WO2015103005A1 - Amphiphilic amine compounds and their use as therapeutic agents and nanocarriers - Google Patents

Abstract

Amphiphilic amine compounds and micelles and compositions containing the same are disclosed. Methods of using the amphiphilic amine compounds in the treatment of hyperproliferative disease such as cancer are disclosed. Methods of using micelles formed from a plurality of the amphiphilic amine compounds as nanocarriers for other bioactive molecules are also disclosed.

Description

AMPHIPHILIC AMINE COMPOUNDS AND THEIR USE AS THERAPEUTIC

AGENTS AND NANO CARRIERS

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 61/923,427, filed January 3, 2014, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present disclosure relates to amphiphilic amine compounds and their use as therapeutic agents and nanocarriers for bioactive molecules.

BACKGROUND OF THE INVENTION

[0003] There are limits to the cytotoxic effects and delivery methods of current antitumor therapies and chemotherapies. The effectiveness of chemotherapy to treat tumors is often hindered by the intrinsic or acquired resistance of cancer cells to antitumor agents. Newer antitumor agents exploit different mechanisms of action to overcome the resistance of tumor cells to chemotherapy and have significantly improved the outcome of many cancers (Sleijfer et al., / Clin Oncol. 2013, 31(15): 1834-41 ; Awada et al., Curr Opin Oncol. 2013, 25(3):296- 304). However, despite the progress made, treating and preventing the reoccurrence of cancer still remains a great challenge as the general and organ- specific toxicities of antitumor therapies limit the doses of drugs that can be safely given, thus reducing the ability to completely kill the population of tumor cells before they develop multidrug resistance.

[0004] Pediatric tumors in particular present a serious challenge to current chemotherapy regimens as they often grow fast and rapidly develop resistance. For example, pediatric cancers such as neuroblastoma, osteosarcoma, sheath nerve tumor, and rhabdomyosarcoma are often diagnosed after they have spread to distant metastatic sites. In spite of the recent treatment protocols involving intensive chemotherapy, surgery, external beam and/or metabolic radiotherapy and hematopoietic stem cell transplant, the long-term prognosis of these diseases in their advanced stages remains poor. In many cases, an apparent complete or partial remission can be achieved initially, however, recurrence of disease within a few months to years is quite common (Smith et al., / Clin Oncol. 2010, 28:2625-2634). Among adult tumors, many types, including breast, colon and lung carcinoma, still pose serious problems to the efficacy of therapeutic treatments due to drug resistance (Jemal et al., J Natl Cancer Inst., 2013, 105: 175-201; Salehan et al., Br J Biomed Sci. 2013, 70(l):31-40). The major aim of cancer therapy should be to achieve maximal tumor cell killing during the first- line treatment, including the elimination of circulating tumor cells and sterilization of the microscopic sites of metastatic foci that often remain after the induction therapy and are responsible for disease relapse.

[0005] Two main approaches to improving anticancer therapy include developing new drugs with specific affinity for tumor cells and using drug-loaded nanometer- sized carriers (nanocarriers) such as liposomes, dendrimers, and micelles. Nanocarriers improve the performance of the passenger drug by allowing for higher payload capacity, prolonged blood circulation times, and increased accumulation in solid tumors, which can result in higher drug concentrations at tumor sites with improved antitumor efficacy and decreased toxicity to normal cells and tissues.

[0006] Among antitumor agents in development, lipophilic amines (Kazmi et al., Drug Metab Dispos. 2013, 41(4):897-905) appear particularly interesting because their mechanism of action differs from classical chemo therapeutic agents. The molecular targets of these compounds are mainly located in lysosomes and mitochondria rather than DNA, RNA or receptor tyrosine kinases (RTKs), which often induce resistance through the expression of p- glycoprotein, multidrug resistance-associated proteins or activation of alternative RTK pathways (Salehan et al., supra; Tredan et al., Journal of the National Cancer Institute. 2007, 99(19): 1441-1454).

[0007] At physiological pH, lipophilic amines are predominantly non-ionized and can passively diffuse across the lipid bilayers of the cell and cellular organelle membranes. Upon entering the acidic environment of the lysosomes or mitochondrial matrix, the amines become ionized and are therefore less able to efficiently diffuse out, resulting in pronounced accumulation within these cellular organelles. As a consequence, the amines cause various physiological and morphological perturbations of the lysosomal and mitochondrial apparatus, including inducing cell death (Sun et al., Cancer Res. 1994, 54(6): 1465-71 ; Duvvuri et al., ACS Chem Biol. 2006, 1(5):309-15; Duvvuri et al., Mol Pharm. 2005, 2(6):440-8; Marceau et al., Toxicol Appl Pharmacol. 2012, 259(1): 1-12). In lysosomes, the accumulation of lipophilic amines has been shown to alter lipid metabolism, induce lipidosis and cause a lysosomal volume increase and various morphological alterations (Arai et al., J Biochem. 2002, 132(4):529-34; Logan et al., J Pharm Sci. 2013, 102(11):4173-80). In mitochondria, the accumulation of lipophilic amines can cause internal membrane damage and increases in reactive oxygen species (ROS) (Edeas et al., Mitochondrion. 2013, 13(5):389-90; Plyavnik et al., Mini Rev Med Chem. 2006, 6(5):533-42). [0008] The effect on mitochondria is specific to cancer cells which, in contrast to normal cells, are characterized by very large mitochondrial membrane potentials (up to 150-160 mV, negative inside), which drive an extensive uptake of the lipophilic amines within the mitochondrial matrix (Sun et al., Cancer Res. 1994, 54(6): 1465-71; Modica-Napolitano et al., Adv Drug Deliv Rev. 2001, 49(l-2):63-70). The effect on lysosomes is similarly quite specific to tumor cells due to their increase in lysosomal enzymes and deregulation of enzymatic functions compared to normal cells, which provide acidification defects and metabolic perturbations that can damage the lysosomal apparatus in the presence of accumulated lipophilic amines (Wilson et al., Cancer Res. 1989, 49(3):507-10; Ndolo et al., PLoS One. 2012;7(l l):e49366).

[0009] In spite of their interesting potential therapeutic properties, a major drawback to the use of lipophilic amines as antitumor agents is their significant lipophilicity, which hinders their aqueous solubilization and limits their bioavailability and therapeutic efficacy. For more effective delivery of chemotherapeutics, lipophilic amines endowed with improved aqueous solubilization are needed to provide increased bioavailability and enhanced antitumor efficacy.

SUMMARY OF THE INVENTION

[0010] The present disclosure is directed to amphiphilic amine compounds that behave as antitumor agents with specific affinity and cytotoxic activity for tumor cells. The

amphiphilic amine compounds can also form micelles that serve as nanocarriers for other bioactive molecules. Figure 1 is a schematic of the assembly of amphiphilic amine compounds into micelles. The disclosure is further directed to methods of using the amphiphilic amine compounds and micelles and compositions containing the compounds to treat hyperproliferative disease, including cancer.

[0011] In one aspect, the disclosure provides a compound comprising a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the ring system is at least tricyclic, contains at least two nitrogen atoms, and contains an azetidine ring or an aziridine ring. In a further aspect, the hydrophilic fused polycyclic hydrocarbon ring system comprises structural formula (I):

wherein x and y are individually selected from the group consisting of 0 to 10; z is 0 or 1; and one or more carbon atoms in structural formula (I) is optionally substituted with one or more groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, x is 3, y is 4, and z is 1. In another aspect, x is 3, y is 4, and z is 0. In another aspect, x is 3, y is 5, and z is 1. In another aspect, x is 3, y is 5, and z is 0. In another aspect, x is 3, y is 3, and z is 1. In another aspect, x is 3, y is 3, and z is 0. The compound may further comprise at least one substituted or unsubstituted Cs_2o alkyl group.

[0012] In another aspect, the compound comprises structural formula (II), (III), or (IV):

wherein Ri and R₂ are individually selected from the group consisting of hydrogen and substituted or unsubstituted Cs_₂o alkyl, and one or more carbon atoms in structural formula (II), (III), or (IV) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, Ri is H and R₂ is Cs_₂o alkyl. In another aspect, Ri is C₅-₂o alkyl and R₂ is hydrogen. In a further aspect, Ri or R₂ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. For example, Ri or R₂ can be tetradecyl.

[0013] In another aspect, the compound comprises structural formula (V), (VI), or (VII):

wherein R₃ is substituted or unsubstituted Cs_2o alkyl, and one or more carbon atoms in structural formula (V), (VI), or (VII) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, R₃ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. For example, R₃ can be tetradecyl.

[0014] In one aspect, the compound is selected from the group consisting of:

[0015] In a second aspect, the disclosure provides a micelle comprising a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the ring system is at least tricyclic, contains at least two nitrogen atoms, and contains an azetidine ring or an aziridine ring. In one aspect, the micelle comprises a hydrophobic hydrocarbon group attached to the hydrophilic fused polycyclic hydrocarbon ring system through a linker. In a further aspect, the hydrophobic hydrocarbon group is substituted or unsubstituted Cs_2o alkyl. In one aspect, the hydrophobic fused polycyclic hydrocarbon ring system comprises structural formula (I):

wherein x and y are individually selected from the group consisting of 0 to 10; z is 0 or 1; and one or more carbon atoms in structural formula (I) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, x is 3, y is 4, and z is 1. In another aspect, x is 3, y is 4, and z is 0. In another aspect, x is 3, y is 5, and z is 1. In another aspect, x is 3, y is 5, and z is 0. In another aspect, x is 3, y is 3, and z is 1. In another aspect, x is 3, y is 3, and z is 0. In one aspect, the micelle further comprises substituted or unsubstituted Cs_2o alkyl.

[0016] In another aspect, the micelle comprises structural formula (II), (III), or (IV):

wherein Ri and R₂ are individually selected from the group consisting of hydrogen and substituted or unsubstituted Cs_₂o alkyl, and one or more of the carbon atoms in structural formula (II), (III), or (IV) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, Ri is H and R₂ is Cs_₂o alkyl. In another aspect, Ri is C₅-₂o alkyl and R₂ is hydrogen. In a further aspect, Ri or R₂ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. For example, Ri or R₂ can be tetradecyl.

[0017] In another aspect, the micelle comprises structural formula (V), (VI) or (VII):

(VI),

wherein R₃ is substituted or unsubstituted Cs_2o alkyl, and one or more carbon atoms in structural formula (V), (VI), or (VII) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, R₃ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. For example, R₃ can be tetradecyl.

[0018] In one aspect, the micelle comprises a plurality of compounds selected from the group consisting of:

and combinations thereof.

[0019] The disclosure also provides a composition comprising a compound or micelle comprising a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the ring system: is at least tricyclic, contains at least two nitrogen atoms, and contains an azetidine ring or an aziridine ring. The composition further comprises a pharmaceutically acceptable excipient. In one aspect, the composition further comprises a therapeutic agent encapsulated within the micelle. The therapeutic agent may be selected from the group consisting of antitumor agents, antineoplastic agents, prodrugs, analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, adrenolytics, adrenocorticoids, adrenomimetics, anticholinergic agents, anticholinesterases,

anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiants, antacids, antidiarrheals, antidotes, antifolics, antipyretics, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, antihelmintics, antibiotics, anticoagulants, antidepressants, antiepileptics, antibacterials, antifungals, antifibrotic agents, anti-infective agents, anti-parasitic agents, antihistamines, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antiprotozoal agents, antiviral agents, cardiac drugs, anxiolytic sedatives, beta- adrenoceptor blocking agents, corticosteroids, cough suppressants, dopaminergics, hemostatics, hematological agents, hypnotics, immunological agents, muscarinics, neurological drugs, bioactive peptides, steroid hormones, nucleic acids, vaccines, anti-protozoan drugs, barbiturates, photosensitizer substances

parasympathomimetics, prostaglandins, radio-pharmaceuticals, sedatives, stimulants, sympathomimetics, vitamins, xanthines, growth factors, hormones, antiprion agents, and combinations thereof.

