaking for a few minutes. The closed vial was then placed in a 40 °C water bath for 1-5 days. After cooling, the vial was opened and placed in a Labconco FreeZone 4.5 L freeze dryer at 0.028 kPa and -48 °C for 1 week. The contents of the vial were then mixed with a water / methanol combination, and the resulting solids were collected by vacuum filtration. The molecular weights of the compounds were found to be 302, 320, 514, and 516.

[0594] Example 14

[0595] A 250 mL round bottom flask was charged with 6 g of vanillin, 3 g of harmine, and 50 mL of ethanol. This dispersion was refluxed at a temperature of about 78 °C for about 4 hours, after which it was allowed to gradually cool to room temperature. The resulting solids were collected by vacuum filtration, rinsed with about 200 mL of cold water, and dried at room temperature to give multiple twinned, racemic crystalline masses. A piece was cut from one of the masses, and gave usable diffraction data, allowing all hydrogen atoms to be located and improved to independent isotropic atoms; both nitrogen atoms also appeared to be protonated. The resulting two-dimensional structure of the 562 compound was determined to be:

[0596]

[0597] Example 15

[0598] In this example, the respective 560 and 562 compounds of Examples 12 and 14 were tested in cell proliferation assays using two pancreatic cancer cell lines, S2-007 and MiaCaPa-2. In each assay, 5 x 10 4 cells were seeded in 96 well plates. After 24 hours incubation, the cells were treated with varying concentrations of the 560 or 562 compounds and allowed to incubate for an additional 72 hour period. Cell proliferation values were determined by the enzyme aminohexosidase assay. The results of these tests are listed in Figure 119 and 120 (560 compound) and Figure 121 and 122 (562 compound). These graphs also provide the IC50 values for each assay.

[0599] Example 16

[0600] In this example, the 560 and 562 compounds of Examples 12 and 14, respectively, were tested using a cell colony formation assay. 500 viable S2-007 and MiaCaPa-2 cells were plated in six well dishes and allowed to grow for 24 hours. The cells were then incubated in the presence or absence of the 560 and 562 compounds for 72 hours. The compound containing media was then removed and the cells were washed in PBS and incubated in complete media for an additional 10 days. The resulting colonies were then washed in PBS and fixed with 10% formalin for 10 minutes at room temperature, then washed with PBS and stained with crystal violet. The colonies from the control and compound supplemented assays were then counted and compared.

[0601] Figure 123 (560 compound, S2-007 cells) demonstrates colony formation at 24, 48 and 72 hours for the control (no 560 compound) and 4 μg and 6 μg of the 560 compound. The 560 compound significantly perturbed colony formation, particularly at the 6 μg level of use.

[0602] Figure 124 (560 compound, MiaPaCa-2 cells) demonstrates colony formation at 24, 48 and 72 hours for the control (no 560 compound) and 3 μg and 5 μg of the 560 compound. The 560 compound significantly perturbed colony formation at both levels of use.

[0603] Figure 125(562 compound, S2-007 cells) demonstrates colony formation at 24, 48 and 72 hours for control (no 562 compound) and 14 μg and 16 μg of 562 compound.

[0604] Figure 126 (562 compound, S2-007 cells) demonstrates colony formation at 24, 48 and 72 hours for control (no 562 compound) and 20 μg and 24 μg of 562 compound.

[0605] Figure 127 (562 compound, MiaPaCa-2 cells) demonstrates colony formation at 24, 48 and 72 hours for control (no 562 compound) and 3 μg, 7 μg, 10 μg and 12 μg of 562 compound. The 562 compound significantly perturbed colony formation, particularly at higher levels of use.

[0606] Example 17

[0607] In this example, the 560 and 562 compounds of Examples 12 and 14, respectively, were used in cell cycle tests on S2-07 and MiaPaCa02 cells. In each case, cells treated with 560 and 562 compounds for 72 hours were trypsinized and suspended in PBS. Single cell suspensions were fixed using pre-chilled 70% ethanol for 3 hours and then permeabilized with PBS containing 0.1% Triton X-100, 1 mg / mL propidium iodide and 2 mg RNase without DNase at room temperature. Flow cytometry assays were then performed using a FASCalibur analyzer (Becton Dickinson) with 10,000 events captured per sample. Results were analyzed using ModFit LT™ software (Verity software House). These results were performed using a SubG0 gate window that was alternately opened (with Sub G0) and closed (without Sub G0) to provide data for quiescent and active state cells.