[0020] The therapeutic agent may be an antitumor agent selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti-androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active agent; an alkylating agent; a retinoid, a carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; an antitumor antibiotic; a nitrosourea, and combinations thereof. In one aspect, the antitumor agent is selected from the group consisting of doxorubicin, etoposide, paclitaxel, fenretinide, and combinations thereof.

[0021] In one aspect, the disclosure provides a method of inhibiting tumor cell growth comprising contacting a cell with a compound, micelle, or composition of the invention, in an amount effective to inhibit growth of the tumor cell. In another aspect, the disclosure provides use of a compound, micelle, or composition of the invention for the preparation of a medicament, wherein the medicament comprises an amount of the compound, micelle, or composition, that is effective for inhibiting growth of a tumor cell. In still another aspect, the disclosure provides a composition for use in inhibiting tumor cell growth comprising a compound, micelle, or composition of the invention in an amount effective to inhibit growth of a tumor cell.

[0022] In another aspect, the disclosure provides a method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of a compound, micelle, or composition of the invention to the subject. In another aspect, the disclosure provides use of a compound, micelle, or composition of the invention for the preparation of a medicament, wherein the medicament comprises an amount of the compound, micelle, or composition that is effective for treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder. In still another aspect, the disclosure provides a composition for use in treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder comprising a compound, micelle, or composition of the invention in an amount effective to treat or prevent a neoplastic, hyperplastic or hyperproliferative disorder. In one aspect, the hyperproliferative disorder is selected from the group consisting of cancer, benign prostate hyperplasia, colorectal neoplasia, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, dermatofibroma, pilar cyst, pyogenic granuloma, and juvenile polyposis syndrome.

[0023] In a preferred aspect, the disclosure provides a method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of a compound, micelle, or composition described herein to the subject. In another aspect, the disclosure provides use of a compound, micelle, or composition of the invention for the preparation of a medicament, wherein the medicament comprises an amount of the compound, micelle, or composition that is effective for treating cancer. In still another aspect, the disclosure provides a composition for use in treating cancer comprising a compound, micelle, or composition of the invention in an amount effective to treat cancer. In one aspect, the cancer is selected from the group consisting of adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell

leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma,

angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney,

craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma,

dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-

Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangio sarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non- small cell carcinoma, non- small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T- lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0024] The foregoing summary is not intended to define every aspect of the invention, and other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawings. The present disclosure is intended to be related as a unified document, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, paragraph, or section of this disclosure. In addition, the disclosure includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated. If aspects of the disclosure are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. Additional features and variations of the disclosure will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 depicts the self-assembly of "RCn" amphiphilic amine compounds into micelles in an aqueous environment.

[0026] Figure 2 depicts the mass spectrum analysis of the compound 2-tetradecyl-4,8- diazatricyclo [6.5.0.0¹ ,⁴] tridecane (RC _{\ 6}) . [0027] Figure 3 depicts western blot analysis of caspase 3, 8 and 9 in SH-SY5Y cells treated with 5 μΜ RC₁₆. Figure 3A shows SH-SY5Y cells treated with RC₁₆ for 2, 8, 12, or 24 hours. Figure 3B shows SH-SY5Y cells treated with RC₁₆ for 4, 6, 12, 24, or 36 hours. M denotes the marker. H3h and H9h denote HeLa cells treated with 5 μΜ staurosporine for 3 hours and 9 hours, respectively, as a positive control of apoptosis. S9h denotes SH-SY5Y cells treated with 5 μΜ staurosporine for 9 hours as a positive control of apoptosis. HPR denotes SH-SY5Y treated with 5 μΜ fenretinide in ethanol for 24 hours as a positive control of apoptosis.

[0028] Figure 4 depicts activation of caspases following treatment with RC₁₆.

[0029] Figure 5 depicts levels of reactive oxygen species (ROS) measured in relative fluorescence units (RFU) in SH-SY5Y, A673, and Osteomet cells following treatment with RC₁₆ for 12 hours or 24 hours. .

[0030] Figure 6 depicts transmission electron microscopy images of tumor and normal cells following treatment with 5 μΜ RC₁₆. Figure 6A depicts SH-SY5Y neuroblastoma cells. Figure 6B depicts normal human fibroblast cells.

[0031] Figure 7 depicts electron microscopy images of the surface of tumor and normal cells following treatment with 5 μg/mL RC₁₆. Figure 7 A depicts SH-SY5Y cells treated with 5 μg/mL RC₁₆. Figure 7B depicts control (untreated) SH-SY5Y cells. Figure 7C depicts normal human fibroblast (HGF 68) cells treated with 5 μg/mL RC₁₆. Figure 7D depicts untreated normal human fibroblast cells.

[0032] Figure 8 depicts the cytotoxicity induced by RC₁₆ in cells with altered sialic acid residues. Figure 8A depicts cytotoxicity in S462TY cells following treatment with 50 mU/mL neuramidase and 3 μΜ RC₁₆ (RC16-NE) or RC₁₆ alone. Figure 8B depicts cytotoxicity in CHLA-20, SK-N-AS, SH-SY5Y, and S462TY cells following treatment with 3 μΜ RC₁₆,3 μΜ sialic acid (SA) or a mixture of 3 μΜ RC₁₆ and 3 μΜ sialic acid (Mix).

[0033] Figure 9 depicts pharmacokinetic data for RC₁₆. Figure 9 A depicts the plasma concentration (log ng/mL) versus time (hours) profile for RC₁₆ after intravenous

administration. Figure 9B depicts the plasma concentration (ng/mL) versus time (hours) profile for RC₁₆ after oral administration.

[0034] Figure 10 depicts the biodistribution of RC₁₆ in nude mice bearing CHLA-20 tumors. Figure 10A depicts whole-body imaging of mice at 12, 24, and 48 hours after intravenous injection of fluorescently-labeled RC₁₆. Figure 10B depicts the fluorescence intensity in tissues and organs at 24, 48, and 72 hours after the intravenous injection.

[0035] Figure 11 depicts cytotoxicity in CHLA-20, SK-N-AS, A673, A549, and S462TY tumors in nude mice measured as relative tumor volume (RTV) following intravenous administration of RC₁₆ and in CHLA-20 cells following oral administration of RC₁₆ compared to control (tumors in untreated mice; CTR).

[0036] Figure 12 depicts body weight in mice following intravenous or oral administration of RC₁₆. Figure 12A depicts the body weight of mice following an intravenous injection of RC₁₆ (Treated) compared to untreated mice (CTR). Figure 12B depicts the body weight of mice following oral administration of RC₁₆ (Treated) compared to untreated mice (CTR).

[0037] Figure 13 depicts survival in a metastatic model following treatment with RC₁₆ and in an untreated control (CTR).

[0038] Figure 14 depicts electron microscopy images of RC₁₆ micelles in an aqueous environment.

[0039] Figure 15 depicts cytotoxic activity of RCie-doxorubicin micelles, free doxorubicin (DOXO), and free RC₁₆. Figure 15A depicts the cytotoxic activity of RCie-doxorubicin micelles (top) and free doxorubicin (bottom). Figure 15B depicts the cytotoxic activity of free RC₁₆.

[0040] Figure 16 depicts cytotoxic activity of RC^-etoposide micelles, free etoposide (ETO), and free RC₁₆. Figure 16A depicts the cytotoxic activity of RC^-etoposide micelles (top) and free etoposide (bottom). Figure 16B depicts the cytotoxic activity of free RC₁₆.

[0041] Figure 17 depicts cytotoxic activity of RCie-paclitaxel micelles, free paclitaxel (PX), and free RC₁₆. Figure 17 A depicts the cytotoxic activity of RC^-paclitaxel micelles (top) and free paclitaxel (bottom). Figure 17B depicts the cytotoxic activity of free RC₁₆.

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure provides amphiphilic amine compounds that have antitumor, e.g., cytotoxic, activity against many different types of hyperproliferative cells, but with lower toxicity to normal tissue compared to other existing drugs. The antitumor activity of the compounds involves alterations in mitochondria and lysosomes. In one aspect, the amphiphilic amine compounds interact with the polysialic acid expressed on the glycoproteins and gangliosides of tumor cell membranes, representing a mechanism of action not present in other antitumor drugs. The amphiphilic amine compounds can be used as therapeutic agents alone and can also serve as nanocarriers for other bioactive molecules. Due to their amphiphilic character, the compounds can spontaneously self-assemble in aqueous solution, forming micelles which can complex hydrophobic or partially hydrophobic drugs. Complexes of the compounds with antitumor agents such as doxorubicin, etoposide, and paclitaxel showed increased antitumor activity in comparison with the free drugs.

[0043] As used herein, the term "antitumor agent" or "chemotherapeutic agent" refers to any compound that is toxic with respect to hyperproliferative cells or inhibits the growth or proliferation of hyperproliferative cells. The term "antitumor agent" includes

chemotherapeutic agents, biologies, antibodies, small molecules, peptides and antisense oligonucleotides. Exemplary antitumor agents include, but are not limited to, an aromatase inhibitor, an anti-estrogen, an anti-androgen, a gonadorelin agonist, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a microtubule active agent, an alkylating agent, a retinoid, a carotenoid, a tocopherol, a cyclooxygenase inhibitor, an MMP inhibitor, a mTOR inhibitor, an antimetabolite, a platin compound, a methionine aminopeptidase inhibitor, a bisphosphonate, an antiproliferative antibody, a heparanase inhibitor, an inhibitor of Ras oncogenic isoforms, a telomerase inhibitor, a proteasome inhibitor, a compound used in the treatment of hematologic malignancies, a Flt-3 inhibitor, an Hsp90 inhibitor, a kinesin spindle protein inhibitor, a MEK inhibitor, an antitumor antibiotic, a nitrosourea, a compound targeting/decreasing protein or lipid kinase activity, a compound targeting/decreasing protein or lipid phosphatase activity, any further anti- angiogenic compound, and combinations thereof. Specific examples of antitumor agents include, but are not limited to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fenretinide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof. Additional examples of antitumor or chemotherapeutic agents are known in the art.

[0044] As used herein, the term "fused polycyclic hydrocarbon ring system" refers to a chemical structure comprising at least two fused hydrocarbon rings. The term "fused" means that each ring in the polycyclic ring system shares at least two atoms, i.e., at least one side of the ring, with at least one other ring in the system. A fused polycyclic hydrocarbon ring system may contain three rings (tricyclic), four rings, five rings, six rings, or more. A fused polycyclic hydrocarbon ring system may be saturated or unsaturated. A fused polycyclic hydrocarbon ring system may contain one or more heteroatoms. One or more carbon atoms in the fused polycyclic hydrocarbon ring system may optionally be substituted with one or more groups selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. Additionally or alternatively, one or more carbon atoms in the fused polycyclic hydrocarbon ring system may optionally be substituted with a linker. The term "substituted with" refers to the replacement of one or more hydrogen atoms attached to a carbon atom with one or more of the aforementioned groups.

[0045] As used herein, the term "alkyl" refers to a straight or branched C_1-2o hydrocarbon chain, including but not limited to heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nondecyl, eicosyl, and combinations thereof. The term Cx_y means the alkyl has between "X" and "Y" carbon atoms. An alkyl may be saturated, i.e., contain only single bonds between carbon atoms, or may optionally contain one or more carbon-carbon double and/or carbon-carbon triple bonds. One or more carbon atoms in an alkyl may optionally be substituted with one or more groups selected from the group consisting of halo, hydroxy, oxo, carboxamido, carboxy, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

[0046] As used herein, the term "heteroatom" refers to an atom that is not carbon that replaces a carbon atom in the backbone of a chemical structure. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and iodine. The prefix "hetero" before a group indicates that the group contains at least one heteroatom.

[0047] As used herein, the term "halo" or "halogen" refers to one or more atoms of fluorine, chlorine, bromine, iodine, and combinations thereof.

[0048] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group. Aryl also refers to bicyclic and tricyclic carbon rings, where one ring is aromatic and the others are saturated, partially unsaturated, or aromatic. [0049] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic aliphatic ring.

[0050] The term "micelle" refers to an aggregate structure comprising a plurality of amphiphilic molecules oriented to provide the structure with a hydrophobic interior region and a hydrophilic exterior surface.

[0051] The term "linker" refers to a carbon-carbon or carbon-heteroatom bond or other chemical moiety connecting the hydrophilic group to the hydrophobic group on an amphiphilic molecule. A linker may include one or more polymers. Exemplary polymers may be linear or branched and include, but are not limited to, polysaccharides, polypeptides, copolymers, polyethylene glycol (PEG), polyoxyethylene glycol, polypropylene glycol, monomethoxy-polyethylene glycol, dextran, hydroxyethyl starch, cellulose, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and combinations and copolymers thereof.

[0052] The term "therapeutically effective" depends on the condition of a subject and the specific compound administered. The term refers to an amount effective to achieve a desired clinical effect. A therapeutically effective amount varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the subject, and ultimately is determined by the health care provider. In one aspect, a therapeutically effective amount of a compound, micelle, or composition of the invention is an amount effective to inhibit growth of hyperproliferative cells, prevent tumor cell metastasis, and/or result in cell death, e.g., via apoptosis or necrosis.

[0053] The present disclosure provides a class of amphiphilic amine compounds having antitumor activity. The disclosure also provides micelles formed from a plurality of the amphiphilic amine compounds. In one aspect, the amphiphilic amine compounds self- assemble into micelles having a hydrophilic exterior and a hydrophobic interior when dispersed in an aqueous solution. In one aspect, the micelles comprises a plurality of compounds of the present disclosure oriented such that the hydrophilic fused polycyclic hydrocarbon ring systems form the exterior surface of the micelles and the hydrophobic hydrocarbon groups reside in the interior of the micelles, as shown in Figure 1.

[0054] The compounds and micelles comprise a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the fused polycyclic hydrocarbon ring system is at least tricyclic, contains at least two nitrogen atoms, and contains an azetidine ring or an aziridine ring. In one aspect, the fused polycyclic hydrocarbon ring system is tricyclic. In another aspect, the fused polycyclic hydrocarbon ring system contains four rings or five rings. In one aspect, the fused polycyclic hydrocarbon ring system contains two nitrogen atoms. In one aspect, the fused polycyclic hydrocarbon ring system contains at least 7 carbon atoms, at least 8 carbon atoms, at least 9 carbon atoms, at least 10 carbon atoms, at least 1 1 carbon atoms, at least 12 carbon atoms, at least 13 carbon atoms, at least 14 carbon atoms, or at least 15 carbon atoms. Preferably, the fused polycyclic hydrocarbon ring system contains 7 to 10 carbon atoms, 8 to 1 1 carbon atoms, 9 to 12 carbon atoms, 9 to 14 carbon atoms, 10 to

13 carbon atoms, 10 to 15 carbon atoms, or 13 to 15 carbon atoms. In a further aspect, one or more carbon atoms in the fused polycyclic ring system is replaced with one or more heteroatoms. In one aspect, one or more carbon atoms in the fused polycyclic hydrocarbon ring system is optionally substituted with one or more groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

[0055] In addition to a hydrophilic fused polycyclic hydrocarbon ring system, the amphiphilic amine compounds or micelles of the present disclosure may comprise a substituted or unsubstituted hydrophobic hydrocarbon group. In one aspect, the hydrophobic hydrocarbon group is a straight or branched alkyl chain. In one aspect, the hydrophobic hydrocarbon group is a straight or branched alkyl chain having 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 1 1 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. In a further aspect, the hydrophobic hydrocarbon group is a straight chain alkyl having 5 to 20 carbon atoms (Cs_2o), 10 to 20 carbon atoms (QO-M), 10 to 15 carbon atoms (Cio-is), 15 to 20 carbon atoms (Ο_15-2ο), 7 to 18 carbon atoms (C_7-18), 10 to 18 carbon atoms (Cio-is), 12 to 18 carbon atoms (C_12-18),

14 to 18 carbon atoms (C_14-18), or 16 to 18 carbon atoms (C_16-18). In one aspect, the hydrophobic hydrocarbon group has a molecular weight less than 281.

[0056] In one aspect, the substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system and substituted or unsubstituted hydrophobic hydrocarbon group are attached via a linker. In one aspect, the linker is a carbon-carbon bond or carbon-heteroatom bond (e.g., carbon-nitrogen), i.e., the substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system and substituted or unsubstituted hydrophobic

hydrocarbon group are directly linked. In another aspect, the linker is a polymer, for example, linear or branched polyethylene glycol (PEG), linear or branched polysaccharide, linear or branched polypeptide, or copolymers thereof. In one aspect, the linker attaches the hydrophobic hydrocarbon group to the azetidine ring or aziridine ring.

[0057] In one aspect, the fused polycyclic hydrocarbon ring system comprises structural formula (I):

wherein x and y are individually selected from the group consisting of 0 to 10, z is 0 or 1, and one or more carbon atoms in structural formula (I) is optionally substituted with one or more groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, x is 3, y is 4, and z is 1. In another aspect, x is 3, y is 4, and z is 0. In another aspect, x is 3, y is 5, and z is 1. In another aspect, x is 3, y is 5, and z is 0. In another aspect, x is 3, y is 3, and z is 1. In another aspect, x is 3, y is 3, and z is 0.

[0058] In one aspect, the compounds and micelles of the present disclosure comprise structural formula (II), (III), or (IV):

wherein Ri and R₂ are individually selected from the group consisting of hydrogen and substituted or unsubstituted Cs_₂o alkyl, and one or more of the carbon atoms in structural formula (II), (III), or (IV) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, Ri is hydrogen, and R₂ is Cs_₂o alkyl. In another aspect, Ri is Cs_₂o alkyl, and R₂ is hydrogen. In still another aspect, Ri is Cs_₂o alkyl, and R₂ is C5-20 alkyl that may be the same as or different from Ri. In one aspect, Ri and/or R₂ is selected from the group consisting of heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nondecyl, eicosyl, and combinations thereof. In preferred embodiments, Ri or R₂ is tetradecyl (C14H29).

[0059] In another aspect, the compounds and micelles of the present disclosure comprise structural formula (V), (VI), or (VII):

(V),

wherein R₃ is substituted or unsubstituted Cs_2o alkyl, and one or more carbon atoms in structural formula (V), (VI) or (VII) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof. In one aspect, R is selected from the group consisting of heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nondecyl, eicosyl, and combinations thereof. In a preferred embodiment, R₃ is tetradecyl.

[0060] The compounds and micelles of the present disclosure may be made by any suitable method known in the art and those described herein. See, e.g., Brandi et al., Chem Rev. 2008, 108(9):3988-4035.

[0061] The present disclosure further includes all possible stereoisomers and geometric isomers of the compounds and micelles comprising structural formula (I), (II), (III), (IV), (V), (VI), or (VII). The present invention includes both racemic compounds and optically active isomers. When a compound of structural formula (I), (II), (III), (IV), (V), (VI), or (VII) is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereo specific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent. Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art. Additionally, in situations where tautomers of the compounds of structural formula (I), (II), (III), (IV), (V), (VI), or (VII) are possible, the present invention is intended to include all tautomeric forms of the compounds. All combinations of formulas (I) to (VII) are contemplated herein. For example, the disclosure provides, but is not limited to, compounds, micelles, and compositions comprising any of the following structures:

and combinations thereof.

[0062] The present disclosure also provides compositions comprising the compounds and/or micelles described herein. In one aspect, a composition comprises a compound and/or micelle described herein and a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient is an aqueous vehicle, carrier, buffer, or diluent.

Optionally, the compound in the composition is in the form of a physiologically acceptable salt, ester, or solvate, which is encompassed by the invention. The term "physiologically acceptable salt" refers to any salt that is pharmaceutically acceptable. Some examples of physiologically acceptable salts include acetate, hydrochloride, hydrobromide, sulfate, citrate, tartrate, glycolate, oxalate, and combinations thereof.

[0063] The particular composition employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound and/or micelle, and by the route of administration. Physiologically-acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders, e.g., lyophilizates, for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Patent No. 5,466,468). Injectable formulations are further described in, e.g., Pharmaceutics and

Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). A composition comprising a compound and/or micelle described herein is, in one aspect, placed within a container, along with packaging material that provides instructions regarding the use of such compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.

[0064] The present disclosure also provides methods of treatment. In one aspect, the disclosure provides a method of inhibiting tumor cell growth comprising contacting a cell with a compound, micelle, or composition described herein in an amount effective to inhibit growth of the tumor cell. In another aspect, the disclosure provides a method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of a compound, micelle, or composition described herein to the subject.

[0065] In one aspect, the disease to be treated is cancer. Examples of treatable cancers include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell

prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy- associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangio sarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non- small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T- lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.

[0066] In another embodiment, the present disclosure provides a method of treating a benign hyperproliferative disorder including but not limited to, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, benign prostate hyperplasia, dermatofibroma, pilar cyst, pyogenic granuloma, and juvenile polyposis syndrome.

[0067] In one aspect of the present methods, a therapeutically effective amount of a compound, micelle, or composition described herein, typically formulated in accordance with pharmaceutical practice, is administered to a subject in need thereof. Whether such a treatment is indicated depends on the individual case and is subject to medical assessment that takes into consideration signs, symptoms, and/or malfunctions that are present, the risks of developing particular signs, symptoms and/or malfunctions, and other factors. A particular administration regimen for a particular subject will depend, in part, upon the compound, micelle, or composition, the amount administered, the route of administration, and the cause and extent of any side effects. The amount administered to a subject (e.g., a mammal, such as a human) in accordance with the invention should be sufficient to effect the desired response over a reasonable time frame. Dosage typically depends upon the route, timing, and frequency of administration. Accordingly, a clinician titers the dosage and modifies the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art.

[0068] Purely by way of illustration, the methods of the disclosure comprise administering, e.g., from about 0.1 g/kg up to about 100 mg/kg or more of compound, micelle, or composition, depending on the factors mentioned above. In other embodiments, the dosage ranges from 1 g/kg up to about 50 mg/kg; or 5 g/kg up to about 25 mg/kg; or 10 g/kg up to about 10 mg/kg. Some conditions require prolonged treatment, which may or may not entail administering lower doses over multiple administrations. If desired, a dose is administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day. The treatment period will depend on the particular condition and may last one day to several days, weeks, months, or years.