[0608] Figure 128 and 128A (Compound 560 S2-007 cells) demonstrates cell cycle results with and without Sub G0 at 24 and 48 hours.

[0609] Figure 129 and 129A (Compound 560 MiaPaCa-2 cells) demonstrates cell cycle results with and without Sub G0 at 24, 48 and 72 hours.

[0610] Figure 130 and 130A(Compound 562 S2-007 cells) demonstrated cell cycle results with and without Sub G0 at 24, 48, and 72 hours.

[0611] Figure 131 and 131A (Compound 562 Mia PaCa-2 cells) demonstrated cell cycle results with and without Sub G0 at 24, 48, and 72 hours.

[0612] Example 18

[0613] Place 2200 mg of benzaldehyde and 1100 mg of harmine in a 50 mL beaker and mix slightly. Heat the beaker until a color change is observed, then transfer the mixture to a 250 mL round bottom flask with the aid of 50 mL of isopropyl alcohol. Next, add 50 μL of 37% HC1, and reflux the mixture for 1.5 hours. After about 1 hour, crystals begin to form. After refluxing, allow the reaction mixture to sit and cool to ambient temperature, and filter using a Buchner funnel, rinsing with cold isopropyl alcohol. The weight of the product collected is about 700 mg, and the color is beet red. Analysis of the compound indicates that it contains approximately 18% of the identified 560 compound (mw = 516) and approximately 80% of a dimer (mw = 604).

[0614] Example 19

[0615] Place 3000 mg of benzaldehyde and 1500 mg of harmine in a 50 mL beaker and mix slightly. Heat the beaker until the color of the mixture changes to light brown. Then transfer the mixture to a 250 mL round bottom flask with the aid of 30 mL of isopropyl alcohol. Next, add 50 μL of 37% HC1, and reflux the mixture for 30 minutes. After refluxing, allow the reaction mixture to sit and cool to ambient temperature, and filter using a Buchner funnel, rinsing with cold isopropyl alcohol. The weight of the product collected is about 1325 mg, and the color is yellow. Analysis of the compound indicates that it contains approximately 80% of the identified 560 compound (mw = 516).

[0616] Example 20

[0617] Place 8000 mg of benzaldehyde and 4000 mg of harmine in a 50 mL beaker and mix slightly. Then transfer the mixture to a 250 mL round bottom flask with the aid of 40 mL of methanol. Next, add 50 μL of 37% HC1, and reflux the mixture for 45 minutes. After refluxing, allow the reaction mixture to sit and cool overnight. After cooling, filter the mixture using a Buchner funnel, rinsing with methanol. The product color is yellow. Analysis of the compound indicates that it contains approximately 93% of the identified 560 compound (mw = 516), with the remainder being unreacted harmine.

[0618] Example 21

[0619] 8000 mg of vanillin and 4000 mg of halamine were placed in a 50 mL beaker. The mixture was heated by applying 40°C water to the outside of the beaker, which initiated the reaction and changed the color of the mixture from yellow to light brown. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 50 mL of isopropanol, causing the mixture to turn yellowish-green. Once all the reactants had dissolved, the color changed from yellowish-green to yellowish-brown. Next, 150 μL of 37% HCl was added, and the mixture was heated until the color of the mixture turned dark brown and a blue precipitate was formed. The mixture was then refluxed for 25 minutes, and the container was cooled with tap water. The reaction mixture was then filtered using a Buchner funnel and washed with isopropanol. The mixture was then dried, producing a grayish-blue color. Analysis of the compound showed that it contained approximately 85% of compound 562 (mw = 562) from Example 14.