[0069] Suitable methods of administering a physiologically-acceptable composition, such as a pharmaceutical composition comprising a compound and/or micelle described herein, are well known in the art. Although more than one route can be used to administer a compound, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition comprising the compound and/or micelle is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation. For example, in certain circumstances, it will be desirable to deliver the pharmaceutical composition orally; through injection or infusion by intravenous, intratumoral, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means; by controlled, delayed, sustained or otherwise modified release systems; or by implantation devices. In one aspect, drug exposure can be optimized by maintaining constant drug plasma concentrations over time. Such a steady-state is generally accomplished in clinical settings by continuous drug infusion at doses depending on the drug clearance and the plasma concentration to be sustained. If desired, the composition is administered regionally via intratumoral, administration, intrathecal administration, intracerebral (intra-parenchymal) administration, intracerebroventricular administration, or intraarterial or intravenous administration targeting the region of interest. Alternatively, the composition is administered locally via implantation of a matrix, membrane, sponge, or another appropriate material onto which the desired compound has been absorbed or encapsulated. Where an implantation device is used, the device is, in one aspect, implanted into any suitable tissue or organ, and delivery of the desired compound is, for example, via diffusion, timed-release bolus, or continuous administration.

[0070] A compound, micelle, or composition described herein can be administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the compound, micelle, or composition can be administered, per dose, in an amount of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams, including all doses between 0.005 and 500 milligrams. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention. In practice, a clinician determines the actual dosing regimen that is most suitable for an individual subject, which can vary with the age, weight, condition and response of the particular subject.

[0071] The compounds and micelles of the present invention may be used in combination with other antitumor therapies. Examples of antitumor therapies that can be used in combination with the compounds and micelles include surgery, radiotherapy (e.g., gamma- radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biologic response modifiers (e.g., interferon, interleukin, tumor necrosis factor (TNF), hyperthermia and cryotherapy), agents to attenuate any adverse effect (e.g., antiemetics), gene therapy, viruses, and any other chemotherapeutic agent.

[0072] In one aspect, the amphiphilic amine compounds can complex other bioactive molecules or antitumor agents including chemotherapeutic agents such as doxorubicin, fenretinide, etoposide and/or paclitaxel. This complexation ability is based on the

amphiphilic character of the compounds triggering a spontaneous self-assembling of molecules in water with formation of micelles, as depicted in the schematic in Figure 1. As it is well known, such micelles with an hydrophobic inner core and an hydrophilic outer shell may encapsulate hydrophobic or partially hydrophobic drugs into their micellar structure (Torchilin VP, Pharm Res. 2007, 24(1): 1-16; Lopez-Davila et al., Curr Opin Pharmacol. 2012; 12(4):414-9), forming micellar assemblates also referred to as complexes.

Complexation of poorly water soluble drugs into the micelles may improve the aqueous solubility of the drugs and consequently their bioavailability and therapeutic efficacy (Orienti et al., Nanomedicine. 2012, 8(6):880-90; Carosio et al., J Pharm Pharmacol. 2012, 64(2):228-36; Orienti et al., Drug Deliv. 2009, 16(7):389-98; Zuccari et al., Drug Deliv. 2009, 16(4): 189-95; Orienti et al, Drug Deliv. 2007, 14(4):209-17; Orienti et al,

Biomacromolecules. 2006, 7(11):3157-63). Moreover, micellar complexation may modify the drug disposition in the body by preventing an uncontrolled drug distribution and promoting drug accumulation in solid tumors where the discontinuity of the capillaries and the lack of an efficient lymphatic drainage favors the entrapment and retention of

nanoparticulate systems such as micelles, liposomes or soluble macromolecules (Torchilin and Lopez-Davila et al., supra).

[0073] In particular, the encapsulation of paclitaxel and etoposide within micelles is desirable because the very low aqueous solubility of these drugs strongly limit their therapeutic potential (Singh et al., Crit Rev The r Drug Carrier Syst. 2009, 26(4):333-72; Ukawala et al., Drug Deliv. 2012, 19(3): 155-67) and the excipients used to improve their solubility may cause serious adverse reactions. Apart from Abraxane® (Celgene Corp., Summit, NJ), which contains nanoparticles of paclitaxel and human serum albumin, all the other currently marketed formulations of paclitaxel and etoposide contain liquid mixtures of Cremophor/Kolliphor EL® (polyethoxylated castor oil, BASF, Florham Park, NJ) and ethanol or benzyl alcohol, polysorbate 80, polyethylene glycol 300, and ethanol, to provide drug solubilisation and avoid drug precipitation in the infusion fluids or in plasma upon infusion. The use of such excipients for drug solubilization have been associated with serious side effects such as pain, inflammation, tissue damage, necrosis at the site of injection, and substantial hemolysis (Strickley RG, Pharm Res. 2004, 21(2):201-30). In addition,

Cremophor EL also causes vasodilation, labored breathing, lethargy, hypotension, and leaching of plasticizers, such as diethylhexylpthalate, from the polyvinylchloride infusion bags/sets.

[0074] The micellar complexation of doxorubicin is also advantageous to modify the biodistribution of the drug in addition to increase its solubility. Doxorubicin induces a strong dose-limiting cardiotoxicity (Berthiaume et al., Cell Biol Toxicol. 2007, 23(1): 15-25), and its encapsulation in nanoparticulate carriers such as micelles or liposomes has proven to decrease its distribution to the heart, resulting in a reduction of the cardiotoxic effect (Mohan et al., Mol Pharm. 2010, 7(6): 1959-73; Sun et al., Biomaterials. 2013, 34(28):6818-28; Tardi et al., J Drug Target. 1996, 4(3): 129-40; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008, 25(1): 1-61). Simultaneously, increased solubility of doxorubicin by micellar complexation has proven to enhance its therapeutic activity (Gao et al., Int J Nanomedicine. 2013, 8:971- 82).

[0075] The compounds and micelles described herein possess strong cytotoxic activity against a variety of cancer types, but with low toxicity to normal cells. Without intending to be bound by any theory, the effects of the compounds and micelles on mitochondria and lysosomes in tumor cells, as well as their interaction with tumor cell-surface sialic acid, appear to contribute to the antitumor activity.

[0076] The particular molecular structure of the amphiphilic amine compounds and micelles may account for the multiple mechanisms of action. The nitrogen atoms in the amphiphilic amine compounds and micelles allow for high water solubility, and the fused polycyclic hydrocarbon ring system allows the compound to partition in biological membranes in the presence of the high aqueous solubility. For example, structural formulas (II), (III), (IV), (V), (VI), and (VII) comprise (a) a 3- or 4-membered ring, (b) a 6-membered ring and (c) a 6-, 7- or 8-membered ring condensed in a tricyclic assembly linked to an alkyl tail. The hydrophobic aliphatic cycles surrounding the amine groups in the tricyclic assembly provide a counter-balance to the hydrophilicity increase triggered by the protonation of the amine groups in an aqueous physiological environment, thus allowing the molecules to partition through biological membranes in the presence of positive charges. The compounds and micelles accumulate in mitochondria based on the high negative transmembrane potential of the mitochondrial inner membrane (negative inside), which favors passage of protonated molecules into the matrix, and by the acidic pH of the intermembrane space, due to the export of hydrogen ions from the matrix, which attracts amine molecules from the cytoplasm inside the mitochondria by the presence of an electrochemical gradient through the outer

mitochondrial membrane (Edeas et al., supra). Moreover, the high content of negatively charged phospholipids such as cardiolipin on the outer mitochondrial membrane further favors accumulation of protonated molecules on the mitochondrial surface (Riedl et al., Chem Phys Lipids. 2011; 164(8):766-81). Similarly, the compounds and micelles can accumulate in the acidic inner environment of lysosomes driven by the same suitable

hydrophilic/hydrophobic balance allowing their partition across the lysosomal membrane and a further protonation inside (Duvvuri et al., Mol Pharm. 2005, 2(6):440-8; Marceau et al., Arai et al., and Logan et al., supra).

[0077] The effects of the amiphiphilic amine compounds and micelles on mitochondria and lysosomes are more pronounced in cancer cells than normal cells, likely due to differences in organelle membrane potentials. Mitochondrial and lysosomal alterations and increases in ROS occurred after treatment with the amphiphilic amine compounds or micelles in cancer cells, but not in normal cells. The finding was in accordance with the observation that the mitochondria of cancer cells, in contrast to normal cells, are characterized by a very large membrane potential across the mitochondrial membrane (up to 150-160 mV, negative inside) driving an extensive uptake of partitionable amines within the mitochondrial matrix (Edease et al., Plyavnik et al., Sun et al., and Modica-Napolitano et al., supra). Similarly, the lysosomes of cancer cells are characterized by an increase in lysosomal enzymes and a deregulation of the enzymatic functions resulting in acidification defects and metabolic perturbations, which may make the lysosomal structure more sensitive to the accumulation of partitionable amines (Wilson et al. and Ndolo et al., supra).

[0078] In one aspect, the amphiphilic amine compounds and micelles interact with sialic acid. The interaction with sialic acid enables selectivity for tumor cells due to higher levels of the polysialylated glycoproteins and gangliosides on their surface (Seifert et al., Arch Biochem Biophys. 2012, 524(l):56-63; Falconer et al., Curr Cancer Drug Targets. 2012, 12(8):925-39; Cantu et al., Chem Phys Lipids. 2011, 164(8):796-810). The molecular interaction of the compounds and micelles with sialic acid may be attributed to the presence of the 3-membered aziridine ring or 4-membered azetidine ring in the polycyclic assembly of the molecules which makes them very reactive towards the sialic acid residues present as polymers (polysialic acid) on the glycoproteins and gangliosides of the tumor cell membranes. Azetidine rings are known to easily react with nucleophiles undergoing acid catalyzed ring opening reactions (Kenis et al., J Org Chem. 2012;77(14):5982-92; Ghorai et al., J Org Chem. 2007, 72(15):5859-62; Vargas-Sanchez et al., Org Lett. 2006, 8(24):5501-4) due to their high ring strain energy. The interaction with polysialic acid can produce structural modifications on the glycoproteins and gangliosides of the tumor cell surface, thus inducing perturbations to the functional properties of the cell membrane that, in addition to the other alterations induced in mitochondria and lysosomes, contribute to antitumor activity. In normal cells, on the contrary, the very limited expression of sialic acid prevents any significant interaction of the amphiphilic amine compound or micelle with the cell surface.

[0079] In one aspect, the amphiphilic amine compounds or micelles of the present disclosure exert their antitumor activity via the induction of apoptosis or necrosis. Apoptotic cells may be identified by histological markers such as nuclear and cytoplasmic condensation and cellular fragmentation. Necrotic cells may be identified by histological markers such as cellular and organelle swelling, chromatin flocculation, loss of nuclear basophilia, degraded cytoplasmic structure, impaired organelle function, increased membrane permeability, and cytolysis. One mechanism for inducing apoptosis and/or necrosis involves the activation of initiator caspases (e.g., caspase-2, caspase-8, caspase-9, caspase-10), executioner caspases (e.g., caspase-3, caspase-6) and also pro-inflammatory caspases (e.g., caspase-1 and caspase - 13). The role of the pro-inflammatory caspases in cell death is likely related to their ability to induce host inflammatory responses in vivo. Caspase activity can therefore be analyzed as a measure of apoptosis and/or necrosis. Other methods for detecting cell death via apoptosis and/or necrosis known in the art are also suitable for measuring the antitumor activity of the compounds or micelles of the present disclosure, including Tdt-mediated dUTP nick-end labeling (TUNEL) assays, in situ end labeling (ISEL) assays, DNA laddering assays, DNA fragmentation assays, fluorescence-activated cell sorting (FACS)/flow cytometry analysis, microscopy analysis, cell staining (e.g., using trypan blue, propidium iodide,7-actinomycin D, Annexin V, Hoescht, fluorescein diacetate-green, DAPI, and/or other dyes known in the art) assays, and enzyme-linked immunosorbent assays (ELISA).