[0620] Example 22

[0621] 4000 mg of vanillin and 2000 mg of halalin were placed in a 50 mL beaker. The mixture was heated by applying 40°C water to the outside of the beaker, which initiated the reaction and changed the color of the mixture from yellow to dark brown. The mixture was then transferred to a 250 mL round-bottom flask with the aid of 50 mL of isopropanol. Once all the reactants dissolved, the color turned dark reddish-brown. Next, 500 μL of 37% HCl was added, and the mixture was heated until the color of the mixture turned dark brown and a bluish precipitate was formed. The mixture was then refluxed for 45 minutes, and the container was cooled with tap water. The reaction mixture was then filtered using a Buchner funnel and washed with isopropanol. The mixture was then dried to give approximately 1500 mg of dark brown product. Analysis of the compound showed that it contained approximately 60% of compound 562 from Example 14 (mw = 562), approximately 23% of the dehydration adduct (mw = 348), and approximately 17% of the dimer (mw = 696).

[0622] Example 23

[0623] 2 g of cuminaldehyde, 1 g of halamine, and 40 mL of ethanol were placed in a 250 mL round-bottom flask. The dispersion was refluxed at approximately 65 °C for about 30 minutes, and then allowed to cool gradually overnight to room temperature. The resulting bottom liquid product was then analyzed. The major compound (referred to herein as product 561) has the following structure:

[0624]

[0625] Example 24

[0626] In this test, the related homalomenin compound 594, homalomenin 3-phenoxybenzaldehyde, was tested using the same cell lines and procedures described in Example 8. The structure of the 594 compound is listed below, and the cell proliferation assay data collected is given in the table below.

[0627]

[0628] Table 16

[0629]

[0630]

[0631] As evidenced by the above data, the 594 compound was effective against a variety of cancer cells.

[0632] Example 25

[0633] In this example, in vitro cell proliferation assays were performed on different cell lines as described in Example 8. In particular, the cell lines are identified in Table 17 below, and the tissue types are: pancreas; endometrium; triple negative breast cancer (TNBC); non-small cell lung cancer (NSCLC); ovary; renal cell carcinoma (RCC); cervical squamous cell; hemagglutinin and neuraminidase (H&N squamous cell); germinal center B-cell like novel diffuse large B-cell lymphoma (GCB-DLBCL); activated B-cell - diffuse large B-cell lymphoma (ABC-DLBCL); human myeloma; human myeloma cell line lymphoblastoid; human myeloma cell line lymphoblastoid myeloma pleural effusion infiltrate; human multiple myeloma; plasma cell leukemia / multiple myeloma Epstein-Barr nuclear antigen-negative (EBNA-negative) and expressing prototype oncogene B-cell lymphoma 2 (BCL2) mRNA; multiple myeloma from peripheral blood type IgD Lmabda.

[0634] In accordance with the present application, these cell lines were tested with a series of homalomenin / aldehyde compounds. The aldehydes reacted with homalomenin are listed in Table 17 by numbers 1-2, 4-15, 17-24, and 27-30, and the corresponding compounds of like number are listed after Table 17. Details are found in the key below. The compounds are identified in the next section of Table 17. In each case, the compound was prepared by reacting one part by weight of homalomenin and two parts by weight of the aldehyde in ethanol at 50°C overnight.

[0635] Key - Tables 17-19

[0636] * Values averaged using 100 μΜ test values

[0637] HMCL Human Myeloma Cell Line

[0638] HMCL-A Human myeloma cell line Lymphoblastoid

[0639] HMCL-B Human myeloma cell line Lymphoblastoid myeloma pleural effusion infiltrate (IgAk)

[0640] HMM Human multiple myeloma

[0641] MM IgD Multiple myeloma from peripheral blood type IgD l mabda

[0642] PCL / MM Plasma cell leukemia / multiple myeloma EBNA-negative and expressing mRNA for proto-oncogene BCL2

[0643] SC Squamous cell

[0644] Table 17

[0645]

[0646]

[0647]

[0648]

[0649]

[0650] Compound:

[0651] #1 - o-Anisaldehyde

[0652]

[0653] #2 - Ethyl vanillin

[0654]

[0655] #4 - Veratraldehyde

[0656]

[0657] #5 - 5-Nitrovanillin

[0658]

[0659] #6 - Vanillyl acetate

[0660]

[0661] #7 - 3-Hydroxy-4-methoxybenzaldehyde

[0662]

[0663] #8 - 2-hydroxy-4-methoxybenzaldehyde

[0664]

[0665] #9 - 3-chloro-4-hydroxy-5-methoxybenzaldehyde

[0666]

[0667] #10 - 3-benzyloxy-4-methoxybenzaldehyde

[0668]

[0669] #11 - 5-hydroxy-3,4-dimethoxybenzaldehyde

[0670]

[0671] #12 - 5-bromo-vanillin

[0672]

[0673] #13 - 2-bromo-3-hydroxy-4-methoxybenzaldehyde

[0674]

[0675] #14 - 3-hydroxy-2-iodo-4-methoxybenzaldehyde

[0676]

[0677] #15 - m-anisaldehyde

[0678]

[0679] #17 - 4-phenoxybenzaldehyde

[0680]

[0681] #18 - biphenyl-3-carboxaldehyde

[0682]

[0683] #19 - 4-fluoro-3-phenoxybenzaldehyde

[0684]

[0685] #20 - 3-fluorobenzaldehyde

[0686]

[0687] #21 - 4-fluorobenzaldehyde

[0688]

[0689] #22 - 3,5-difluorobenzaldehyde

[0690] #23 - 2,4,5-trifluorobenzaldehyde

[0691]

[0692] #24

[0693]

[0694] #27 - 4-chlorobenzaldehyde

[0695]

[0696] #28 - perillaldehyde

[0697]

[0698] #29 - cuminic aldehyde

[0699]

[0700] #30 - cyclohexanecarboxaldehyde

[0701]

[0702] Example 26

[0703] In this example, a compound was produced via a reaction between the fused bicyclic compound, 1-methyl-3,4-dihydroisoquinoline and vanillin, as described in Example 25. The compound was tested against the same cell lines as Example 25, giving the following results.

[0704] Table 18

[0705]

[0706] Example 27

[0707] In this example, 200 mg of 4-methyl-6,7-dihydrothieno[3,2-c]pyridine of the following formula

[0708]

[0709] was reacted with an excess of vanillin in methanol at 100 °C by microwave heating the reaction mixture for 30 minutes. Surprisingly, a spirocyclic solid compound was recovered having the following formula

[0710]

[0711] which is a MW 588.82 species that hydrogen bonds with a MW 151.14 species. Note that the MW 588.82 species includes a single vanillin moiety and three 4-methyl-6,7-dihydrothieno[3,2-c]pyridine moieties. This spirocyclic compound, designated HRM05, was tested against a variety of different tissue types by the in vitro cell proliferation test described above, with the following results:

[0712] Table 19

[0713]