[0080] Tumor cell growth also can be analyzed to determine the antitumor activity of the compounds or micelles of the present disclosure. Tumor mass, volume, and/or length can be assessed using methods known in the art such as calipers, ultrasound imaging, computed tomography (CT) imaging, magnetic resonance imaging (MRI), optical imaging (e.g., bioluminescence and/or fluorescence imaging), digital subtraction angiography (DSA), positron emission tomography (PET) imaging and/or other imaging analysis. Tumor cell proliferation can also be analyzed using cellular assays that measure, e.g., DNA synthesis, metabolic activity, antigens associated with cell proliferation, and/or ATP.

[0081] The compounds and micelles of the present disclosure share some properties with other lipophilic amine type antitumor drugs, such as Delocalized Lipophilic Cations (DLCs) and lysosomotropic drugs. DLCs such as rhodamine, MKT-077 F16 and dequalinium are characterized by the presence of quaternary ammonium ions and amines in highly

hydrophobic molecular structures able to delocalize the positive charge in the molecular backbone. The antitumor activity of DLCs is based on their ability to accumulate into the mitochondria of tumor cells. However, a major drawback to their use as antitumor drugs is their high lipophilicity, which restrains their aqueous solubilization and thus their

bioavailability (Wang et al., Mol Aspects Med. 2010, 31(l):75-92; Kurtoglu et al., Mol Nutr Food Res. 2009, 53(l):68-75). Lysosomotropic drugs such as chloroquine and MDL 72527 are amines endowed with hydrophilic/hydrophobic balance suitable for drug partition through the biological membranes and accumulation in lysosomes (Kimura et al., Cancer Res. 2013; 73(l):3-7). Due to their limited antitumor efficacy as single agents, chloroquine and MDL 72527 are currently employed as chemosensitizers in tumor treatments in association with other antitumor drugs in the presence of multidrug resistance (Agostinelli et al., Int J Oncol. 2007, 31(3):473-84; Funk et al., Mol Pharm. 2012, 9(5): 1384-95.). Despite having some similarities to other drugs, the amphiphilic amine compounds and micelles of the present disclosure also provide increased bioavailability, improved tumor specificity, and other advantages that are lacking with currently available antitumor agents. For example, the generalized nature of the effects of the amphiphilic amine compounds and micelles of the present disclosure may have a distinct advantage over drugs that attack specific molecular targets, as it may be more difficult for cells to develop resistance. Cells are able to develop resistance to drugs that bind DNA, RNA or RTKs through the expression of P-glycoprotein, multidrug resistance-associated proteins, activation of alternative RTK pathways, or acquisition of non-binding mutations (Salehan et al. and Tredan et al., supra).

[0082] The present disclosure will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

Examples

[0083] The following examples describes the preparation and confirmation of compounds and micelles of the present disclosure as antitumor agents and nanocarriers for bioactive molecules.

[0084] Statistical analysis: All data points represent mean + S.D., n=6 for all the experiments. All the experiments were repeated at least three times. Representative data are shown. Statistical significance among the mean values was analyzed via an unpaired two- tailed Student t-test assuming equal variance. The significance (P-value) was set at the nominal level of 0.05 or less. For Annexin V staining, the mean values and standard deviations were calculated for untreated and treated cells at different concentrations in combination with the statistical analysis determined by Mann-Whitney test. Example 1

Characterization of the Synthesized Compounds

[0085] Amphiphilic amine compounds 1 to 7 were synthesized in a one-step procedure set out in Scheme 1 and were characterized by 1 H NMR, 13 C NMR, mass spectra, and elemental analysis. Compounds 1 to 7 were synthesized by mixing l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and the appropriate alkyl bromide in the presence of an enoxy catalyzer. All reagents and solvents employed are commercially available (e.g., from Sigma- Aldrich, St. Louis, MO, or Fluka Chemie GmbH, Buchs, Switzerland).

[0086] Amphiphilic amine compounds (denoted "RC) were synthesized following the procedure in Scheme 1. A mixture of DBU and the appropriate alkene (in a 1:3 mole ratio) in N-methylpyrrolidone was stirred at room temperature for 4 hours in the presence of 2- [2- (ethenyloxy)ethoxy]ethan-l-ol (0.2 M) as a catalyst. The addition of diethylether induced the precipitation of a solid residue that was purified by flash chromatography using a mixture of methanol/water (9: 1) as eluent. The final products were characterized by 1 H NMR, 13 C NMR, mass spectra, and elemental analysis.

Scheme 1

(CH₂)_n-CH₃

(i) N-methylpyrrolidone (NMP), 2-[2-(ethenyloxy)ethoxy]ethan-l-ol (CH₂=CH-(0- CH₂CH₂)₂"OH) as a catalyst, 4 hours, room temperature

[0087] Compound 1 (n=4; (RC₇): 2-pentyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 58% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.86 (t, 3H), 1.22-1.38 (m, 8 H), 1.40 (m, 2H), 1.47 (m, 2H), 1.68 (m, 2H), 1.74-1.79 (m, 2H), 1.92-1.98 (t, 2H), 2.56 (dt, 1H), 2.80 (t, 2H), 2.83 (t, 2H), 2.85 (t, 2H), 2.87 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 14.6, 20.9, 22.5, 28.6, 29.6, 30.3, 32.9, 37.3, 44.4, 53.5, 59.0, 67.1 ppm; ESI-MS (m/z): 250 M+:Anal.Calcd for Ci₆H₃oN₂: C 76.74, H 12.07, N 11.19; found: C 76.83, H 12.21, N 11.25.

[0088] Compound 2 (n=7; (RC₁₀): 2-octyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 63% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.86 (t, 3H), 1.23-1.38 (m, 14 H), 1.41 (m, 2H), 1.45 (m, 2H), 1.67 (m, 2H), 1.74-1.79 (m, 2H), 1.91-1.98 (t, 2H), 2.56 (dt, 1H), 2.79 (t, 2H), 2.82 (t, 2H), 2.85 (t, 2H), 2.86 ppm (dd, 2H);^1JC NMR (DMSO, 600MHz): δ 14.2, 21.0, 22.4, 27.8, 28.9, 30.6, 32.5, 37.2, 45.1, 52.9, 60.7, 68.0 ppm; ESI-MS (m/z): 292 M+:Anal.Calcd for C₁₉H₃₆N₂: C 78.02, H 12.41, N 9.58; found: C 78.15, H 12.10, N 9.62.

[0089] Compound 3 (n=9; (RC₁₂): 2-decyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 60% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.86 (t, 3H), 1.23-1.39 (m, 18 H), 1.42 (m, 2H), 1.47 (m, 2H), 1.69 (m, 2H), 1.73-1.80 (m, 2H), 1.92-1.97 (t, 2H), 2.55 (dt, 1H), 2.81 (t, 2H), 2.83 (t, 2H), 2.86 (t, 2H), 2.88 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 14.0, 21.7, 23.0, 28.2, 29.7, 30.3, 32.2, 37.7, 44.9, 51.8, 59.8, 67.5 ppm; ESI-MS (m/z): 320 M+:Anal.Calcd for C₂₁H₄₀N₂: C 78.68, H 12.58, N 8.74; found: C 78.60, H 12.63, N 8.82.

[0090] Compound 4 (n=l 1; (RC₁₄): 2-dodecyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 61% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.85 (t, 3H), 1.24-

1.38 (m, 22 H), 1.40 (m, 2H), 1.45 (m, 2H), 1.68 (m, 2H), 1.75-1.79 (m, 2H), 1.90-1.98 (t, 2H), 2.57 (dt, 1H), 2.79 (t, 2H), 2.84 (t, 2H), 2.87 (t, 2H), 2.87 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 13.9, 19.8, 22.0, 28.6, 29.8, 31.0, 32.6, 37.2, 43.9, 51.8, 60.4, 67.1 ppm; ESI-MS (m/z): 348 M+:Anal.Calcd for C₂₃H₄₄N₂: C 79.24, H 12.72, N 8.04; found: C 79.12, H 12.67, N 7.98.

[0091] Compound 5 (n=13, (RC₁₆): 2-tetradecyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 65% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.86 (t, 3H), 1.23-

1.39 (m, 26 H), 1.40 (m, 2H), 1.47 (m, 2H), 1.68 (m, 2H), 1.74-1.79 (m, 2H), 1.92-1.98 (t, 2H), 2.56 (dt, 1H), 2.80 (t, 2H), 2.83 (t, 2H), 2.85 (t, 2H), 2.87 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 14.1, 20.9, 22.8, 28.7, 29.7, 30.0, 32.1, 37.5, 44.6, 52.5, 60.0, 67.5 ppm; ESI-MS (m/z): 377 M+:Anal.Calcd for C₂5H₄₈N₂: C 79.72, H 12.84, N 7.44; found: C 78.15, H 12.10, N 7.62.

[0092] Compound 6 (n=15; (RC₁₈): 2-hexadecyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 67% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.87 (t, 3H), 1.24-

1.40 (m, 30 H), 1.42 (m, 2H), 1.48 (m, 2H), 1.69 (m, 2H), 1.73-1.78 (m, 2H), 1.90-1.96 (t, 2H), 2.54 (dt, 1H), 2.79 (t, 2H), 2.84 (t, 2H), 2.86 (t, 2H), 2.88 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 14.0, 20.2, 22.5, 28.2, 29.9, 30.6, 32.8, 37.3, 44.0, 51.9, 61.1, 67.0 ppm; ESI-MS (m/z): 404 M+:Anal.Calcd for C27H52N2: C 80.13, H 12.95, N 6.92; found: C 80.15, H 12.99, N 6.89. [0093] Compound 7 (n=17. (RC₂₀): 2-octadecyl-4,8-diazatricyclo[6.5.0.0¹,⁴]tridecane): Yellow semisolid: 64% yield; 1H NMR (DMSO, 600 MHz, free base): δ 0.87 (t, 3H), 1.22- 1.39 (m, 34 H), 1.43 (m, 2H), 1.48 (m, 2H), 1.66 (m, 2H), 1.77-1.80 (m, 2H), 1.90-1.98 (t, 2H), 2.57 (dt, 1H), 2.81 (t, 2H), 2.85 (t, 2H), 2.87 (t, 2H), 2.90 ppm (dd, 2H); ¹³C NMR (DMSO, 600MHz): δ 13.8, 19.9, 22.4, 28.0, 29.5, 31.3, 32.8, 37.0, 43.7, 51.8, 59.6, 67.2 ppm; ESI-MS (m/z): 432 M+:Anal.Calcd for C₂₉H₅₆N₂: C 80.48, H 13.04, N 6.47; found: C 80.45, H 13.12, N 6.59.

[0094] The results of the characterization analysis on each compound confirmed the structures reported in Scheme 1, as shown for RC₁₆ in Figure 2.

Example 2

In Vitro Cytotoxicity of the Synthesized Compounds

[0095] In vitro cytotoxicity: Time-dependent and dose-dependent cytotoxicity was observed in the presence of all the RC compounds of the series (Table 1), with differences in activity exhibited among the different RC compounds. RC₁₆ (Compound 5 from Scheme 1) was the most active RC compound tested against all the pediatric tumor cell lines analyzed.

[0096] The human pediatric tumor cell lines tested were: neuroblastoma (CHLA-20, SH- SY5Y, SK-N-AS), osteosarcoma (Osteomet/143.98.2), malignant peripheral nerve sheath tumor (S462.TY), rhabdomyosarcoma (A673 and RH41) and lymphoma (Ramos).

[0097] Cells were plated in 96- well tissue culture plates at a density of 1 x 10³ cells/well, allowed to attach for 24 hours, and then left untreated or treated with growth medium containing different concentrations of RC compounds dissolved in PBS. After different time periods, the cell vitality was determined by a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazoliumbromide (MTT) assay, according to the manufacture's instruction (Promega, Madison, WI). Briefly, the cell culture medium was substituted with fresh medium containing 0.5 mg/mL of MTT. After incubation for 5 hours at 37 °C, the absorbance was measured at 570 nm with a microplate reader (Model 680, Bio-Rad Life Science Research Group, Hercules, CA). The cytotoxicity of the RC compounds against the different cell lines was reported as the micromolar concentration required to reduce cell survival to 50% (IC₅₀). TABLE 1

RC₇ RC₁₂ RC₁₄ RCl6 RC₁₈ RC₂₀

Tumor 24 h 48h 24 h 48h 24 h 48h 24 h 48h 24 h 48h 24 h 48h 24 h 48h cell lines

A673 5.73 4.11 2.60 2.02 1.82 1.65 1.27 1.13 0.80 0.65 1.03 0.98 1.22 1.35

CHLA-20 8.21 6.57 4.62 3.43 3.50 2.16 2.59 1.87 1.50 1.18 2.30 1.76 2.67 1.92

Osteomet 4.51 3.78 2.55 1.68 1.98 1.24 1.31 0.78 0.50 0.32 0.92 0.65 1.09 0.83

SH-SY5Y 6.20 4.96 3.02 2.24 2.78 1.80 2.30 1.72 1.20 0.82 1.90 1.33 1.95 1.50

SK-N-AS 5.21 3.43 2.14 1.56 1.87 0.71 1.20 0.63 0.88 0.67 1.23 0.75 1.60 0.90

TY 5.62 3.90 2.39 1.78 2.00 1.09 1.81 0.95 1.11 0.76 1.67 0.88 1.83 1.16

Ramos 4.55 3.12 2.10 1.03 1.18 0.74 0.96 0.22 0.10 0.04 0.83 0.17 0.97 0.20

RH41 5.78 4.01 2.85 2.03 1.75 1.16 1.36 0.97 0.25 0.15 1.01 0.82 1.68 1.12

P-values<0.05. Data are representative of three independent experiments.

[0098] Due to its improved antitumor activity, RC₁₆ was further evaluated on a set of adult tumor cell lines which were selected for being particularly resistant to the antitumor treatments in clinical use: mammary carcinoma (MDA-MB-231), osteosarcoma (MG-63), colon carcinoma (WiDr) and lung carcinoma (A549). A murine neuroblastoma (Neuro-2A) cell line was also used.

[0099] Against the adult tumor cells, RC₁₆ was very active with IC₅₀ values in the low micromolar range (1 μΜ to 10 μΜ, Table 2). In contrast, RC₁₆ exhibited very low cytotoxicity with respect to normal cells. The IC₅₀ values for RC₁₆ for human keratinocytes, human fibroblasts and HUVECs were more than ten-fold higher than those for tumor cells (Table 2).

TABLE 2

P-values<0.05. Data are representative of three independent experiments.

Example 3

RCi6 Induces Apoptosis in Tumor Cells

[00100] An Annexin V binding assay was performed to distinguish and quantitatively determine the percentage of dead, viable, apoptotic and necrotic cells in SH-SY5Y,

Osteomet/143.98.2 and A673 cells treated with 5 μΜ RC₁₆ for 15 hours and 24 hours.

[00101] SH-SY5Y, A673 and Osteomet/143.98.2 cells were stained with Annexin V- FITC/7AAD according to the manufacturer's instructions (eBiosciences Inc., San Diego, CA). Briefly, adherent cells were seeded in 6- well plates at a concentration of 4.5 x

10⁵cells/well and after 24 hours, treated with 5 μΜ RC₁₆ for 15 hours and 24 hours. At the end of the treatment, the cells were detached, washed twice with cold PBS and then suspended in Annexin binding buffer containing 5 μΐ_^ of Annexin V-FITC and 5 μΐ_^ of 7AAD. The samples were gently vortexed and incubated for 15 minutes at 25 °C in the dark. Finally, 400 μΐ_^ of binding buffer were added to each tube, and the samples were analyzed by BD LSR II (BD Biosciences, San Jose, CA) with the analysis performed by FlowJo software version 10.0.5.

[00102] The data indicated an apoptotic mode of cell death in all the cell lines analyzed and a different rapidity of cell death depending on the cell type. Differences between control and treated cells were statistically significant at any time of treatment (p-value = 0.016 versus control). Flow cytometric (FACS) analysis showed that the percentage of cells in apoptosis following treatment with 5 μΜ RC₁₆ increased over time with differences among the cell lines SH-SY57, A673, and Osteomet. At 15 hours, the extent of apoptosis was higher in A673 and lower in Osteomet. After 24 hours, all the cells showed about 100% apoptosis. To further confirm the apoptotic nature of cell death induced by RC₁₆ in tumor cells, the involvement of caspases in the death process was evaluated. Both western blot analysis and fluorimetric assays were performed on SH-SY5Y cells in the presence of RC₁₆ .

[00103] Western blot analysis was performed according to standard procedures. Briefly, cells with or without RC₁₆ treatment were washed with PBS and lysed on ice for 30 minutes in lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined with the Bio-Rad protein assay kit. Fifty micrograms of total protein was electrophoresed on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes, blocked for 1 hour with 50 mM Tris buffer, pH 7.5, containing 0.15 M NaCl, 0.05% Tween 20 (TBST) and 5% (wt/vol) nonfat dry milk and probed overnight at 4 °C with TBST containing primary antibodies. After three 10-minute washes in TBST, the filters were incubated with HRP-conjugated secondary antibody in the blocking buffer for 1 hour. After three 10-minute washes in TBST, proteins were detected by enhanced chemiluminescence detection reagents (Amersham Biosciences, Pittsburgh, PA). Antibodies for caspases 3, 8, 9, and PARP (Cell Signaling Technology, Danvers, MA) were used for immunoblotting. Blots were stripped and reprobed with β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX) as the loading control.

[00104] Detection of caspase activity was evaluated by ApoFluor^® Green Apoptosis Detection kits specific for: caspase- 1 and caspase-4; caspase-2, caspase-3 and caspase-7: caspase-6, caspase-8, caspase-9, caspase- 10, and caspase- 13 (MP Biochemicals, Verona, Italy), according to the manufacturer's instructions. Briefly, cells were detached with EDTA and centrifuged at 400 x g for 5 minutes at room temperature. Cell supernatants were removed, and the pellets were resuspended in a buffer containing the appropriate caspase- specific fluorescent probe. After 1 hour of incubation, samples were washed and analyzed by flow cytometry (FACScan, Becton Dickinson, San Jose, CA) equipped with a 15-mW argon ion laser at 488 nm (16).

[00105] The western blot analysis indicated activation of caspases 3, 8 and 9 after exposure to RC₁₆, as shown in Figures 3A and 3B. In the fluorimetric assays, different commercially available kits for single caspases (i.e., caspase-1, caspase-2, caspase-3, caspase-6, caspase-8, caspase-9, caspase- 10, and caspase-13) were used to measure the increase in fluorescence after 1, 2, 4, 6, 12, 24, and 36 hours in SH-SY5Y cells exposed to RC₁₆. As shown in Figure 4, all the foregoing caspases were already activated at 4 hours and became more active thereafter in treated tumor cells with respect to controls. In particular, the activation of the caspases involved in apoptosis initiation (caspase-2, caspase-8, caspase- 9, caspase-10) and in apoptosis execution (caspase-3, caspase-6) was observed. The proinflammatory caspases (caspase-1 and caspase-13) were also activated. The role of caspases in cell death is mainly related to their ability to induce inflammatory processes in vivo with massive activation of inflammatory cells, thus their contribution to the whole mechanism activating cell death may be observed in vivo. The experiments clearly confirmed the apoptotic nature of cell death induced by RC₁₆.

Example 4

RCi6 Induces Oxidative Stress in Tumor Cells

[00106] The intracellular reactive oxygen species (ROS) increased after treatment with RC₁₆ in all the cell lines analysed. Intracellular ROS were measured by OxiSelect™

Intracellular ROS Assay Kit (Cell Biolabs Inc, San Diego, CA) following the manufacturer's instructions. Briefly, cells (1 x 10⁵/mL) were washed twice in PBS and incubated with 5 μΜ 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for 1 hour at 37 °C. After PBS washes, the medium was changed with fresh medium containing 5 μΜ RC₁₆ for 15 hours and 24 hours. At the end of the treatment, samples were washed with PBS, and a mixture of medium and cell lysis buffer (1: 1) was added for 5 minutes at 37 °C. The fluorescence was read by a fluorimetric plate reader (Spectra max M2, Molecular Devices, Sunnyvale, CA). The excitation wavelength was 480 nm and the emission wavelength was 530 nm.

[00107] The highest increase was observed in A673 cells where the ROS levels increased to more than twice the control values after 12 hours of cell exposure to RC₁₆, as shown in Figure 5. In SH-SY5Y and Osteomet, the ROS increase occurred after longer time periods (24 hours) of cell exposure to RC₁₆. The increase in ROS levels in cancer cells following treatment demonstrated that RC₁₆ can accumulate within mitochondria and selectively induce oxidative stress, which can lead to mitochondrial damage and ultimately cell death.

[00108] Transmission electron microscopy (TEM) studies were carried out on SH-SY5Y cells and HF cells incubated with 5 μΜ RC₁₆ for 24 hours. Cells were seeded on cover glasses for 24 hours and then the medium was replaced with fresh medium containing 5 μΜ

RC₁₆ for 24 hours. At the end of the treatment, the samples were washed with PBS, fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 hours at 4 °C and subsequently post- fixed with 1% OsO₄ in 0.1 M phosphate buffer for 1 hour at room temperature. After several washes, the samples were dehydrated in an acetone series (70%, 90%, 100%) and embedded in Epon resin (Fluka; Sigma- Aldrich). Thin sections were collected on nickel grids, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (Philips CM10; FEI, Eindhoven, Netherlands). Images were recorded using a Megaview III digital camera (FEI). TEM was also used to verify the formation of micelles in an aqueous environment due to the amphiphilic nature of the RC molecules. An aqueous solution of RCi₆ (20 μΜ) was stained with 2% (w/v) phosphotungstic acid for 3 minutes on a copper grid and subsequently visualized under TEM. As a comparison, TEM images were also recorded from SH-SY5Y cells treated with the same RC₁₆ concentration (20 μΜ) according with the previously described procedure for cells.

[00109] SH-SY5Y cells treated with RC₁₆ for 24 hours showed a polygonal morphology in which nucleus and nucleolus were well preserved while cytoplasm was characterized by several vacuoles of different diameter, as shown in Figure 6A. At higher magnification, damaged mitochondria with no clear cristae were easily detected. Late autophagy vacuoles (lysosome-like vacuoles) with materials at different stage of degradation, damaged rough endoplasmic reticulum (RER) and multilamellar bodies connected with phospholipid degradation were also observed. Normal human fibroblast cells treated with RC₁₆ at the same concentration and time showed a fibroblastic shape morphology and a well-preserved nucleus, as shown in Figure 6B. Several small vacuoles were detected in the cytoplasm. At higher magnification, normal mitochondria were observed, while some autophagy vacuoles connected with an early process were detected. The observed damage to the inner mitochondrial membranes and observed alterations of the lysosomes in tumor cells was attributed to the accumulation of RC₁₆ within the organelles driven by the

hydrophilic/hydrophobic balance of the molecule. The increased sensitivity to RC₁₆ of the organelles in cancer cells contributes to the compound' s antitumor activity.