[0714] TBD = To Be Determined

[0715] While the anti-cancer properties of the compositions of the present application have been demonstrated against certain cancers, it is believed that the present application can be applicable to almost all cancers, such as the following: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Acute Myeloid Leukemia, Childhood; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; Adolescent, Cancer; AIDS-Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Appendix Cancer; Astrocytoma, Childhood; Atypical Teratoid Tumor / Rhabdoid Tumor, Childhood, Central Nervous System; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma, and Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Central Nervous System Atypical Teratoid Tumor / Rhabdoid Tumor, Childhood; Brain Tumor, Central Nervous System Embryonal Tumors, Childhood; Brain Tumor, Astrocytoma, Childhood; Brain Tumor, Craniopharyngioma, Childhood; Brain Tumor, Ependymoblastoma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Medulloepithelioma, Childhood; Brain Tumor, Pineal Parenchymal Tumors of Intermediate Differentiation, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors and Pinealoblastoma, Childhood; Brain and Spinal Cord Tumors, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Tumors, Childhood; Burkitt Lymphoma; Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma of Unknown Primary; Central Nervous System Atypical Teratoid Tumor / Rhabdoid Tumor, Childhood; Central Nervous System Embryonal Tumors, Childhood; Central Nervous System Lymphoma, Primary; Cervical Cancer; Cervical Cancer, Childhood; Childhood Cancers; Spinal Cord Tumor, Childhood; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer, Childhood; Craniopharyngioma, Childhood; Cutaneous T-Cell Lymphoma; Embryonal Tumors, Central Nervous System, Childhood; Endometrial Cancer; Ependymoblastoma, Childhood; Ependymoma, Childhood; Esophageal Cancer; Esophageal Cancer, Childhood; Esthesioneuroblastoma, Childhood; Ewing Sarcoma Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Gastrointestinal Stromal Tumors (GIST); Gastrointestinal Stromal Tumor, Childhood; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma, Adult; Glioma, Childhood Brain Stem; Hairy Cell Leukemia; Head and Neck Cancer; Heart Cancer, Childhood; Hepatocellular (Liver) Cancer, Adult (Primary);Hepatocellular (liver) cancer, childhood (primary); histiocytosis, Langerhans cell; Hodgkin lymphoma, adult; Hodgkin lymphoma, childhood; hypopharyngeal cancer; intraocular melanoma; islet cell tumor (endocrine pancreas); Kaposi Sarcoma; kidney (renal cell) cancer; kidney cancer, childhood; Langerhans cell histiocytosis; laryngeal cancer; laryngeal cancer, childhood; leukemia, acute lymphoblastic, adult; leukemia, acute lymphoblastic, childhood; leukemia, acute myeloid, adult; leukemia, acute myeloid, childhood; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; lip and oral cavity cancer; liver cancer, adult (primary); liver cancer, childhood (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoma, AIDS-related; lymphoma, Burkitt; lymphoma, cutaneous T-cell; lymphoma, Hodgkin, adult; lymphoma, Hodgkin, childhood; lymphoma, non-Hodgkin, adult; lymphoma, non-Hodgkin, childhood; lymphoma, primary central nervous system (CNS); macroglobulinemia, Waldenstrom; osteosarcoma and malignant fibrous histiocytoma of bone; medulloblastoma, childhood; medulloepithelioma, childhood; melanoma; melanoma, intraocular (eye); merkel cell carcinoma; mesothelioma, adult malignant; mesothelioma, childhood; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome, childhood; multiple myeloma / plasma cell neoplasms; mycosis fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative neoplasms; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple; myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; nasopharyngeal cancer, childhood; neuroblastoma; non-hodgkin lymphoma, adult; non-hodgkin lymphoma, childhood; non-small cell lung cancer; oral cancer, childhood; oral cavity cancer and lip cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pancreatic cancer, childhood; pancreatic cancer, islet cell tumors; papillomatosis, childhood; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pineal parenchymal tumors of intermediate differentiation, childhood; pinealoblastoma and supratentorial primitive neuroectodermal tumors, childhood; pituitary tumor; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma, childhood; pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell cancer; respiratory tract carcinoma with changes in chromosome 15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland cancer; salivary gland cancer, childhood; sarcoma, Ewing sarcoma family of tumors; kaposi's sarcoma; sarcoma, soft tissue, adult; sarcoma, soft tissue, childhood; sarcoma, uterine; sezary syndrome; skin cancer (non-melanoma); skin cancer, childhood; skin cancer (melanoma); skin cancer, merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma, adult; soft tissue sarcoma, childhood; squamous cell carcinoma; squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; stomach (gastric) cancer, childhood; supratentorial primitive neuroectodermal tumors, childhood; t-cell lymphoma, cutaneous; testicular cancer; testicular cancer, childhood; throat cancer; thymoma and thymic carcinoma; thymoma and thymic carcinoma, childhood; thyroid cancer; thyroid cancer, childhood; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor, pregnancy; cancer of unknown primary site, adult; cancer of unknown primary site, childhood; rare childhood cancers; ureter and renal pelvis, transitional cell cancer; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vaginal cancer, childhood; vulvar cancer; waldestrom's macroglobulinemia; wilms' tumor; women's cancers.

Claims

1. An anticancer composition comprising a therapeutic compound having the structure or wherein the -O-R3groups can independently be located at any position on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, each R2is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, each R3group is independently selected from the group consisting of C1-C4 alkyl groups, and the phenoxy groups can be substituted at any position on the benzyl ring, and wherein the notation ---- refers to the fact that one or both of the following can optionally be present: one or two non- fused double bonds, located at one or both of the valence-allowing positions around either of the six-membered N-containing rings; or either of the N-containing rings is free of non-fused double bonds, and each R1is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups; or a therapeutic compound having the structure wherein each R4is independently selected from the group consisting of absent, H, OH, and C1-C4 alkyl groups, each R5is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, each R6group is independently located anywhere around the corresponding terminal phenyl group, and is selected from the group consisting of C1-C4 alkoxy groups, R7and R8are connected anywhere around the phenyl ring, and are independently selected from the group consisting of H, OH, and C1-C4 alkoxy groups, with the proviso that R7and R8are not both H, R7is OH, R8is a C1-C4 alkoxy group, and each R9is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, and wherein the notation ---- refers to the fact that zero, one, or two non-conjugated double bonds can optionally be present, located in one or both of the valence-allowing positions around either of the six-membered N-containing rings; or either of the N-containing rings is free of non-conjugated double bonds, and each R4is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups.

2. The composition of claim 1 comprising additional ingredients selected from the group consisting of active agents, preservatives, buffers, salts, carriers, excipients, diluents, and other pharmaceutically acceptable ingredients, and combinations thereof.

3. The composition of claim 1, the structure being 。 4. The composition of claim 1, the structure being 。 5. The composition of claim 1, the therapeutic compound having the structure 。 6. The composition of claim 1, wherein the structure is 。 7. A compound having the structure wherein the -O-R3groups can independently be located anywhere on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of absent, H, OH, and a C1-C4 alkyl group, each R2is independently selected from the group consisting of H, OH, and a C1-C4 alkyl group, each R3group is independently selected from the group consisting of a C1-C4 alkyl group, and wherein the notation ---- refers to the fact that one or both non-fused double bonds can optionally be present, located in one or both valence-allowed positions around either or both of the six-membered N-containing rings; or either or both of the N-containing rings do not contain a non-fused double bond, and each R1is independently selected from the group consisting of H, OH, and a C1-C4 alkyl group.

8. The compound of claim 7, the structure being 。 9. A compound having the structure ， wherein each R4is independently selected from the group consisting of absent, H, OH, and C1-C4 alkyl groups, each R5is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, each R6group is independently located anywhere around the corresponding terminal phenyl group, and is selected from the group consisting of C1-C4 alkoxy groups, R7and R8are connected anywhere around the phenyl ring, and are independently selected from the group consisting of H, OH, and C1-C4 alkoxy groups, with the proviso that R7and R8are not both H, R7is OH, R8is a C1-C4 alkoxy group, and each R9is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups, and wherein the notation ---- refers to the fact that zero, one, or two non-conjugated double bonds can optionally be present, located in one or both of the valence-allowing positions around either of the six-membered N-containing rings; or either of the N-containing rings is free of non-conjugated double bonds, and each R4is independently selected from the group consisting of H, OH, and C1-C4 alkyl groups.

10. The compound of claim 9, the structure being 。 11. The compound of claim 9, the compound having the structure 。 12. A compound having the structure ， wherein the -O-R3groups can independently be located anywhere on the terminal phenyl groups, wherein each R1is independently selected from the group consisting of absent, H, OH, and a C1-C4 alkyl group, each R2is independently selected from the group consisting of H, OH, and a C1-C4 alkyl group, each R3group is independently selected from the group consisting of a C1-C4 alkyl group, and the phenoxy groups can be substituted anywhere on the benzyl ring, and wherein the notation ---- refers to the fact that one or both of the following can optionally be present: one or two non- fused double bonds, located in one or both of the valence-allowing positions around either of the six-membered N-containing rings; or either of the N-containing rings is free of non-fused double bonds, and each R1is independently selected from the group consisting of H, OH, and a C1-C4 alkyl group.

13. The compound of claim 12, wherein the structure is 。 14. Use of the composition of any one of claims 1-6 in the manufacture of a medicament for treating a human cancer patient.

15. The use of claim 14, the therapeutic compound comprising two harmine moieties covalently bonded to a single moiety derived from an aldehyde compound.

16. The use of claim 15, the aldehyde compound selected from the group consisting of vanillin, benzaldehyde, cinnamaldehyde, o-vanillin, phenoxybenzaldehyde, and mixtures thereof.

17. Use of a composition for the manufacture of a medicament for anticancer therapeutic applications, the composition being according to any one of claims 1-6.

18. A composition for use in the treatment of human cancer, the composition being according to any one of claims 1-6.

19. A pharmaceutical composition for use in the treatment of cancer, comprising a therapeutically effective amount of the composition of any one of claims 1-6 and a pharmaceutically acceptable carrier.

Images