Example 5

Interaction of RCi6 with Sialic Acid

[00110] Neuraminidase was used to cleave the sialic acid residue from the sialylated glycoproteins and gangliosides of the tumor cell surface. Cells were seeded on 96-well plates at a density of 2 x 10⁴ cells/plate and after 24 hours, they were exposed to 50 mU/ml neuraminidase Type VI from Clostridium perfrigens (Sigma- Aldrich) for 3 hours. Cells were washed 3 times with PBS and then treated with RC₁₆. The cytotoxicity was evaluated after 24 hours by MTT assay and compared to cells not treated with neuraminidase. A different experiment aimed at testing the same hypothesis was performed by evaluating the

cytotoxicity of RC₁₆ in the presence of sialic acid added to the culture medium in

stoichiometric amount with respect to RC₁₆. Cells were seeded on 96-well plates at a density of 2 x 10⁴ cells/plate and after 24 hours, they were treated with a mixture of RC₁₆ and sialic acid (3 μΜ:3 μΜ) and compared with cells treated with pure RC₁₆ or pure sialic acid at the same concentration (3 μΜ) of the mixture. After 24 hours, the cytotoxicity was evaluated by MTT assay.

[00111] RC₁₆ micelles accumulated on tumor cell membranes, but not normal cells, as shown in Figures 7A to 7D. The pre-treatment of cells with neuraminidase to decrease the presence of sialic acid on the glycoproteins and gangliosides of the cell surface also decreased the cytotoxic activity of RC₁₆, as shown in Figure 8A, supporting the hypothesis of an interaction of RC₁₆ with the sialic acid residue of the cell membrane as a contribution to its whole mechanism of action. The addition of sialic acid to the culture medium, to interact with RC₁₆ and decrease its availability towards the sialic acid present on the glycoproteins and gangliosides of the tumor cell surface, also decreased the RC₁₆ cytotoxic activity, as shown in Figure 8B.

Example 6

In Vivo Activity of RCi₆

[00112] The maximum tolerated dose (MTD) of RC₁₆ in mice was determined. Athymic nude female mice at 6 to 12 weeks of age were injected via the tail vein with RC₁₆ aqueous solutions at increasing doses starting with 0.5 mg/kg and escalating the doses by 0.25 mg/kg until MTD was achieved. Each dose was tested on a cohort of 5 mice in individual independent experiments. MTD was defined as the highest dose that could be given resulting in no drug-related moribund state or death, with temporary body weight loss within 20%. Other signs of toxicity such as mouse behavior, movement and diarrhea were also monitored during MTD testing.

[00113] The MTD of RC₁₆ by intravenous administration was 4 mg/kg. At the MTD, 100% survival was obtained with no body weight loss with respect to the controls.

Moreover, no signs of toxicity such as altered mouse behavior, movement and diarrhea were observed.

[00114] Pharmacokinetic data was obtained after RC₁₆ administration in mice, as shown in Figure 9. Female athymic nude mice were treated with RC₁₆ by oral or intravenous route at doses of 2 mg/kg and 1 mg/kg respectively. Three mice at each time point were sacrificed at 2 hours, 4 hours, 6 hours, 12 hours, 24 hours and 48 hours after administration. Blood samples were removed from the chest cavity, and the RC₁₆ concentration in the blood was determined by ESTMS. The mean plasma concentration versus time profile of RC₁₆ was generated using a least squares nonlinear regression. After intravenous administration, a biexponential disposition of RC₁₆ in the blood was observed with a rapid initial distribution (ti/2a = 0.65 hours) followed by an extensive elimination (tmp = 12.33 hours) (Figure 9A). The values for t _a and t p represent half-lives in the initial distribution phase and in the subsequent elimination phase, respectively. The initial concentration of RC₁₆ in the plasma was 16.66 μg/mL, which corresponded to the injected dose. After oral administration, the blood concentration of RC₁₆ versus time was characterized by an absorption phase until a peak at 4 hours followed by an elimination phase characterized by a t = 11.07 hours (Figure 9B). The pharmacokinetic parameters of RC₁₆ following intravenous administration into nude mice are summarized in Table 3.

TABLE 3

P-values<0.05.

[00115] A biodistribution study was carried out in athymic nude mice bearing CHLA-20 tumors. The mice received intravenous injections of RC₁₆ labelled with CellVue® Maroon (eBioscience, Inc.) at a mole ratio of dye to RC₁₆ of 1 : 100 through the tail vein at a dose of 1 mg/kg. At 12, 24 and 48 hours after injection, the drug distribution was visualized by the In Vivo Imaging System (IVIS) 200 (excitation/emission filter sets: 760/800 nm; Xenogen Corp., Alameda, CA). The mice were then euthanized, and the organs were removed and weighed for quantitative optical imaging performed by the IVIS system with the same filter sets (excitation/emission: 760/800 nm).

[00116] IVIS images were taken at 12, 24 and 48 hours after intravenous injection of RC₁₆ labelled with CellVue® Maroon in nude mice bearing CHLA-20 tumors. The images showed an extensive distribution of fluorescence in the whole body at 12 hours and a gradual decrease of distribution at 24 hours and 48 hours with a persistence of fluorescence in the liver and the gastrointestinal system, as shown in Figure 10A. For a quantitative evaluation of biodistribution, the organs were removed from the euthanized mice at 12 hours, 24 hours and 48 hours after intravenous injection of the labelled RC₁₆ and were imaged immediately after dissection, as shown in Figure 10B. All images were acquired for 5 seconds and processed using the Xenogen Living Image software (Biochem Biophy Res Commun 2010; 394:348-35). The fluorescence intensity (photons/second, (p/s)) was calculated per unit weight of each organ (Biomaterials 2009; 30:2929-2939). At each time point, the stomach, intestine and liver showed the highest fluorescence intensity. All the organs showed a decrease in RC₁₆ content over time except the tumors, whose RC₁₆ levels remained nearly constant over time.

[00117] In vivo efficacy was analyzed in female athymic nude mice, 6 to 8 weeks old, weighing 20 to 25 grams (Charles River Laboratories). These mice were subcutaneously implanted in the right flank with 5 x 10⁶ cells/mouse in a 100 μL· volume of serum- free medium mixed with 100 μL· Matrigel™ (Becton Dickinson) (12 mice per group). Animals were monitored and tumor dimensions were measured every 2 to 3 days in two perpendicular directions using calipers. Tumor volume (mm 3 ) was defined as follows: (Wi 2 x W₂) x (π/6), where Wi and W₂ are the largest and smallest tumor diameters (mm), respectively. The mice were then randomized into groups of 6 animals for each tumor type. When the tumors reached a mean volume of 150 mm , the animals were treated with RC₁₆ or vehicle (PBS) alone, administered slowly through the tail vein or gavaged in a volume of 200 μL·. RC₁₆ was administered at the dose of 1 mg/kg 3 times a week for 3 weeks or at the dose of 2 mg/kg/day for 3 weeks by gavage. After suspension of the treatments, the tumors were monitored for 5 days. Relative tumor volume (RTV) was calculated according to the following formula: RTV = TV_n/TVo, where TV_n is the tumor volume at day n and TVo is the tumor volume at day 0. The average percentage of body weight change was used as an indicator for tolerability. Toxicity was defined as body weight loss of 20% or more and/or mortality.

[00118] Administration of RC₁₆ to mice bearing human tumor xenografts demonstrated a strong antitumor activity by both intravenous and oral administration, as shown in Figures 11 and 12. No weight loss nor signs of toxicity were observed in any case during the treatment period, indicating good tolerability of RC₁₆ both by intravenous administration and gavage. In the metastatic model of neuroblastoma disease, the mice showed a significant increase in the mean survival time with respect to the control, as shown in Figure 13, indicating a strong activity of RC₁₆ after a single intravenous administration. At the end of the experiment (90 days), about 50% of the treated mice were healthy and alive and showed no evidence of macroscopic disease at necropsy.

[00119] In vivo experiments were also carried out on a metastatic model of neuroblastoma. IA/J mice were slowly injected through the tail vein with Neuro 2 A murine neuroblastoma cells (0.2 x 10⁶) dispersed in PBS. After 5 days from the injection, the mice were treated once with RC₁₆ injected through the tail vein in a volume of 200 μΐ_^ at doses of 20 μg or 40 μg/mouse. After treatment, the animals were monitored for survival.

Example 7

Electron Microscopy

[00120] RC₁₆ micelles were detected by Scanning electron microscopy (SEM). SH-SY5Y cells were seeded on holders for 24 hours, and then the medium was replaced with fresh medium containing 20 μΜ RC₁₆ for 1 hour. At the end of the treatment, the samples were washed with PBS, fixed in 2.5% glutaraldehyde, and then dehydrated in an acetone series (70%, 90%, 100%). The dried specimens were sputter-coated with gold and examined by SEM (Hitachi SU-70; Hitachi Instruments, Schaumburg, IL). The aqueous solution of RC₁₆ (20 μΜ) was desiccated at room temperature on the sample holder, coated with gold and examined by SEM.

[00121] The formation of micelles, triggered by the spontaneous self-assembling of the amphiphilic RC₁₆ molecules in an aqueous solution, was detected by electron microscopy. SEM and TEM images of RC₁₆ in aqueous solutions, as shown in Figure 14, and RC₁₆ in culture medium in the presence of SH-SY5Y cells, as shown in Figure 7, demonstrated the presence of micelles of spherical shape and regular surface. A measure of their mean diameter, performed by TEM, provided values in the nanometric range (20 nm to 30 nm).

Example 8

Encapsulation of Bioactive Molecules

[00122] An aqueous solution of RC₁₆ (10 mM, lmL) was mixed with an ethanol solution of doxorubicin, paclitaxel or etoposide (ImM, 1 mL). After stirring for 12 hours at 25 °C in a sealed container, the mixture was dialyzed through a 5000 Da MW cutoff dialysis membrane against 100 mL of water. The dialysis was carried out for 48 hours by changing the external medium every 8 hours. The internal RC₁₆-drug complex was freeze-dried and the solid residue obtained was weighed and spectrophotometrically analysed to establish the percentage of drug content in the final complex. The complexes were also evaluated for their cytotoxicity in CHLA-20 in comparison with the free drugs at the same concentration.

[00123] The RC₁₆ micelles were able to encapsulate exemplary antitumor drugs. RC compounds comprising hydrophobic straight chain alkyl tail groups containing more than 16 carbons demonstrated less stability as micelles in water due to the increased lipophilicity of the tail chains. The encapsulation efficiency, calculated as the weight percentage of drug in the final dried complex, was: doxorubicin (2.3%) (DOXO) > paclitaxel (1.8%) (PTX) > etoposide 1.0%) (ETO).

[00124] The DOXO, PTX and ETO retained their cytotoxic activity when complexed with RC₁₆. CHLA-20 cells were seeded in 96-well plates (2 x 10⁴ cells/plate) and after 24 hours, they were treated with the RC^-drug complexes (3 μΜ) or with the free drugs at

concentrations corresponding to those present in the complexes. After 48 hours, the cytotoxicity was evaluated by a MTT assay and expressed as percent control (% CTR). As a comparison, the cells were also treated with pure R_Ci6 (3 μΜ). The cytotoxic activity of the RC^-drug complex was higher than the free drug and the pure RC₁₆ at the corresponding concentrations of the complex (Table 4). The most significant improvement in cytotoxicity for the drug-loaded RC₁₆ micelle compared to the free drug was observed for doxorubicin, as shown in Figures 15 to 17.

TABLE 4

P-values<0.05. Data are representative of three independent experiments.

[00125] The foregoing Examples demonstrated that compounds, micelles, and

compositions of the present disclosure possess strong cytotoxic activity against cell lines derived from multiple cancer types. RC₁₆ is characterized by an intermediate alkyl tail length and exhibited the strongest cytotoxic activity among the compounds tested. RC₁₆ exhibited significant antitumor activity in several cancer models by both intravenous and oral routes. Mechanistic studies suggested that RC₁₆ binds to sialic acid residues on the cell membrane and induces cell death via mitochondrial and lysosomal damage. The IC₅₀ of the compound RCi₆ for normal cells was ten-fold higher than for tumor cells, indicating that RC₁₆ is significantly less toxic to normal cells. Additionally, RC₁₆ exhibited significant antitumor effects in vivo using several human xenografts and in a metastatic model of murine neuroblastoma. RC₁₆ fully suppressed tumor growth by both intravenous and oral administration routes and strongly improved the survival in the metastatic model.

[00126] RC₁₆ was also evaluated as a nanocarrier for bioactive molecules. The amphiphilic character of RC₁₆ provided a spontaneous molecular self-assembling in water with formation of nanomicelles allowing complexation of doxorubicin, etoposide and paclitaxel. The multiagent micelles significantly improved the in vitro antitumor activity of the drugs through enhancement of their solubility in water and improved drug availability. Thus, RC₁₆ and related amphiphilic amine compounds, micelles, and compositions may be useful as antitumor therapies.

[00127] The present disclosure provides a multimechanistic antitumor drug class that shows promise for cancer therapy. The amphiphilic amine compounds bind to and induce cytotoxicity in tumor cells by several mechanisms of action, with differential effects on normal cells, resulting in a clinically significant therapeutic window. Furthermore, the amphiphilic amine compounds are useful for encapsulating traditional cytotoxic

chemotherapies, thereby enhancing their efficacy and reducing side effects.

[00128] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED:

1. A compound comprising a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the ring system: is at least tricyclic; contains at least two nitrogen atoms; and contains an azetidine ring or an aziridine ring.

2. The compound of claim 1, wherein the hydrophilic fused polycyclic hydrocarbon ring system comprises structural formula (I):

wherein x and y are individually selected from the group consisting of 0 to 10; z is 0 or 1 ; and one or more carbon atoms in structural formula (I) is optionally substituted with one or more groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

3. The compound of claim 2, wherein x is 3, y is 3, 4, or 5, and z is 1.

4. The compound of any of the preceding claims, comprising at least one substituted or unsubstituted Cs_2o alkyl group.

5. The compound of any of the preceding claims, wherein the compound comprises structural formula (II), (III), or (IV):

wherein Ri and R₂ are individually selected from the group consisting of hydrogen and substituted or unsubstituted Cs_₂o alkyl, and one or more carbon atoms in structural formula (II), (III), or (IV) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

6. The compound of claim 5, wherein Ri is H and R₂ is C₅-₂o alkyl.

7. The compound of claim 5, wherein Ri is Cs_₂o alkyl and R₂ is hydrogen.

8. The compound of any of claims 5-7, wherein Ri or R₂ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

9. The compound of any of claims 5-8, wherein Ri or R₂ is tetradecyl.

10. The compound of claim 2, wherein x is 3, y is 3, 4, or 5, and z is 0.

11. The compound of any of claims 1, 2 or 10, wherein the compound comprises structural formula (V), (VI), or (VII):

wherein R₃ is substituted or unsubstituted Cs_2o alkyl, and one or more carbon atoms in structural formula (V), (VI), or (VII) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

12. The compound of claim 11, wherein R₃ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

13. The compound of claim 11 or 12, wherein R is tetradecyl.

14. The compound of any of the preceding claims, wherein the compound is selected from the group consisting of:

15. A composition comprising the compound of any of the preceding claims and a pharmaceutically acceptable excipient.

16. A micelle comprising a substituted or unsubstituted hydrophilic fused polycyclic hydrocarbon ring system, wherein the ring system: is at least tricyclic; contains at least two nitrogen atoms; and contains an azetidine ring or an aziridine ring.

17. The micelle of claim 16, wherein the micelle comprises a hydrophobic hydrocarbon group attached to the hydrophilic fused polycyclic hydrocarbon ring system through a linker.

18. The micelle of claim 17, wherein the hydrophobic hydrocarbon group is substituted or unsubstituted C5-20 alkyl.

19. The micelle of any of claims 16-18, wherein the hydrophobic fused polycyclic hydrocarbon ring system comprises structural formula (I):

(I), wherein x and y are individually selected from the group consisting of 0 to 10; z is 0 or 1 ; and one or more carbon atoms in structural formula (I) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocyclo alkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

20. The micelle of claim 19, wherein x is 3, y is 3, 4, or 5, and z is 1.

21. The micelle of any of claims 16-20, wherein the micelle comprises substituted or unsubstituted C5-20 alkyl.

22. The micelle of any of claims 16-21, wherein the micelle comprises structural formula (II), (III), or (IV):

wherein Ri and R₂ are individually selected from the group consisting of hydrogen and substituted or unsubstituted Cs_₂o alkyl, and one or more of the carbon atoms in structural formula (II), (III), or (IV) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

23. The micelle of claim 22, wherein Ri is H and R₂ is C₅-₂o alkyl.

24. The micelle of claim 22, wherein Ri is Cs_₂o alkyl and R₂ is hydrogen.

25. The micelle of any of claims 22-24, wherein Ri or R₂ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

26. The micelle of any of claims 22-25, wherein Ri or R₂ is tetradecyl.

27. The micelle of claim 19, wherein x is 3, y is 3, 4, or 5, and z is 0.

28. The micelle of any of claims 16-19 or 27, wherein the micelle comprises structural formula (V), (VI), or (VII):

I),

wherein R₃ is substituted or unsubstituted Cs_2o alkyl, and one or more of the carbon atoms in structural formula (V), (VI), or (VII) is optionally substituted with one or more side groups individually selected from the group consisting of alkyl, hydroxy, oxo, halo, carboxamido, aryl, carboxy, heteroaryl, cycloalkyl, heterocycloalkyl, alkoxy, amino, azo, nitro, cyano, alkylamino, imino, thio, and combinations thereof.

29. The micelle of claim 28, wherein R₃ is selected from the group consisting of pentyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

30. The micelle of any of claims 27-29, wherein R is tetradecyl.

31. The micelle of any of claims 16-30, wherein the micelle comprises a plurality of compounds selected from the group consisting of

and combinations thereof.

32. A composition comprising the micelle of any one of claims 16-31 and a pharmaceutically acceptable excipient.

33. The composition of claim 32, further comprising a therapeutic agent encapsulated within the micelle.

34. The composition of claim 33, wherein the therapeutic agent is selected from the group consisting of antitumor agents, antineoplastic agents, prodrugs, analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, adrenolytics, adrenocorticoids, adrenomimetics, anticholinergic agents, anticholinesterases, anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiants, antacids, antidiarrheals, antidotes, antifolics, antipyretics, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, antihelmintics, antibiotics, anticoagulants, antidepressants, antiepileptics, antibacterials, antifungals, antifibrotic agents, anti-infective agents, anti-parasitic agents, antihistamines, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antiprotozoal agents, antiviral agents, cardiac drugs, anxiolytic sedatives, beta- adrenoceptor blocking agents, corticosteroids, cough suppressants, dopaminergics, hemostatics, hematological agents, hypnotics, immunological agents, muscarinics, neurological drugs, bioactive peptides, steroid hormones, nucleic acids, vaccines, anti-protozoan drugs, barbiturates, photo sensitizer substances

parasympathomimetics, prostaglandins, radio-pharmaceuticals, sedatives, stimulants, sympathomimetics, vitamins, xanthines, growth factors, hormones, antiprion agents, and combinations thereof.

35. The composition of claim 34, wherein the therapeutic agent is an antitumor agent selected from the group consisting of an aromatase inhibitor; an anti-estrogen; an anti- androgen; a gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active agent; an alkylating agent; a retinoid, a carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound; a methionine aminopeptidase inhibitor; a bisphosphonate; an antiproliferative antibody; a heparanase inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin spindle protein inhibitor; a MEK inhibitor; an antitumor antibiotic; a nitrosourea, and combinations thereof.

36. The composition of claim 35, wherein the antitumor agent is selected from the group consisting of doxorubicin, etoposide, paclitaxel, fenretinide, and combinations thereof.

37. A method of inhibiting tumor cell growth comprising contacting a cell with the micelle, composition, or compound of any of the preceding claims in an amount effective to inhibit growth of the tumor cell.

38. A method of treating or preventing a neoplastic, hyperplastic or hyperproliferative disorder in a subject in need thereof comprising administering a therapeutically effective amount of the micelle, composition, or compound of any of claims 1-36 to the subject.

39. The method of claim 38, wherein the hyperproliferative disorder is selected from the group consisting of cancer, benign prostate hyperplasia, colorectal neoplasia, benign soft tissue tumors, bone tumors, brain and spinal tumors, eyelid and orbital tumors, granuloma, lipoma, meningioma, multiple endocrine neoplasia, nasal polyps, pituitary tumors, prolactinoma, pseudotumor cerebri, seborrheic keratoses, stomach polyps, thyroid nodules, cystic neoplasms of the pancreas, hemangiomas, vocal cord nodules, polyps, and cysts, Castleman disease, chronic pilonidal disease, dermatofibroma, pilar cyst, pyogenic granuloma, or juvenile polyposis syndrome.

40. A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the micelle, composition or compound of any of claims 1- 36 to the subject.

41. The method of claim 39 or 40, wherein the cancer is selected from the group consisting of adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, astrocytoma, atypical teratoid rhabdoid tumor, B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-cell lymphoma, basal cell carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, Brenner tumor, Brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, carcinoma in situ, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid plexus papilloma, clear-cell sarcoma of the kidney, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse large B-cell lymphoma,

dysembryoplastic neuroepithelial tumor, dysgerminoma, embryonal carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetus in fetu, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, germ cell tumor, gestational choriocarcinoma, giant cell fibroblastoma, giant cell tumor of the bone, glial tumor, glioblastoma multiforme, glioma, gliomatosis cerebri, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastric cancer, hairy cell leukemia, hemangioblastoma, head and neck cancer, hemangiopericytoma, hematological malignancy, hepatoblastoma, hepatosplenic T-cell lymphoma, Hodgkin's lymphoma, non-

Hodgkin's lymphoma, invasive lobular carcinoma, intestinal cancer, kidney cancer, laryngeal cancer, lentigo maligna, lethal midline carcinoma, leukemia, leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangio sarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myelogeous leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non- small cell carcinoma, non- small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant triton tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary carcinoma of the breast, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, merkel cell cancer, mesothelioma, metastatic urothelial carcinoma, mixed Mullerian tumor, mucinous tumor, multiple myeloma, muscle tissue neoplasm, mycosis fungoides, myxoid liposarcoma, myxoma, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, ocular cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, optic nerve tumor, oral cancer, osteosarcoma, ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pinealoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, precursor T- lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, preimary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, pseudomyxoma periotonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumor, sex cord-gonadal stromal tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, soot wart, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Sezary's disease, small intestine cancer, squamous carcinoma, stomach cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachal cancer, urogenital cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, visual pathway glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor.