Septins inhibitors for cancer treatment

Abstract

The present disclosure provides novel compounds, compositions, and methods for modulating one or more septin proteins, such as Septin-2. Such compounds and compositions are useful in the treatment of cancer, such as endometrial cancer, pancreatic cancer, lung cancer, breast cancer, or ovarian cancer, such as serous and clear cell ovarian cancer.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 894,424, filed on August 30, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] There is a continuing need for treatment options for the treatment of cancers of the liver, lung, kidney, and pancreas. Similarly, for patients with advanced cancers such as ovarian and endometrial cancers, effective treatment options are limited. Sequential secondary and tertiary treatments do not add a meaningful response other than adding toxicity that debilitates many lives. Therefore, to improve the survival rate of patients diagnosed with these deadly malignancies, it is necessary to identify molecular targets that promote tumorigenesis and develop targeted therapies against such driver genes.

[0003] Septins are cytoskeleton - like GTP - binding proteins that are essential for several biological processes such as cytokinesis, cell migration, chromosome dynamics, and protein secretion. (Dolat et al. (2014) J Cell Biol 207(2):225 - 235, Mostowy and Cossart (2012) Nat Rev Mol Cell Biol 13(3):183 - 194, and Tokhtaeva et al. (2015) J Biol Chem 290(9):5280 - 5297). Changes in septin expression and gene mutations have been identified in multiple malignancies.

Summary of the Invention

[0004] The present invention provides compounds, compositions, kits, products, and methods for using them. Exemplary compounds, along with their structures, are provided herein. Exemplary methods of use are described herein and include, but are not limited to, inhibiting or reducing the expression level and / or activity of septin proteins, inhibiting or reducing the survival and / or proliferation of cells expressing septin, and inhibiting or reducing the survival rate and / or proliferation of cancer cells expressing septin, in vitro, ex vivo, or in vivo, thereby preventing or treating, e.g., reducing and / or delaying, the symptoms of cancer, tumors, metastasis, and other neurological and / or psychological disorders or conditions. Exemplary cancers, tumors, metastasis, disorders or conditions include, but are not limited to, gynecological cancers, pancreatic cancer, endometrial cancer, liver cancer, kidney cancer, blood cancers, central nervous system (CNS) cancers, and others described herein.

[0005] The present invention provides compounds as modulators of septin proteins. Such modulators can reduce or increase the expression and / or activity of septin in vitro, ex vivo, and / or in vivo. In some embodiments, such modulators can reduce or antagonize the expression and / or activity of septin.

[0006] The septin proteins described herein are any one or combination of septin proteins known in the art, e.g., septin-1, septin-2, septin-3, septin-4, septin-5, septin-6, septin-7, septin-8, septin-9, septin-10, septin-11, septin-12, and / or septin-14. In some embodiments, the septin protein is septin-2, septin-4, septin-9, and / or septin-14. In some embodiments, the septin protein is septin-2 and septin-9. In some embodiments, the septin protein is septin-2.

[0007] In one embodiment, the present invention provides, for example, novel compounds, compositions, and methods of using them for inhibiting septin proteins. In certain embodiments, the compounds can be used in methods for treating cancer, such as ovarian cancer, for example, ovarian clear cell carcinoma.

[0008] Disclosed herein is a compound of formula (I):

Chemical formula

[0009] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0010] In some embodiments, the compounds described herein have the following structure:

Chem.

[0011] In some embodiments, X at the position of the compounds described herein 2 is N. In some embodiments, X of the compounds described herein 1 and X 3 at both positions are C.

[0012] In some embodiments, the compounds described herein have the structure of formula (II):

Chem.

[0013] In some embodiments, the compounds described herein have the structure of formula (III):

Chem.

[0014] In some embodiments, the compounds described herein have the structure of formula (IV):

Chem.

[0015] In some embodiments, for the compounds described herein, R 1 is F, Cl, Br, I, NO2, (C=O)-R 11 , (C=O)-OR 11 , (C=O)-NHR 11 , (C=O)-N(R 11 )2, or C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one, two, three, or more of F, Cl, Br, I, or phenyl.

[0016] In some embodiments, for the compounds described herein, R 2 is F, Cl, Br, I, NO2, (C=O)-R 11 , (C=O)-OR 11 , (C=O)-NHR 11 , (C=O)-N(R 11)2, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl.

[0017] In some embodiments, for the compounds described herein, R 1 and R 2 each independently is F, Cl, Br, I, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl.

[0018] In some embodiments, for the compounds described herein, R 12 is an ortho substituent.

[0019] In some embodiments, for the compounds described herein, each R 12 is F, Cl, Br, I, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, or I.

[0020] In some embodiments, for the compounds described herein, each R 11 independently is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C 10 aryl or 5- to 10-membered heteroaryl, wherein the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C6-C 10 aryl or 5- to 10-membered heteroaryl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, (C=O)-(C1-C6 alkyl), (C=O)-O(C1-C6 alkyl), (C=O)-NH(C1-C6 alkyl), or (C=O)-N(C1-C6 alkyl)2.

[0021] In some embodiments, the compounds described herein are forchlorfenuron (FCF). In some embodiments, the compounds described herein are not FCF. For example, the compounds described herein include FCF analogs but do not include FCF.

[0022] In some embodiments, for the compounds described herein, i) R 2 is not phenoxy, or ii) R 3 is not imidazolyl or pyrimidinyl.

[0023] In some embodiments, the compounds described herein have the following structure:

Chemical formula

Chemical formula

[0024] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0025] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0026] The present invention provides a composition, preferably a pharmaceutical composition, comprising at least one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

[0027] The present invention also provides a kit comprising at least one of the compounds described herein, or a pharmaceutically acceptable salt thereof, or at least one of the compositions described herein, preferably a pharmaceutical composition, and optionally, instructions for use of at least one of the above compounds, a pharmaceutically acceptable salt thereof, or at least one of the above compositions.

[0028] The present invention also provides a product comprising a unit dose of at least one of the compounds described herein, or a pharmaceutically acceptable salt thereof, or at least one of the compositions described herein, preferably a pharmaceutical composition, in a suitable packaging for use of at least one of the above compounds, a pharmaceutically acceptable salt thereof, or at least one of the above compositions.

[0029] In some embodiments, the compounds, compositions, preferably pharmaceutical compositions, kits, or products described herein can: i) induce the destruction of septin filaments, ii) reduce the activity of septin proteins, iii) reduce the activity of intracellular septin, actin and / or tubulin proteins, iv) disrupt the intracellular localization of septin proteins, v) reduce the viability or proliferation of cells expressing septin proteins, vi) arrest the progression of the cell cycle, vii) induce cell apoptosis, viii) reduce the secretion of HE (e.g., HE4) from cells, ix) reduce the intracellular expression of HER2, or x) treat a subject having a disease or disorder characterized by an increase in the expression or activity of septin proteins when below a therapeutically effective amount. In some embodiments, the cells are cancer cells.

[0030] The present invention also provides a method for reducing the activity of a septin protein, which method comprises contacting the septin protein with a compound, composition, preferably a pharmaceutical composition, kit, or article of manufacture described herein.

[0031] The present invention also provides a method for reducing the activity of a septin protein within a cell, which method comprises contacting the septin protein with a compound, composition, preferably a pharmaceutical composition, kit, or article of manufacture described herein.

[0032] The present invention also provides a method for reducing the viability of a cell, which method comprises contacting the cell with a compound, composition, preferably a pharmaceutical composition, kit, or article of manufacture described herein.

[0033] In some embodiments, the compound or composition disrupts the cellular location of the septin protein, reduces cell proliferation, arrests the progression of the cell cycle (e.g., in the S phase), induces cell apoptosis, decreases HE4 secretion from the cell, and reduces HER2 expression in the cell (preferably without reducing EGFR expression). In some embodiments, the cell is a cancer cell. In some embodiments, the cell overexpresses a septin protein such as septin-2 and / or septin-9.

[0034] Cancer types for treatment using the compounds, compositions, preferably pharmaceutical compositions, kits, or articles of manufacture described herein include pancreatic cancer, breast cancer, lung cancer (e.g., small cell and non-small cell lung cancer), kidney (renal) cancer, liver cancer, ovarian cancer, endometrial cancer, basal cell cancer, biliary tract cancer, bladder cancer, bone cancer, brain and / or CNS cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, fibroma, head and neck cancer, gastric cancer, intraepithelial neoplasia, laryngeal cancer, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), lymphoma (e.g., Hodgkin and non-Hodgkin lymphoma), melanoma, oral cancer (e.g., lip, tongue, mouth, and pharyngeal cancer), prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, and at least one of the cancers described herein such as other carcinomas and sarcomas, or any combination thereof. In some embodiments, the cancers described herein include ovarian cancer, endometrial cancer, kidney (renal) cancer, lung cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, brain cancer, neuroblastoma, breast cancer, or blood cancers such as leukemia and / or lymphoma. In some embodiments, the cancer includes pancreatic cancer, breast cancer, lung cancer, kidney cancer, liver cancer, ovarian cancer, or endometrial cancer, or any combination thereof. In some embodiments, the cancer includes ovarian clear cell carcinoma (OCCC).

[0035] Optionally, when administering the compounds, compositions, preferably pharmaceutical compositions, kits, or articles of manufacture described herein to cells, a second agent or therapy capable of reducing cell viability is also administered either simultaneously with, before, or after administration of the compounds, compositions, preferably pharmaceutical compositions, kits, or articles of manufacture described herein.

[0036] The present invention also provides a method of treating cancer in a subject in need thereof (preferably a subject expressing or overexpressing a septin protein), the method comprising administering to the subject a therapeutically effective amount of a compound, composition, preferably a pharmaceutical composition, kit, or article of manufacture described herein. In some embodiments, the compound or composition disrupts the cellular localization of septin proteins, reduces cell proliferation, arrests the progression of the cell cycle (e.g., at the S phase), induces cell apoptosis, reduces HE4 secretion from cells, and reduces HER2 expression in cells (preferably without reducing EGFR expression). In some embodiments, the cells are cancer cells. In some embodiments, the cells overexpress septin proteins such as septin-2 and / or septin-9.

[0037] The compounds and compositions described herein are delivered to the subject via any one of the routes that are deemed appropriate by a physician and / or are known in the art. Exemplary delivery routes include intrathecal, intravenous (IV), subcutaneous, oral, transdermal patch, nasal aerosol, and the like. In some embodiments, the route of administration includes the least intrusive delivery modalities such as oral or IV administration.

[0038] The subject is one of any of the subjects described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, non-human primate, mouse, rat, hamster, guinea pig, monkey, ape, rabbit, cat, dog, horse, pig, lion, tiger, or wolf. BRIEF DESCRIPTION OF THE DRAWINGS

[0039]

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Figure 13

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure provides compounds, compositions, and methods for modulating the expression or activity of septin proteins such as septin-2. The design, synthesis, and screening of novel molecules, such as aryl ureas, containing pyridines with various substitutions are disclosed herein. These exemplary molecules disclosed in the examples potently disrupt the assembly of septin-2 in tumor cells and reduce cell proliferation in the micronanomolar range. Compounds described herein, such as diaryl ureas, were also able to reduce pancreatic tumor tissue mass and other tumor models.

[0041] Septin-2 is a filament-forming cytoskeletal GTPase that forms filamentous oligomeric structures with Septin-7, Septin-6, Septin-9, Septin-4, and other septins. The filamentous structure mediated by Septin-2 is essential for the structural integrity and motility of sperm tails during postmeiotic differentiation (Kuo et al (2015) J Cell Sci 128(5):923-934). Septin-2 is required for the normal organization of the actin cytoskeleton, maintains polyglutamylated microtubules, promotes efficient vesicle transport, and plays a role in the biosynthesis of polarized columnar epithelia by preventing the binding of MAP4 to tubulin. Septin-2 is required for the progression of mitosis. Septin-2 forms a scaffold at the central plane of the mitotic spindle necessary to maintain localization at kinetochores and chromosome congression. During anaphase, Septin-2 is involved in chromosome segregation and spindle elongation. Septin-2 plays a role in ciliogenesis and cell motility. In cilia, Septin-2 acts by regulating the assembly of a structural complex (B9 complex) by localizing the TMEM231 protein. Cilia are required for the integrity of the diffusion barrier at the base of primary cilia that prevents the diffusion of transmembrane proteins between the cilium and the plasma membrane. Septin-2 plays a role in the internalization of two intracellular microbial pathogens such as Listeria monocytogenes and Shigella flexneri. Overexpression of Septin-2 promotes tumorigenesis in serous and clear cell ovarian cancers (Cantillo et al. (2017) Gynecologic Oncology 145:Supplement 1,pp.123-124).

[0042] Septins assemble into hetero-oligomers to generate higher-order scaffold structures such as filaments, bundles, and rings inside cells. They are cytoskeletal components similar to actin filaments, microtubules, and intermediate filaments. Septins are known to be involved in diverse essential cellular mechanisms, including the control of diffusion barriers, protein localization, extracellular membrane fusion, autophagosome regulation, lysosome homeostasis maintenance, mitochondrial fission, and the biogenesis of membranous organelles and multivesicular bodies (Kartmann and Roth (2001) J Cell Sci 114(Pt 5):839-844, Caudron and Barral (2009) Dev Cell 16(4):493-506, Bridges and Gladfelter (2015) J Biol Chem 290(28):17173-17180, Pagliuso et al. (2016) EMBO Rep 17(6):858-873, Sirianni et al. (2016) EMBO Rep 17(7):1029-1043, Mostowy et al. (2010) Cell Host Microbe 8(5):433-444, Traikov et al. (2014) PLoS One 9(11):e109372, and Dolat and Spiliotis (2016) J Cell Biol 214(5):517-527). Septins are structurally related to the RAS oncogene, and their expression levels have been found to vary in several types of cancer, such as kidney, lung, colorectal, skin, brain, endometrial, ovarian, and breast cancers (Cerveira et al. (2011) Biol Chem 392(8-9):713-724, Connolly et al. (2011) Biol Chem 392(8-9):725-738, and Angelis and Spiliotis (2016) Front Cell Dev Biol 4:122).Furthermore, abnormal septin expression has been associated with neurodegenerative / neuromuscular diseases, blood disorders, infertility, and developmental disorders (Dolat et al. (2014) Biol Chem 395(2):123-141, and Marttinen et al. (2015) Mol Neurodegener 10:16).

[0043] The present disclosure provides compounds for modulating the expression or activity of septin proteins such as septin-2. In some preferred embodiments, such compounds can reduce or inhibit the expression or activity of septin proteins such as septin-2.

[0044] Disclosed herein are exemplary compounds of formula (I):

Chemical formula

[0045] In some embodiments, X in the compound of formula (I) 2 is N. In some embodiments, both X 1 and X 3 are C. In some embodiments, X in the compound of formula (I) 2 is N, and both X 1 and X 3 are C.

[0046] In some embodiments, R in the compound of formula (I) 2 is not phenoxy. In some embodiments, R in the compound of formula (I) 3 is not imidazolyl or pyrimidinyl. In some embodiments, R in the compound of formula (I) 2 is not phenoxy, and R 3 is not imidazolyl or pyrimidinyl.

[0047] In some embodiments, X in the compound of formula (I) 2 is N, both X 1 and X 3 are C, R 2 is not phenoxy, and R 3 is not imidazolyl or pyrimidinyl.

[0048] In some embodiments, the compound of formula (I) is forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is not forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof.

Chemical formula

[0049] In some embodiments, the compound of formula (I) has the structure of formula (II):

Chemical formula

[0050] In some embodiments of the compounds of formula (I) and / or (II), R 3 , R 4 , and R 5 each independently is H, F, Cl, Br, I, NO2, (C=O)-R 11 , (C=O)-OR 11 , (C=O)-NHR 11 , (C=O)-N(R 11 )2, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl.

[0051] In some embodiments, R 2 in the compound of formula (II) is not phenoxy. In some embodiments, R 3 in the compound of formula (II) is not imidazolyl or pyrimidinyl. In some embodiments, R 2 in the compound of formula (II) is not phenoxy and R 3 is not imidazolyl or pyrimidinyl.

[0052] In some embodiments, the compound of formula (II) is forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (II) is not forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof.

[0053] In some embodiments, the compound of formula (I) has the structure of formula (III):

Chemical formula

[0054] In some embodiments of the compounds of formula (I), (II), and / or (III), each of R 6 , R 7 , R 9 and R 10 is independently H, F, Cl, Br, I, NO2, (C=O)-R 11 , (C=O)-OR 11 , (C=O)-NHR 11 , (C=O)-N(R 11 )2, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl.

[0055] In some embodiments, R 2 in the compound of formula (III) is not phenoxy.

[0056] In some embodiments, the compound of formula (III) is forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (III) is not forchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof.

[0057] In some embodiments, the compound of formula (I) has the structure of formula (IV):

Chemical formula

[0058] In some embodiments, R in the compound of formula (III) 2 is not phenoxy.

[0059] In some embodiments of the compounds of formula (I), (II), (III), and / or (IV), R 1 is F, Cl, Br, I, NO2, (C=O)-R 11 , (C=O)-OR 11 , (C=O)-NHR 11 , (C=O)-N(R 11 )2, or C1-C6 alkyl, and the above C1-C6 alkyl is optionally substituted with one, two, three, or more of F, Cl, Br, I, or phenyl. In some embodiments, R 1 is F, Cl, Br, or I. For example, R 1 can be F.

[0060] In some embodiments of the compounds of formula (I), (II), (III), and / or (IV), R 2 is F, Cl, Br, I, NO2, (C═O)-R 11 , (C═O)-OR 11 , (C═O)-NHR 11 , (C═O)-N(R 11 )2, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl. In some embodiments, R 2 is C1-C6 alkyl optionally containing 1, 2, 3, or more substitutions by F, Cl, Br, or I. In some embodiments, R 2 is C1-C3 alkyl optionally containing 1, 2, or 3 substitutions by F or Cl. For example, R 2 can be CF3.

[0061] In some embodiments of the compounds of formula (I), (II), (III), and / or (IV), each of R 1 and R 2 is independently F, Cl, Br, I, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, I, or phenyl. In some embodiments, R 1 is F, Cl, Br, or I, and R 2 is C1-C3 alkyl optionally containing 1, 2, or 3 substitutions by F or Cl.

[0062] In some embodiments of the compounds of formula (IV), each R 12 is F, Cl, Br, I, or C1-C6 alkyl, wherein the C1-C6 alkyl optionally contains 1, 2, 3, or more substitutions by F, Cl, Br, or I. In some embodiments, each R 12 is F, Cl, or Br. In some embodiments, each R12 is Cl. In some embodiments, R 12 is an ortho substituent.

[0063] In some embodiments, the compound of formula (IV) is fluchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (IV) is not fluchlorfenuron (FCF), or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments, the compounds described herein include analogs of fluchlorfenuron (FCF). In some embodiments, the compounds described herein have the following structure:

Chemical formula

Chemical formula

[0065] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0066] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0067] In some embodiments, the compounds described herein have the following structure:

Chemical formula

[0068] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the present disclosure (e.g., a compound of formula (I), (II), (III), and / or (IV)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0069] In certain embodiments, the pharmaceutical composition comprises one or more additional therapeutic agents.

[0070] A pharmaceutical composition comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof can be prepared with one or more pharmaceutically acceptable excipients selected according to conventional practice. Tablets can contain excipients including glidants, fillers, binders, etc. Aqueous compositions can be prepared in a sterile form and can generally be isotonic if intended for delivery other than oral administration. All compositions can optionally contain excipients as described in Rowe et al, Handbook of Pharmaceutical Excipients, 6 th edition, American Pharmacists Association, 2009. Excipients can include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid, etc. In certain embodiments, the composition is provided as a solid dosage form including a solid oral dosage form.

[0071] The compositions include those suitable for various routes of administration, including oral administration. The compositions may be provided in unit dosage forms and can be prepared by any of the well-known methods in the pharmaceutical art. Such methods include the step of associating an active ingredient (e.g., a compound of the present disclosure or a pharmaceutically acceptable salt thereof) with one or more pharmaceutically acceptable excipients. The compositions are prepared by uniformly and intimately associating the active ingredient with a liquid excipient or a subdivided solid excipient or both, and then shaping the product, if necessary. The techniques and formulation are generally described in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams and Wilkins, Philadelphia, Pa., 2006.

[0072] The compositions described herein suitable for oral administration can be presented as discrete units (unit dosage forms) including, but not limited to, capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient. In one embodiment, the pharmaceutical composition is a tablet.

[0073] The pharmaceutical compositions disclosed herein comprise one or more of the compounds disclosed herein, or pharmaceutically acceptable salts thereof, together with pharmaceutically acceptable excipients and optionally other therapeutic agents. The pharmaceutical compositions containing the active ingredient can be in any form suitable for the intended method of administration. For example, when used for oral use, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs can be prepared. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions can contain one or more excipients including sweetening, flavoring, coloring and preserving agents to provide a palatable preparation. Tablets containing the active ingredient admixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets are acceptable. These excipients can be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium or sodium phosphate, granulating and disintegrating agents such as corn starch or alginic acid, binding agents such as cellulose, microcrystalline cellulose, starch, gelatin or acacia, and lubricants such as magnesium stearate, stearic acid, or talc. Tablets can be uncoated or coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be used alone or in combination with waxes.

[0074] The amount of active ingredient that can be combined with the inert ingredients to produce the dosage form can vary depending upon the intended subject of treatment and the particular mode of administration. For example, in some embodiments, dosage forms for oral administration to humans can contain from about 1 to 1000 mg of the active substance formulated with a suitable and convenient amount of pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutically acceptable excipient varies from about 5% to about 95% of the total composition (weight:weight).

[0075] In certain embodiments, a composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof in one variation does not contain an agent that affects the rate at which the active ingredient is metabolized. Thus, it is understood that in one aspect, a composition comprising a compound of the present disclosure does not contain an agent that affects (e.g., slows, inhibits, or delays) the metabolism of any other active ingredient administered separately, sequentially, or simultaneously, either with the compound of the present disclosure or separately from the compound of the present disclosure.

[0076] Thus, it is understood that in one aspect, any of the methods, kits, articles of manufacture, etc. detailed herein do not contain an agent that affects (e.g., slows, inhibits, or delays) the metabolism of any other active ingredient administered separately, sequentially, or simultaneously, either with the compound of the present disclosure or separately from the compound of the present disclosure.

[0077] The present disclosure further provides a compound of the present disclosure for administration as a single active ingredient in a pharmaceutically acceptable composition, which can be prepared by conventional methods known in the art, for example, by binding the active ingredient to a pharmaceutically acceptable therapeutically inert organic and / or inorganic carrier or excipient, or by mixing them therewith.

[0078] Another possibility is the use of a compound of the present disclosure as a second or other active ingredient having a synergistic effect with other active ingredients in a known drug, or the administration of a compound of the present disclosure together with such a drug.

[0079] The compounds of the present disclosure can also be used in the form of a prodrug or other suitably modified form that releases the active ingredient in vivo.

[0080] The present disclosure provides methods of using a compound of the present disclosure or a pharmaceutically acceptable salt thereof.

[0081] For example, in some embodiments, a method of reducing or inhibiting the activity of a septin protein comprises contacting the septin protein with a compound or composition of the present disclosure, or a pharmaceutically acceptable salt thereof. The septin protein can be any one or combination of septin proteins known in the art, for example, septin-1, septin-2, septin-3, septin-4, septin-5, septin-6, septin-7, septin-8, septin-9, septin-10, septin-11, septin-12, and / or septin-14. In some embodiments, the septin protein is septin-2, septin-4, septin-9, and / or septin-14. In some embodiments, the septin protein is septin-2 and septin-9. In some embodiments, the septin protein is septin-2.

[0082] In some embodiments, the present disclosure provides a method of inhibiting cell viability and / or proliferation, comprising contacting a cell with a compound or composition of the present disclosure, or a pharmaceutically acceptable salt thereof.

[0083] Further disclosed herein is a method of treating a disease or disorder, comprising administering to a subject in need of treatment for the disease or disorder a therapeutically effective amount of a compound or composition of the present disclosure, or a pharmaceutically acceptable salt thereof. In some embodiments, the disease or disorder is cancer.

[0084] Generally, the cancers that can be treated with the compounds of the present disclosure or pharmaceutically acceptable salts thereof are cancers having abnormal expression or activity of septin proteins as described above. In some embodiments, the cancer includes lung cancer, ovarian cancer, renal cancer, urothelial cancer, endometrial cancer, colorectal cancer, skin cancer, brain cancer, breast cancer, liver cancer, pancreatic cancer, or any combination thereof. In some embodiments, the cancer includes lung cancer, ovarian cancer, renal cancer, endometrial cancer, liver cancer, pancreatic cancer, or any combination thereof. In some embodiments, the cancer includes ovarian cancer, endometrial cancer, pancreatic cancer, or any combination thereof. In some embodiments, the cancer includes ovarian cancer such as ovarian clear cell carcinoma.

[0085] The present disclosure provides a kit comprising a compound or composition of the present disclosure, or a pharmaceutically acceptable salt thereof. Optionally, the kit further includes, for example, instructions for use for treating cancer. The instructions for use are generally written instructions, but an electronic storage medium (such as a magnetic disk or optical disk) containing the instructions can also be used.

[0086] The present disclosure also provides a pharmaceutical kit comprising one or more containers containing a compound of the present disclosure or a pharmaceutically acceptable salt thereof. Optionally, associated with such containers is a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, and this notice reflects the approval by the government agency for manufacture, use, or sale for human administration. Each component (if there are two or more components) can be packaged in separate containers or, to the extent cross-reactivity and shelf life permit, several components can be combined in one container. The kit can be in unit dosage form, bulk package (such as a multiple-dose package), or sub-unit dosage. Optionally, the kit contains a plurality of unit doses of the compound and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy (such as a hospital pharmacy and a dispensing pharmacy).

[0087] Also provided are articles of manufacture containing a unit dosage of a compound of the present disclosure or a pharmaceutically acceptable salt thereof, in packaging suitable for use in the methods described herein. Suitable packaging is known in the art and includes, for example, vials, containers, ampoules, bottles, jars, flexible packaging, and the like. Optionally, the article of manufacture is further sterilized and / or sealed.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Dashes at the front or end of a chemical group are for convenience in indicating the point of attachment to the parent moiety, and the chemical group can be represented with or without one or more dashes without losing its normal meaning. "C" u-v " or "C" u -C" v " and the like as prefixes indicate that the group that follows has u to v carbon atoms, where u and v are integers. For example, "C" 1-6 alkyl" or "C1-C6 alkyl" indicates that the alkyl group has 1 to 6 carbon atoms.

[0089] "Alkyl" is a straight or branched saturated monovalent hydrocarbon. For example, an alkyl group can have 1 to 10 carbon atoms (i.e., C" 1-10 alkyl) or 1 to 8 carbon atoms (i.e., C" 1-8 alkyl) or 1 to 6 carbon atoms (i.e., C" 1-6 alkyl) or 1 to 4 carbon atoms (i.e., C" 1-4It can have an alkyl group. Examples of the alkyl group include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH(CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), and octyl (-(CH2)7CH3).

[0090] "Alkenyl" is a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon double bond. For example, the alkenyl group has 2 to 8 carbon atoms (i.e., C 2-8 alkenyl) or 2 to 6 carbon atoms (i.e., C 2-6(alkenyl) or 2 to 4 carbon atoms (i.e., C 2-4 can have an alkenyl group. Examples of alkenyl groups include, but are not limited to, ethenyl (-CH=CH2), allyl (-CH2CH=CH2), and -CH2-CH=CH-CH3.

[0091] "Alkynyl" is a linear or branched monovalent hydrocarbon radical having at least one carbon-carbon triple bond. For example, an alkynyl group can have 2 to 8 carbon atoms (i.e., C 2-8 (alkynyl) or 2 to 6 carbon atoms (i.e., C 2-6 (alkynyl) or 2 to 4 carbon atoms (i.e., C 2-4 can have an alkynyl group. Examples of alkynyl groups include, but are not limited to, ethynyl (-C≡CH), propargyl (-CH2C≡CH), and -CH2-C≡C-CH3.

[0092] As used herein, "halo" or "halogen" refers to fluoro (-F), chloro (-Cl), bromo (-Br), and iodo (-I).

[0093] As used herein, "aryl" refers to a single all-carbon aromatic ring, or a polycondensed all-carbon ring system in which at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Aryl includes phenyl radicals. Aryl also includes polycondensed ring systems having about 9 to 20 carbon atoms in which at least one ring is aromatic and the other rings are aromatic or non-aromatic (i.e., carbocyclic). Such polycondensed ring systems are optionally substituted on any carbocyclic moiety of the polycondensed ring system with one or more (e.g., 1, 2, or 3) oxo groups. The rings of the polycondensed ring system can be connected to each other via fusion, spiro, and bridge bonds, where possible, given the valence requirements. When referring to a member of an aryl of a particular atom range (e.g., 6- to 10-membered aryl), it should also be understood that the atom range is with respect to all ring atoms of the aryl. For example, 6-membered aryl includes phenyl, and 10-membered aryl includes naphthyl and 1,2,3,4-tetrahydronaphthyl. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0094] As used herein, "heteroaryl" refers to a single aromatic ring having at least one atom other than carbon in the ring, the atom being selected from the group consisting of oxygen, nitrogen and sulfur, and "heteroaryl" also includes a polycondensed ring system having at least one such aromatic ring, which polycondensed ring system is further described below. Thus, "heteroaryl" includes a single aromatic ring having from about 1 to 6 carbon atoms and from about 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Optionally, when the ring is aromatic, sulfur and nitrogen atoms are also present in oxidized forms. Exemplary heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidinyl, oxazolyl or furyl. "Heteroaryl" also includes a polycondensed ring system (e.g., a ring system containing two rings) in which the heteroaryl group as defined above is condensed with one or more rings selected from heteroaryl (e.g., to form 1,8-naphthyridinyl), heterocycle (e.g., to form 1,2,3,4-tetrahydro-1,8-naphthyridinyl), carbocycle (e.g., to form 5,6,7,8-tetrahydroquinolyl) and aryl (e.g., to form indazolyl) to form a polycondensed ring system. Thus, heteroaryl (single aromatic ring or polycondensed ring system) has from about 1 to 9 carbon atoms and from about 1 to 6 heteroatoms in the heteroaryl ring. Such a polycondensed ring system is optionally substituted with one or more (e.g., 1, 2, 3 or 4) oxo groups on the carbocyclic or heterocyclic moiety of the condensed ring. The rings of the polycondensed ring system can be connected to each other via condensation, spiro, and bridge bonds where possible by valence requirements. It should be understood that the individual rings of the polycondensed ring system are optionally connected to each other in any order. The point of attachment of the heteroaryl or heteroaryl polycondensed ring system can be any suitable atom of the heteroaryl or heteroaryl polycondensed ring system including carbon and heteroatoms (e.g., nitrogen). When referring to a member of a particular atomic range of heteroaryl (e.g., 5- to 10-membered heteroaryl), the atomic range is with respect to all ring atoms of the heteroaryl and is understood to include carbon and heteroatoms.For example, 5-membered heteroaryl includes thiazolyl, and 10-membered heteroaryl includes quinolinyl. Exemplary heteroaryl includes, but is not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrazolyl, thienyl, indolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furyl, oxadiazolyl, thiadiazolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, indazolyl, quinoxalinyl, quinazolinyl, 5,6,7,8-tetrahydroisoquinolinyl benzofuranyl, benzimidazolyl, thianaphthenyl, pyrrolo[2,3-b]pyridinyl, quinazolinyl-4(3H)-one, and triazolyl.

[0095] "The compounds of the present disclosure" include the compounds disclosed herein. For example, the compounds of the present disclosure include the compounds of the examples, and the compounds of formulas (I), (II), (III), and (IV).

[0096] As used herein, "treatment" or "treating" or "to treat" refers to an approach for obtaining a beneficial or desired result. For the purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviation of symptoms and / or reduction in the degree of symptoms, and / or prevention of worsening of symptoms associated with a disease or condition. In one embodiment, "treatment" or "treating" includes one or more of the following: a) inhibiting a disease or condition (e.g., reducing one or more symptoms resulting from the disease or condition and / or reducing the degree of the disease or condition), b) delaying or preventing the onset of one or more symptoms associated with a disease or condition (e.g., stabilizing the disease or condition, delaying the worsening or progression of the disease or condition), and c) alleviating a disease or condition, e.g., causing regression of clinical symptoms, improving the disease state, delaying the progression of the disease, enhancing the quality of life, and / or extending survival.

[0097] As used herein, "delaying" with reference to the onset of a disease or condition means deferring, interfering with, delaying, retarding, stabilizing and / or postponing the onset of the disease or condition. This delay can be of various lengths depending on the medical history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention in that the individual does not develop the disease or condition.

[0098] As used herein, "prevent", "prevention" or "preventing" refers to a regimen that protects against the onset of a disease or disorder such that clinical symptoms of the disease do not develop. Thus, "prevention" is related to the administration of a therapy (e.g., administration of a therapeutic agent) to a subject prior to the time at which signs of the disease are detectable in the subject. The subject can be an individual at risk of developing a disease or disorder, such as an individual having one or more risk factors known to be associated with the development or onset of the disease or disorder. It is also understood that prevention does not require a 100% success rate.

[0099] As used herein, "modulating" or "modulation" of the activity of a protein, such as a septin protein, refers to a change in activity such that the activity is increased or decreased. In some embodiments, the modulation decreases the activity.

[0100] As used herein, "reduce" or "reduction" or "reducing" refers to the effect of decreasing a particular level (e.g., the expression level and / or activity or function of a protein and / or mRNA). The resulting reduced level is about 99%, 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 1% or less of the previous level, or any percentage not listed above that is less than 100%. Specifically, the resulting reduced level may be zero, which means that it cannot be detected experimentally (e.g., with respect to the expression level and / or activity or function). Terms such as "reduce" as used herein are interchangeable with terms such as "inhibit".

[0101] As used herein, "subject" refers to mammals, such as, but not limited to, animals within the phylum Chordata, including primates (humans, other great apes (hominids), gibbons (hylobatids), Old World monkeys (cercopithecids), New World monkeys (platyrrhines, including at least Callitrichid, Cebid, Aotid, Pitheciid, Atelid), tarsiers and Strepsirrhine (including at least primates such as lemurs and lorises)), Glires (rabbits, hares, pikas, and rodents (including at least rodents such as mice, springhares, beavers, guinea pigs, and squirrels)), carnivores such as cats (including at least cats, Asiatic linsang, African palm civet, viverroids, hyenas, and mongooses (feliform)), carnivores such as dogs (including at least dogs, bears, lesser pandas, skunks, weasels, minks, ferrets, raccoons, sea lions, walruses, seals), hamsters, voles, moles, equines (including at least horses and llamas), pigs (including at least pigs and peccaries), camels, whales, dolphins, bats, and marsupials, etc. In some embodiments, the subject described herein is a domestic animal such as a pig, cow, horse, equine, dog, cat, rat, mouse, etc., a non-human primate such as a cynomolgus monkey or chimpanzee, or a human. In some embodiments, the subject is a human. In some embodiments, the subject is an animal including, but not limited to, mice, rats, hamsters, guinea pigs, monkeys, apes, rabbits, cats, dogs, horses, pigs, lions, tigers, wolves, etc.

[0102] As used herein, "at-risk individual" refers to an individual at risk of developing a condition to be treated. An "at-risk" individual may or may not have a detectable disease or condition, and may or may not exhibit a detectable disease prior to treatment with the methods described herein. "At-risk" means that the individual has one or more so-called risk factors, which are measurable parameters known in the art that correlate with the development of a disease or condition. An individual having one or more of these risk factors is more likely to develop a disease or condition than an individual not having these risk factors.

[0103] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount of a compound that, when administered to a subject for treating a disease, is effective to elicit the desired biological or medical response, including an amount sufficient to effect such treatment of the disease. The effective amount will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated. The effective amount can include a range of amounts. As understood in the art, the effective amount can be one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired therapeutic endpoint. The effective amount can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be administered in an effective amount if, in combination with one or more other agents, a desired or beneficial result is possible or achieved. The appropriate dosage of any co-administered compound can optionally be reduced due to the combined action of the compounds (e.g., additive or synergistic effect).

[0104] "Pharmaceutically acceptable excipients" include, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent approved by the U.S. Food and Drug Administration as acceptable for use in humans or livestock.

[0105] Also provided are pharmaceutically acceptable salts, hydrates, solvates, tautomers, polymorphs, and prodrugs of the compounds described herein. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials useful in preparing pharmaceutical compositions suitable for veterinary or human pharmaceutical use.

[0106] The compounds described herein can be prepared and / or formulated as pharmaceutically acceptable salts or, where appropriate, as the free base. Pharmaceutically acceptable salts are non-toxic salts of the free base form of the compounds having the desired pharmacological activity. These salts can be derived from inorganic or organic acids or bases. For example, a compound containing a basic nitrogen can be prepared as a pharmaceutically acceptable salt by contacting the compound with an inorganic or organic acid. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, and mandelates. A list of other suitable pharmaceutically acceptable salts is described in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Wiliams and Wilkins, Philadelphia, Pa., 2006.

[0107] Examples of "pharmaceutically acceptable salts" of the compounds disclosed in this specification include salts derived from suitable bases such as alkali metals (e.g., sodium, potassium), alkaline earth metals (e.g., magnesium), ammonium, and NX4 + (wherein X is C1-C4 alkyl). Also included are base addition salts such as sodium salts or potassium salts.

[0108] As used herein, the term "cancer" refers to the uncontrolled cell growth of organs that disrupt the normal functions of the body's organs and systems. Cancer cells that move from their original location and seed important organs can ultimately lead to the death of the subject through the decline in the function of the affected organs. Cancer cells are cells that divide and proliferate abnormally. In some cases, it is possible to distinguish cancer cells from their normal counterparts based on the profile of genes and proteins expressed, as well as the levels of their expression. Genes commonly affected in cancer cells include oncogenes. Cancer-related mutations lead to a decrease or complete loss of their expression. Otherwise, the mutations cause an increase in expression or the expression of an activated variant of the normal counterpart.

[0109] The term "tumor" is usually equivalent to neoplasm, literally meaning "new growth", and is used interchangeably with "cancer". A "tumorous disorder" is any disorder related to cell growth, particularly neoplasms. A "neoplasm" is an abnormal mass of tissue that persists and grows after the removal of the carcinogenic factor that initiated its appearance. There are two types of neoplasms: benign and malignant. Almost all benign tumors are encapsulated and non-invasive. In contrast, malignant tumors are rarely encapsulated and invade adjacent tissues by infiltrative, destructive growth. After this infiltrative growth, there is a possibility that tumor cells will be transplanted to sites discontinuous with the original tumor.

[0110] Metastasis is an area of cancer cells that is different from the location of the primary tumor and is caused by the seeding of cancer cells from the primary tumor to other parts of the body. At the time of diagnosis of the primary tumor mass, the subject can be monitored for the presence of metastasis. Metastasis is most frequently detected by monitoring for specific symptoms, alone or in combination with magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function tests, chest X-rays, and bone scans.

[0111] The compounds and compositions of the present invention can be used to treat cancer in a subject. In some embodiments, the cancer includes gynecological cancers (such as ovarian cancer), pancreatic cancer, endometrial cancer, liver cancer, kidney cancer, blood cancer, central nervous system (CNS) cancer, or any combination thereof. Examples of some important CNS cancers include, but are not limited to, neuroblastoma, medulloblastoma, peripheral malignant schwannoma, ependymoma, chraniopharyngioma, astrocytoma, meningioma, germ cell tumor, glioma, mixed glioma, choroid plexus tumor, anaplastic oligodendroglioma, peripheral primitive neuroectodermal tumor, primitive neuroectodermal tumor (PNET), CNS lymphoma, pituitary adenoma, and schwannoma. In some embodiments, the astrocytoma is grade I, grade II, grade III, or grade IV. Astrocytomas can be of low or high grade. Astrocytomas can be juvenile pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, or glioblastoma multiforme. In some embodiments, the oligodendroglioma is a mixed glioma (oligoastrocytoma) or anaplastic oligodendroglioma. In one preferred embodiment, the cancer includes neuroblastoma.

[0112] Cancers that can be treated by the compounds and compositions of the present invention include, but are not limited to, basal cell cancer, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, fibroma, head and neck cancer, gastric cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, leukemia including chronic lymphocytic leukemia, liver cancer, lung cancer (e.g., small cell and non-small cell), lymphoma including Hodgkin and non-Hodgkin lymphoma, melanoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, and other carcinomas and sarcomas.

[0113] Exemplary cancers for which the compounds and compositions of the present invention are intended for treatment include, but are not limited to, lobular carcinoma, follicular adenocarcinoma (also called adenocystic carcinoma, adenomyoepithelioma, cribriform carcinoma, and cylindroma), carcinoma adenomatosum, adenocarcinoma, adrenocortical carcinoma, alveolar carcinoma, alveolar epithelial carcinoma (also called bronchioloalveolar carcinoma, alveolar epithelioma, and pulmonary adenomatosis), basal cell carcinoma, carcinoma basocellulare (also called basaloma or basal cell tumor, and trichoblastoma), basaloid carcinoma, basal squamous cell carcinoma, breast cancer, bronchioloalveolar epithelial carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma (also called cholangioma and bile duct carcinoma), choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, reticular carcinoma, cutaneous carcinoma, cylindrical carcinoma, columnar cell carcinoma, ductal carcinoma, carcinoma durum, fetal carcinoma, medullary carcinoma, epibulbar carcinoma, epidermoid carcinoma, carcinoma epitheliale adenoide, ulcerating carcinoma, carcinoma fibrosum, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, giant cell, adenocarcinoma, granular cell carcinoma, trichoblastoma, hemocytoid carcinoma, hepatocellular carcinoma (also called hepatoma, malignant liver cancer, and liver cancer), Hürthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher’s carcinoma, Kulchitzky-cell carcinoma, lenticular carcinoma, lenticular carcinoma, carcinomalenticulare), lipomatous cancer, lymphoepithelial cancer, carcinoma mastitoides, carcinoma medullare, medullary carcinoma, carcinoma melanodes, melanotic carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucinous epidermoid carcinoma, carcinoma mucosum, mucous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, carcinoma nigrum, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prostate carcinoma, renal cell carcinoma of the kidney (referred to as adenocarcinoma of the kidney and hypernephroid carcinoma), precursor cell carcinoma, carcinoma sarcomatodes, scheinderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, ellipsoid cell carcinoma tumor, spindle cell carcinoma, spongy carcinoma, squamous cell carcinoma, spinous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma vilosum are included.

[0114] Exemplary sarcomas are rare mesenchymal neoplasms that occur in bone and soft tissue. Various types of sarcomas are recognized and include the following: for example, liposarcoma (including myxoid liposarcoma and pleomorphic liposarcoma), leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor (also called malignant Schwannoma, neurofibrosarcoma, or neurogenic sarcoma), Ewing tumor (Ewing sarcoma of bone, extraskeletal (i.e., non-osseous) Ewing sarcoma, and primitive neuroectodermal tumor [PNET]), synovial sarcoma, angiosarcoma, hemangiosarcoma, lymphangiosarcoma, Kaposi sarcoma, hemangioblastoma, fibrosarcoma, desmoid tumor (also called aggressive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma (MFH), perivascular cell tumor, malignant mesenchymoma, alveolar soft part sarcoma, epitheloid sarcoma, clear cell sarcoma, fibrous histiocytoma of childhood, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma) - skeletal and extraskeletal, as well as chondrosarcoma.

[0115] The compounds and compositions of the present invention can also be used to treat refractory cancers. A refractory cancer is a cancer that is resistant to the normal standard treatments that are prescribed. Thus, a subject being treated according to the present invention for a refractory cancer may already have been exposed to another treatment for that cancer. Alternatively, if the cancer is likely to be refractory (e.g., if an analysis of the cancer cells or the subject's medical history is available), the subject may not yet have been exposed to another treatment. Examples of refractory cancers include, but are not limited to, leukemia, melanoma, renal cell cancer, colon cancer, liver (hepatic) cancer, pancreatic cancer, non-Hodgkin lymphoma, and lung cancer.

[0116] The compounds and compositions of the present invention can also be used to treat immunogenic cancers. Immunogenic cancers are cancers that may express immunogens on their surface or upon cell death. These immunogens are an in vivo endogenous source of cancer antigens, and their release can be utilized by the methods of the present invention for treating cancer. Exemplary immunogenic cancers include melanoma and renal cell carcinoma, Mantel Cell Lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia, Burkitt lymphoma, myeloma, immunocytoma, acute promyelocytic leukemia, chronic myeloid / acute lymphoblastic leukemia, acute leukemia, B-cell acute lymphoblastic leukemia, undifferentiated large cell leukemia, myelodysplastic syndrome / acute myeloid leukemia, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), common (pre-B) acute lymphocytic leukemia, melanoma, T-cell lymphoma, leukemia, B-cell lymphoma, epithelial malignancies, lymphoid malignancies, gynecological cancers, cholangiocarcinoma, and pancreatic ductal adenocarcinoma.

[0117] The compounds and compositions of the present invention can also be used to treat angiogenesis. Angiogenesis is an abnormal and rapid proliferation of endothelial cells, resulting in the persistent and non-decaying formation of abnormal new blood vessels. Angiogenesis that persists for months or years can support cancer growth and progression and can damage various organs and tissues such as, for example, the eye, skin, heart, blood vessels, lung, gastrointestinal tract, and urogenital tract.

[0118] As used herein, the term "inhibiting angiogenesis" refers to a reduction in the number or density of abnormal microvessels upon treatment with a compound described in the present invention, or by targeting septin-2 or other septin family proteins individually or collectively. The most widely used methods in the clinical setting rely on histochemical or immunohistochemical staining of blood vessels (microvessels) in a biopsy (open or needle) or specimen. Characteristics of angiogenesis that can be examined include, for example, blood vessel density and / or the morphology and / or thickness of the perivascular cuff. The area of microvessel density in a histological biopsy or specimen is quantified. Areas with high microvessel density ("hot spots") may, for example, contain the most tumor cells and / or have the highest potential for metastasis. One technique for determining microvessel density is to measure the intercapillary distance. Another method of assessing angiogenesis is to measure the thickness of the perivascular cuff. An increase in the thickness of the perivascular cuff is associated with the progression of angiogenesis and may indicate disease worsening.

[0119] Inhibition of angiogenesis, which is one aspect of the present invention, can be evaluated by measuring the blood, serum, plasma, or tissue level of angiogenesis (angiogenic) factors, or the level of angiogenic factors that function as surrogate markers of angiogenesis. Exemplary angiogenic factors that can function as surrogate markers of angiogenesis include, but are not limited to, angiogenin, angiopoietin-1, Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF), follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF) / scatter factor (SF), interleukin-8 (IL-8), leptin, midkine, placental growth factor, platelet-derived endothelial cell growth factor (PD-ECGF), platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN), progranulin, proliferin, transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), and vascular endothelial growth factor (VEGF) / vascular permeability factor (VPF). Image processing techniques are also useful for the evaluation of angiogenesis. Suitable image processing techniques or devices include CT, spiral CT, micro-CT, multiple energy computed tomography (MECT), single detector CT (SDCT), multi-detector CT (MDCT), volume CT (VCT), MRI, micro-MR, X-ray, spiral X-ray, PET, non-invasive devices such as near-infrared / optical, as well as other non-invasive scanning techniques and devices that can be used outside the body of the subject or non-invasively inserted into body cavities. Angiogenesis can also be imaged by CT angiography (CTA), tomosynthesis, X-ray microangiography, and other techniques. As one of the angiogenesis image processing techniques, it includes the use of a microbubble-based contrast agent (SonoVue) combined with ultrasonic and contrast-specific imaging modalities that detect perfusion changes in tumor microvascular perfusion. Other angiogenesis image processing techniques include color Doppler and mammography.Color Doppler imaging can show angiogenesis in tumors such as breast cancer. Mammography can reveal the angiogenic margins of breast tumors. Extensive images or radiological signs can be enhanced by dyes.

[0120] Angiogenesis can also be evaluated in a subject by a process that includes introducing at least one contrast agent into the region of interest of the body. For example, a contrast agent for detecting blood vessels can be injected into the blood vessels. A small amount of the contrast agent can be introduced locally to enhance the detection of blood vessels in a particular region of interest of the body. Alternatively, the contrast agent can be provided in an amount sufficient to enhance the detection of blood vessels in a broad body region or the entire body of the subject. Structural data can be acquired about the subject's body or about one or more target organs, such as the lungs, heart, breast, colon, etc., a part of an organ, or another target volume of the subject's body. The target volume can be any part of the subject's body, such as the extremities, abdomen, torso, neck, head, or any part thereof. Other methods or techniques for evaluating angiogenesis not described herein can be used for the purposes of the present invention. Methods and techniques for evaluating angiogenesis are known to those of skill in the art.

[0121] The use of the compounds and compositions described in the present invention can be combined with other therapies such as radiation therapy, surgery, conventional chemotherapy, etc., or can be combined with a combination with one or more additional therapies.

[0122] The compounds and compositions described in the present invention are administered alone as pharmaceutical compositions or in combination with one or more other active ingredients or agents in therapeutically effective and physiologically acceptable amounts. Such other active ingredients include, but are not limited to, glutathione antagonists, angiogenesis inhibitors, chemotherapeutic agents, and antibodies (e.g., cancer antibodies). The compounds and compositions described in the present invention and the other active ingredients or agents are administered simultaneously or sequentially. When the compounds and compositions described in the present invention are administered simultaneously with another active agent or in combination with another active ingredient, the compounds and compositions and the other active ingredients are administered in the same or separate formulations but simultaneously. When the administration of the other active agent and the septin target compound is temporally separated, the other active agents are administered sequentially with each other and with the compounds and compositions of the present invention. The time interval between administrations is minutes, hours, days, or more hours.

[0123] Examples of glutathione antagonists include, but are not limited to, buthionine sulfoximine, cyclophosphamide, ifosfamide, actinomycin D, and N-(4-hydroxyphenyl)retinamide (4-HPR).

[0124] Examples of angiogenesis inhibitors include, but are not limited to, 2-methoxyestradiol (2-ME), AG3340, angiostatin, antithrombin III, anti-VEGF antibody, batimastat, bevacizumab (Avastin), BMS-275291, CAI, canstatin, captopril, cartilage-derived inhibitor (CDI), CC-5013, celecoxib (CELEBREX®), COL-3, combretastatin, combretastatin A4 phosphate, dalteparin (FRAGMIN®), EMD 121974 (silenid), endostatin, erlotinib (TARCEVA®), gefitinib (Iressa), genistein, halofuginone hydrobromide (TEMPOSTATIN™), Id1, Id3, IM862, imatinib mesylate, interferon-alpha, interleukin 12, lavendustin A, LY317615 or AE-941 (NEOVASTAT™), marimastat, maspin, medroxyprogesterone acetate, Meth-1, Meth-2, Neovastat, osteopontin cleavage product, PEX, pigment epithelial growth factor (PEGF), platelet factor 4, prolactin fragment, proliferin-related protein (PRP), PTK787 / ZK 222584, recombinant human platelet factor 4 (rPF4), restin, squalamine, SU5416, SU6668, slamine, taxol, tecogalan, thalidomide, thrombospondin, TNP-470, troponin I, vasostatin, VEG1, VEGF-Trap, and ZD6474. In some embodiments, the angiogenesis inhibitor is a VEGF antagonist. The VEGF antagonist can be a VEGF binding molecule. The VEGF binding molecule includes a VEGF antibody or an antigen-binding fragment thereof. An example of a VEGF antagonist is NeXstar.

[0125] Chemotherapeutic agents can be used as additional active ingredients for the compounds and compositions of the present invention. Such chemotherapeutic agents include, but are not limited to, DNA-damaging agents, which include topoisomerase inhibitors (e.g., etoposide, lamptothecin, topotecan, teniposide, mitoxantrone), antimicrotubule agents (e.g., vincristine, vinblastine), antimetabolites (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, cladribine), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), and DNA strand break-inducing agents (e.g., bleomycin, doxorubicin, daunorubicin, idarubicin, mitomycin C). Chemotherapeutic agents include synthetic, semi-synthetic, and naturally derived drugs. Important chemotherapeutic agents include, but are not limited to, the following: acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, adriamycin, aldosterone, alitretinoin, allopurinol sodium, altretamine, ambomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, annonaceous acetogenin, anthramycin, asimicin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bexarotene, bicalutamide, bisantrene hydrochloride, bisnafide dimethylsulfate, bizelesin, bleomycin sulfate, brequinar sodium, carbomethoxyl, broxuridine, bratasin, busulfan, cabergoline, actinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carboquone hydrochloride, carzelesin, cerivastatin, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine,DACA (N-[2-(dimethyl-amino)ethyl]acridine-4-carboxamide), dactinomycin, daunorubicin hydrochloride, daunomycin, decitabine, denileukin diftitox, dexormaplatin, desaguanine, desaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drostanolone propionate, duazomycin, edatrexate, efloxatin hydrochloride, elsamitrucin, enloplatin, empromate, epipropidine, epirubicin hydrochloride, erbucol, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, ethiodized oil I 131, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, 5-FdUMP, fluorocitabine, fosquidone, fostriecin sodium, FK-317, FK-973, FR-66979, FR-900482, gemcitabine, gemcitabine hydrochloride, gemtuzumab ozogamicin, gold Au 198, goserelin acetate, guanacone, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, mitansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, methoxsalen, methopterin, meturedepa, mitindomide, mitocarcin, mitocromin,Mitogillin, Mitomalcin, Mitomycin, Mitomycin C, Mitosper, Mitotane, Mitoxantrone Hydrochloride, Mycophenolic Acid, Nocodazole, Nogalamycin, Oprelvekin, Oxaliplatin, Oxisuran, Paclitaxel, Pamidronate Disodium, Pegaspargase, Peliomycin, Pentostatin, Pepromycin Sulfate, Perfosfamide, Pipobroman, Piposulfan, Pirarubicin Hydrochloride, Plicamycin, Promestane, Porfimer Sodium, Porfiromycin, Prednimustine, Procarbazine Hydrochloride, Puromycin, Puromycin Hydrochloride, Pyrazofurin, Riboprine, Rituximab, Logretimide, Rolliniastatin, Safingol, Safingol Hydrochloride, Samarium / Lexidronam, Semustine, Simtrazene, Sparfosate Sodium, Sparsomycin, Spirogermanium Hydrochloride, Spiroprimycin, Spiroplatin, Squamocin, Squamotacin, Streptozocin, Strontium Chloride Sr89, ThioTEPA, Taliomycin, Taxane, Taxoid, Tecogalan Sodium, Tegafur, Teloxantrone Hydrochloride, Temoporfin, Teniposide, Teloxiron, Testolactone, Thiamiprine, Thioguanine, Thiotepa, Thymitaq, Thiazofurin, Tirapazamine, Tomudex, TOP-53, Topotecan Hydrochloride, Toremifene Citrate, Trastuzumab, Trestolone Acetate, Triciribine Phosphate, Trimethoprim, Trimethoprim Glucuronide, Triptorelin, Tributyltin Chloride, Uracil Mustard, Uredepa, Valrubicin, Bapreotide, Verteporfin, Vinblastine, Vinblastine Sulfate, Vincristine, Vincristine Sulfate, Vindesine, Vindesine Sulfate, Vineropidine Sulfate, Vinglycinate Sulfate, Vinleurosine Sulfate, Vinorelbine Tartrate, Vinrosidine SulfateVinzolidine sulfate, borozole, zinoplatin, dinostatin, zorubicin hydrochloride, 2-chlorodeoxyadenosine, 2'-deoxyformycin, 9-aminocamptothecin, raltitrexed, N-propargyl-5,8-dideazafolic acid, 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, 2-chloro-2'-deoxyadenosine, anisomycin, trichostatin A, hPRL-G129R, CEP-751, linomide, sulfur mustard, nitrogen mustard (mechlorethamine), cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-methyl-N-nitrosourea (MNU), N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU), N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nitrosourea (fotemustine), streptozotocin, diacarbazine (DTIC), mitozolomide, temozolomide, thiotepa, mitomycin C, AZQ, adozelesin, cisplatin, carboplatin, ormaplatin, oxaliplatin, C1-973, DWA2114R, JM216, JM335, Bis(platinum), tomudex, azacitidine, cytarabine, gemcitabine, 6-mercaptopurine, 6-thioguanine, hypoxanthine, teniposide, 9-aminocamptothecin, topotecan, CPT-11, doxorubicin, daunomycin, epirubicin, darbicin, mitoxantrone, losoxantrone, dactinomycin (actinomycin D), amsacrine, pyrazoloacridine, all-trans retinol, 14-hydroxyretinol, all-trans retinoic acid, N-(4-hydroxyphenyl)retinamide, 13-cis retinoic acid, 3-methyl TTNEB, 9-cis retinoic acid, fludarabine (2-F-ara-AMP), and 2-chlorodeoxyadenosine (2-Cda).

[0126] Other chemotherapeutic agents intended to be combined with the compounds and compositions of the present invention include, but are not limited to, the following: 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adespenol, adozelesin, aldesleukin, ALL-TK antagonist, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, anti-androgen, prostate cancer, anti-estrogen, antineoplaston, antisense oligonucleotide, glycidic acid aphidicolin, apoptosis gene modulator, apoptosis regulator, appurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azaseron, azatoxin, azatyrosine, baccatin III derivative, baranol, batimastat, BCR / ABL antagonist, benzochlorin, benzoyl staurosporine, beta-lactam derivative, beta-alethine, beta-clamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinyl spermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, broxuridine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivative (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine, carboxamide-amino-triazole, carboxamide triazole, CaRest M3, CARN 700,Chondrogenesis inhibitors, calpain, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorins, chloroquinequinone sulfonamide, cicaprost, cis-porphyrin, cladribine analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, classin A, cyclopentanthraquinone, cycloplatam, cypemycin, cytarabine oxophosphate, cytolytic factors, cytostatic, dacliximab, decitabine, dehydrodidemnin B, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspirostatin, discodermolide, docosanol, drotrecogin, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, econumycin, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epirubicin, epothilones (A, R = H, B, R = Me), epothilone, epristeride, estramustine analogs, estrogen agonists, estrogen antagonists, ethynidazole, etoposide, etoposide 4'-phosphate (etopophos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, formestane, fostriecin, fostmestin, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix,Gelatinase inhibitor, gemcitabine, glutathione inhibitor, hepsulfam, heregulin, hexamethylenebisacetamide, homoharringtonine (HHT), hypericin, ibandronic acid idarubicin, idoxifene, idramantone, ilmofosine, irostat, imidazoacridone, imiquimod, immunostimulatory peptide, insulin-like growth factor-1 receptor inhibitor, interferon agonist, interferon, interleukin, yobenguan, iododoxorubicin, ipomeanol, 4-, irinotecan, iroplact, ilsogladine, isobengazole, isohomohalicondrin B, itacetron, jasplakinolide, kahalalide F, lamellarinin-N triacetate, lanreotide, reynamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprorelin + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compound, lissoclinamide 7, lobaplatin, lombricin, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, lytic peptide, maytansine, mannan protein A, marimastat, masoprocol, maspin, matrilysin inhibitor, matrix metalloproteinase inhibitor, menogaril, melvalone, metelethrin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, millimostim, mismatched double-stranded RNA, mitracin, mitoguazone, mitolactol, mitomycin analog, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotropin, mopidamol, multidrug resistance gene inhibitor,Multiple tumor suppressor 1-based therapy, mustard anticancer agent, mycaperoxide B, mycobacterial cell wall extract, myriaporone, N-acetyl dinarine, N-substituted benzamide, naphthalene, nagrestip, naloxone + pentazocine, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, nerydronic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulator, nitroxide antioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone, oligonucleotide, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel analog, paclitaxel derivative, palauamine, palmitoyl lysophosphatidic acid, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, perdesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitor, picibanil, pilocarpine hydrochloride, pirarubicin, pirimtrex, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compound, platinum-triamine complex, podophyllotoxin, porfimer sodium, porphyromycin, propylbisacridone, prostaglandin J2, proteasome inhibitor, protein A-based immunomodulator, protein kinase C inhibitor, multiple protein kinase C inhibitors, microalgae, protein tyrosine phosphatase inhibitor, purine nucleoside phosphorylase inhibitor, purpurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonist,raltitrexed, lamotrigine, ras farnesyl protein transferase inhibitor, ras inhibitor, ras-GAP inhibitor, retiliptin demethylation, rhenium Re186 etidronate, lysokin, ribozyme, RII retinamide, rogletimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetic, semustine, senescence derived inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single-chain antigen-binding protein, schizophyllan, sobuzoxan, sodium borocaptate, sodium phenylacetate, solverol, somatomedin-binding protein, sonermin, sparfosic acid, spicamycin D, spiro-mustine, splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, stipiamide, stromelysin inhibitor, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramine, swainsonine, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellurapyrylium, telomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide, thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonistThymotrinan, thyroid stimulating hormone, ethyl ethio purpurin tin, tilapazamine, titanocene dichloride, topotecan, topsentin, tamoxifen, pluripotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, triciribine, trimethoprim, tryptoreline, Tropisetron, turosteride, tyrosine kinase inhibitor, tilostatin, UBC inhibitor, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonist, vapreotide, variolin B, vector system, erythrocyte gene therapy, velaresol, veramine, verdin, verteporfin, vinorelbine, vinxaltine, vitaxin, borosole, zanoterone, zeniplatin, zilascorb, and dinostatin stimalamer.

[0127] Other chemotherapeutic agents intended to be combined with the compounds and compositions of the present invention include, but are not limited to, anti-proliferative agents (e.g., Piritrexim Isothionate), anti-benign prostatic hyperplasia agents (e.g., Citoglucoside), agents for treating benign prostatic hyperplasia (e.g., Tamsulosin Hydrochloride), prostate growth inhibitors (e.g., Pentomone), and radioactive substances: Fibrinogen 1 125, Fluorodeoxyglucose F 18, Fluorodopa F 18, Insulin I 125, Insulin I 131, Iobenguane I 123, Iopamidate Sodium I 131, Iodoantipyrine I 131, Iodocholesterol I 131, Iohippurate Sodium I 123, Iohippurate Sodium I 125, Iohippurate Sodium I 131, Iodopyracet I 125, Iodopyracet I 131, Iofetamine Hydrochloride I 123, Iomethin I 125, Iomethin I 131, Iotalamate Sodium I 125, Iotalamate Sodium I 131, Iothyrosine I 131, Liothyronine I 125, Liothyronine I 131, Merisoprol Acetate Hg 197, Merisoprol Hg 203, Merisoprol Hg 197, Methyl Iodobenzo Guanine (MIBG-I131 or MIBG-I123), Selenomethionine Se 75, Technetium Tc 99m Antimony Trisulfide Colloid, Technetium Tc 99m Bicisate, Technetium Tc 99m Disofenin, Technetium Tc 99m Etidronate, Technetium Tc 99m Exametazine, Technetium Tc 99m Furofosmin, Technetium Tc 99m Gluceptate, Technetium TC 99m Lidofenin, Technetium Tc 99m Mebrofenin, Technetium Tc 99m Medronate, Technetium Tc 99m Medronate Disodium, Technetium Tc 99m Meretiazone, Technetium Tc 99m Oxidronate, Technetium Tc 99m Pentetate, Technetium Tc 99m Pentetate Calcium Trisodium, Technetium Tc 99m Sestamibi, Technetium TcIncluded are 99m siboroxime, technetium Tc 99m saccimer, technetium Tc 99m sulfur colloid, technetium Tc 99m teboroxime, technetium Tc 99m tetrofosmin, technetium Tc 99m thiatide, thyroxine I 125, thyroxine I 131, tolpovidone I 131, triolein I 125, and triolein I 131. MIBG-I131 and MIBG-I123 are particularly preferred chemotherapeutic agents for co-administration with the compounds and compositions of the present invention.

[0128] Another category of chemotherapeutic agents intended to be combined with the compounds and compositions of the present invention includes one or more anti-cancer adjuvants, such as tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline), non-tricyclic antidepressants (e.g., sertraline, trazodone, and citalopram), Ca ++Antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine), calmodulin inhibitors (e.g., prenylamine, trifluoperazine and clomipramine), amphotericin B, triparanol analogs (e.g., tamoxifen), antiarrhythmic agents (e.g., quinidine), antihypertensive agents (e.g., reserpine), thiol depleters (e.g., buthionine and sulfoximine) and multiple drug resistance reducing agents, such as Cremaphor EL etc. are included, but not limited thereto. Other chemotherapeutic agents that can be combined with the agents described in the present invention include: annonaceous acetogenins, asimicin, rolliniastatin, guanacone, squamocin, bullatacin, squamotacin, taxanes, paclitaxel, gemcitabine, methotrexate FR-900482, FK-973, FR-66979, FK-317, 5-FU, FUDR, FdUMP, hydroxyurea, docetaxel, discodermolide, epothilone, vincristine, vinblastine, vinorelbine, meta-pac, irinotecan, SN-38, 10-OH camptothecin, topotecan, etoposide, adriamycin, flavopiridol, cis-Pt, carboplatin, bleomycin, mitomycin C, mitramycin, capecitabine, cytarabine, 2-Cl-2'deoxyadenosine, fludarabine-PO4, mitoxantrone, mitozolomide, pentostatin, and tomudex.

[0129] One important class of chemotherapeutic agents intended to be combined with the compounds and compositions of the present invention is taxanes (e.g., paclitaxel and docetaxel). For example, tamoxifen or the aromatase inhibitor Arimidex (i.e., anastrozole) combined with the compounds and compositions of the present invention is particularly useful for breast cancer and gynecological cancers (such as ovarian cancer).

[0130] As an important aspect of the present invention, the compounds and compositions of the present invention are administered in combination with an antibody. Examples of antibodies that can be used as other active ingredients according to the present invention include, but are not limited to, anti-CD20 antibodies (including monoclonal antibodies, or mAbs), rituximab, rituxan (trademark), tositumomab-bexarotene, anti-HER2 antibodies, trastuzumab, herceptin (trademark), MDX-210, anti-CA125 mAb, oregovomab, B43.13, Ovarex (trademark), Breva-Rex, AR54, GivaRex, ProstaRex, anti-EGF receptor mAb, IMC-C225, abraxane (trademark), anti-EGF receptor mAb, MDX-447, gemtuzumab ozogamicin, mylotarg, CMA-676, anti-CD33 (Wyeth Pharmaceuticals), anti-tissue factor protein (TF), ior-c5, anti-c5 antibody, anti-EGF receptor mAb, MDX-447, anti-17-1A mAb, edrecolomab, panorex, anti-CD20 mAb (Y-90 labeled), ibritumomab tiuxetan (IDEC-Y2B8), zevalin, anti-idiotype mAb mimetic of ganglioside GD3 epitope, BEC2, anti-HLA-Dr10 mAb (131I LYM-1), Oncolym (trademark), anti-CD33 humanized mAb (SMART M195), Zamyl (trademark), anti-CD52 humAb (LDP-03), CAMPATH, anti-CD1 mAb, anti-t6 antibody, anti-CAR (complement activation receptor) mAb, MDX-11, humanized bispecific mAb conjugate (complement cascade activator), MDX-22, OV103 (Y-90 labeled antibody), serogovab, OncoScint (trademark), anti-17-1A mAb, 3622W94, anti-VEGF (RhumAb-VEGF), bevacizumab, Avastin (trademark), anti-TAC (IL-2 receptor) humanized antibody (SMART), daclizumab, zenapax, anti-TAG-72 partially humanized bispecific antibody, MDX-220, anti-idiotype mAb mimetic of high molecular weight proteoglycan (I-Mel-1), MELIMMUNE-1, anti-idiotype mAb mimetic of high molecular weight proteoglycan (I-Mel-2), MELIMMUNE-2, anti-CEAAb(hMN14), CEACide (trademark), Pretarget (trademark) radiolabeled targeting agents, hmAbH11 scFv fragment (NovomAb-G2), H11 scFv, anti-DNA or DNA-related protein (histone) mAb and conjugates, TNT (e.g., Cotara (trademark)), Gliomab-H mAb, GNI-250 mAb, anti-EGF receptor mAb, EMD-72000, anti-CD22 humanized Ab, LymphoCide, anti-CD33 mAb conjugate with non-Hodgkin's calicheamicin (CMA 676), gemtuzumab ozogamicin, Mylotarg (trademark), Monopharm-C, colon, anti-idiotype human mAb against GD2 ganglioside, 4B5, melanoma, anti-EGF receptor humanized Ab, ior egf / r3, anti-ior c2 glycoprotein mAb, BABS (biosynthetic antibody binding site) protein, anti-FLK-2 / FLT-3 mAb, mAb / small molecule conjugate, TAP (tumor-activated prodrug), anti-GD-2 bispecific mAb, MDX-260, anti-nuclear autoantibody (binding monoclonal antibody), ANA Ab, anti-HLA-DR Ab (SMART 1D10 Ab), Remitogen (trademark), SMART ABL 364 Ab, anti-CEA I131-labeled mAb, and ImmuRAIT-CEA, are included.

[0131] Other antibodies for combination with the compounds and compositions of the present invention include, but are not limited to, anti-TNFα antibodies such as infliximab (Remicade) and etanercept (Enbrel) for rheumatoid arthritis and Crohn's disease, palivizuma, anti-RSV antibody for pediatric subjects, bevacizumab, alemtuzumab, Campath-1H, BLyS-mAb, fSLE, anti-VEGF2, anti-Trail receptor, B3 mAb, m170 mAb, mAB BR96, and Abx-Cbl mAb. The present invention encompasses several classes of antibodies and fragments thereof, including but not limited to antibodies directed against cancer antigens (described herein), cell surface molecules, stromal cell molecules, extracellular matrix molecules, and tumor vasculature-related molecules.

[0132] Cell surface molecules are molecules expressed on the surface of cells. In addition to the extracellular domain, it may further include a transmembrane domain and a cytoplasmic domain. Examples include HLA markers such as HER2, CD20, CD33, EGF receptor, HLA-DR, CD52, CD1, CEA, CD22, GD2 ganglioside, FLK2 / FLT3, VEGF, VEGFR, etc.

[0133] Stromal cell molecules are molecules expressed by stromal cells. Examples include, but are not limited to, FAP and CD26.

[0134] Extracellular matrix molecules are molecules found in the extracellular matrix. Examples include, but are not limited to, collagen, glycosaminoglycan (GAG), proteoglycan, elastin, fibronectin, and laminin.

[0135] Tumor vasculature-related molecules are molecules expressed by the vasculature of a tumor (i.e., solid tumors, not systemic cancers such as leukemia). Similar to cancer antigens, tumor vasculature-related molecules can be expressed by normal vasculature, but by virtue of their presence on the vasculature of a tumor, they are suitable targets for anti-cancer therapy. In some cases, tumor vasculature-related molecules are expressed at higher levels in the tumor vasculature than in normal vasculature. Examples include, but are not limited to, endoglin (see U.S. Patent No. 5,660,827), ELAM-1, VCAM-1, ICAM-1, ligands reactive with LAM-1, MHC class II antigens, aminophospholipids such as phosphatidylserine and phosphatidylethanolamine (such as those described in U.S. Patent No. 6,312,694), VEGFR1 (Flt-1) and VEGFR2 (KDR / Flk-1), and other tumor vasculature-related molecules as described in U.S. Patent No. 5,776,427. Antibodies against endoglin are described in U.S. Patent No. 5,660,827 and include TEC-4 and TEC-11, and antibodies that recognize the same epitopes as these antibodies. Antibodies against aminophospholipids are described in U.S. Patent No. 6,312,694. Antibodies that inhibit VEGF are described in U.S. Patent 6,342,219 and include 2C3 (ATCC PTA1595). Other antibodies specific for the tumor vasculature include antibodies that react with complexes of growth factors and their receptors, such as complexes of FGF and FGFR or TGFβ and TGFβR. Antibodies of this latter class are described in U.S. Patent No. 5,965,132 and include GV39 and GV97.

[0136] It should be understood that the antibodies included in the present invention also include the antibodies expressly described herein and antibodies that bind to the same epitopes as those described herein.

[0137] The compounds and compositions described in the present invention can achieve excellent results in combination with apoptosis antibodies. Exemplary apoptosis antibodies include, but are not limited to, anti-BAX antibodies (which antagonize their BAX antigens derived from human, murine, and / or different animal sources), anti-flax antibodies, anti-Fas / Fas ligand antibodies, anti-granzyme antibodies (e.g., anti-granzyme B antibodies), anti-BCL antibodies, anti-cytochrome C antibodies, antibodies that antagonize TRADD, TRAIL, TRAFF, and / or DR3, anti-BIM antibodies, anti-PARP antibodies, anti-caspase antibodies, anti-CD29, PL18-5 PanVera, anti-CD29, PL4-3 PanVera, anti-CD41a, PT25-2 PanVera, anti-CD42b, PL52-4 PanVera, anti-CD42b, GUR20-5 PanVera, anti-CD42b, WGA-3 PanVera, anti-CD43, 1D4 PanVera, anti-CD46, MCP75-6 PanVera, anti-CD61, PL11-7 PanVera, anti-CD61, PL8-5 PanVera, anti-CD62 / P-slctn, PL7-6 PanVera, anti-CD62 / P-slctn, WGA-1 PanVera, anti-CD154, 5F3 PanVera, and anti-CD1, anti-CD2, anti-CD3, anti-CD4, anti-CD5, anti-CD6, anti-CD7, anti-CD8, anti-CD9, anti-CD10, anti-CD11, anti-CD12, anti-CD13, anti-CD14, anti-CD15, anti-CD16, anti-CD17, anti-CD18, anti-CD19, anti-CD20, anti-CD21, anti-CD22, anti-CD23, anti-CD24, anti-CD25, anti-CD26, anti-CD27, anti-CD28, anti-CD29, anti-CD30, anti-CD31, anti-CD32, anti-CD33, anti-CD34, anti-CD35, anti-CD36, anti-CD37, anti-CD38, anti-CD39, anti-CD40 anti-CD41, anti-CD42, anti-CD43, anti-CD44, anti-CD45, anti-CD46, anti-CD47, anti-CD48, anti-CD49, anti-CD50, anti-CD51, anti-CD52, anti-CD53, anti-CD54, anti-CD55, anti-CD56, anti-CD57, anti-CD58, anti-CD59, anti-CD60, anti-CD61, anti-CD62, anti-CD63, anti-CD64, anti-CD65, anti-CD66, anti-CD67, anti-CD68, anti-CD69, anti-CD70, anti-CD71, anti-CD72, anti-CD73, anti-CD74, anti-CD75,Anti-CD76, Anti-CD77, Anti-CD78, Anti-CD79, Anti-CD80, Anti-CD81, Anti-CD82, Anti-CD83, Anti-CD84, Anti-CD85, Anti-CD86, Anti-CD87, Anti-CD88, Anti-CD89, Anti-CD90, Anti-CD91, Anti-CD92, Anti-CD93, Anti-CD94, Anti-CD95, Anti-CD96, Anti-CD97, Anti-CD98, Anti-CD99, Anti-CD100, Anti-CD101, Anti-CD102, Anti-CD103, Anti-CD104, Anti-CD105, Anti-CD106, Anti-CD107, Anti-CD108, Anti-CD109, Anti-CD110, Anti-CD111, Anti-CD112, Anti-CD113, Anti-CD114, Anti-CD115, Anti-CD116, Anti-CD117, Anti-CD118, Anti-CD119, Anti-CD120, Anti-CD121, Anti-CD122, Anti-CD123, Anti-CD124, Anti-CD125, Anti-CD126, Anti-CD127, Anti-CD128, Anti-CD129, Anti-CD130, Anti-CD131, Anti-CD132, Anti-CD133, Anti-CD134, Anti-CD135, Anti-CD136, Anti-CD137, Anti-CD138, Anti-CD139, Anti-CD140, Anti-CD141, Anti-CD142, Anti-CD143, Anti-CD144, Anti-CD145, Anti-CD146, Anti-CD147, Anti-CD148, Anti-CD149, Anti-CD150, Anti-CD151, Anti-CD152, Anti-CD153, Anti-CD154, Anti-CD155, Anti-CD156, Anti-CD157, Anti-CD158, Anti-CD159, Anti-CD160, Anti-CD161, Anti-CD162, Anti-CD163, Anti-CD164, Anti-CD165, Anti-CD166, Anti-CD167, Anti-CD168, Anti-CD169, Anti-CD170, Anti-CD171, Anti-CD172, Anti-CD173, Anti-CD174, Anti-CD175, Anti-CD176, Anti-CD177, Anti-CD178, Anti-CD179, Anti-CD180, Anti-CD181, Anti-CD182, Anti-CD183, Anti-CD184, Anti-CD185, Anti-CD186, Anti-CD187, Anti-CD188, Anti-CD189, Anti-CD190, Anti-CD191, Anti-CD192, Anti-CD193, Anti-CD194, Anti-CD195, Anti-CD196, Anti-CD197, Anti-CD198, Anti-CD199, Anti-CD200, Anti-CD201, Anti-CD202, Anti-CD203, Anti-CD204, Anti-CD205, Anti-CD206, Anti-CD207, Anti-CD208, Anti-CD209, Anti-CD210, Anti-CD211, Anti-CD212, Anti-CD213, Anti-CD214, Anti-CD215, Anti-CD216, Anti-CD217, Anti-CD218, Anti-CD219, Anti-CD220, Anti-CD221Antibodies such as anti-CD222, anti-CD223, anti-CD224, anti-CD225, anti-CD226, anti-CD227, anti-CD228, anti-CD229, anti-CD230, anti-CD231, anti-CD232, anti-CD233, anti-CD234, anti-CD235, anti-CD236, anti-CD237, anti-CD238, anti-CD239, anti-CD240, anti-CD241, anti-CD242, anti-CD243, anti-CD244, anti-CD245, anti-CD246, anti-CD247, anti-CD248, anti-CD249, anti-CD250 are included.

[0138] Other exemplary human chemokine antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, human CNTF antibody, human eosinotaxin antibody, human epithelial neutrophil activating peptide-78, human exodus antibody, human GRO antibody, human HCC-1 antibody, human I-309 antibody, human IP-10 antibody, human I-TAC antibody, human LIF antibody, human liver-expressed chemokine antibody, human lymphotoxin antibody, human MCP antibody, human MIP antibody, induced human monokine by IFN-gamma antibody, human NAP-2 antibody, human NP-1 antibody, human platelet factor 4 antibody, human RANTES antibody, human SDF antibody, and human TECK antibody.

[0139] Other exemplary chemokine antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, human B cell attracting mouse chemokine antibody, chemokine-1 antibody, mouse eosinotaxin antibody, mouse exodus antibody, mouse GCP-2 antibody, mouse KC antibody, mouse MCP antibody, mouse MIP antibody, mouse RANTES antibody, rat chemokine antibody, rat chemokine antibody, rat CNTF antibody, rat GRO antibody, rat MCP antibody, rat MIP antibody, and rat RANTES antibody.

[0140] Exemplary cytokine / cytokine receptor antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, human biotinylated cytokine / cytokine receptor antibodies, human IFN antibodies, human IL antibodies, human leptin antibodies, human oncostatin antibodies, human TNF antibodies, human TNF receptor family antibodies, mouse biotinylated cytokine / cytokine receptor antibodies, mouse IFN antibodies, mouse IL antibodies, mouse TNF antibodies, mouse TNF receptor antibodies, mouse anti-CCR4 antibodies, rat biotinylated cytokine / cytokine receptor antibodies, rat IFN antibodies, rat IL antibodies, and rat TNF antibodies.

[0141] Exemplary ECM antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, collagen / procollagen, laminin, collagen (human), laminin (human), procollagen (human), vitronectin / vitronectin receptor, vitronectin (human), vitronectin receptor (human), fibronectin / fibronectin receptor, fibronectin (human), and fibronectin receptor (human).

[0142] Exemplary growth factor antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, human growth factor antibodies, mouse growth factor antibodies, and porcine growth factor antibodies.

[0143] Other exemplary growth factor antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, baculovirus antibodies, cadherin antibodies, complement antibodies, Clq antibodies, von Willebrand factor antibodies, Cre antibodies, HIV antibodies, influenza antibodies, human leptin antibodies, mouse leptin antibodies, mouse CTLA-4 antibodies, human CTLA-4 antibodies, P450 antibodies, and RNA polymerase antibodies.

[0144] Exemplary neurobiological antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, amyloid antibodies, GFAP antibodies, human NGF antibodies, human NT-3 antibodies, and human NT-4 antibodies.

[0145] Additional exemplary antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, antibodies described in references such as the MSRS catalog of primary antibodies and the Linscott Directory.

[0146] In some preferred embodiments of the present invention, antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, Avastin (bevacizumab), BEC2 (mitumomab), Bexxar (tositumomab), Campath (alemtuzumab), CeaVac, Herceptin (trastuzumab), IMC-C225 (cetuximab), LymphoCide (epratuzumab), MDX-210, Mylotarg (gemtuzumab ozogamicin), Panorex (edrecolomab), Rituxan (rituximab), Theragyn (pemtumomab), Zamyl, and Zevalin (ibritumomab tiuxetan). The present invention also encompasses antibody fragments thereof.

[0147] In some preferred embodiments, the cancer antigen is VEGF, anti-idiotype mAb (GD3 ganglioside mimic), CD20, CD52; anti-idiotype mAb (CEA mimic), ERBB2, EGFR, CD22, ERBB2 X CD65 (fcγRI), EpCam, PEM, and CD33.

[0148] Antibodies administered in combination with the compounds and compositions described in the present invention include, but are not limited to, monoclonal or polyclonal antibodies that can be prepared by conventional methodologies. They may be further isolated or may be present in ascites. Such antibodies can be further engineered to produce chimeric or humanized antibodies, as discussed in more detail below.

[0149] Importantly, as is well known in the art, only the paratope, which is a small part of the antibody molecule, is involved in the binding of the antibody to its epitope (see generally Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York, Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). For example, the pFc’ and Fc regions are effectors of the complement cascade but are not involved in antigen binding. Antibodies in which the pFc’ region has been enzymatically cleaved or produced without the pFc’ region are called F(ab’)2 fragments and retain both antigen-binding sites of the intact antibody. Similarly, antibodies in which the Fc region has been enzymatically cleaved or produced without the Fc region are called Fab fragments and retain one of the antigen-binding sites of the intact antibody molecule. Further, the Fab fragment is composed of the covalently linked antibody light chain and a portion of the antibody heavy chain designated Fd. The Fd fragment is the major determinant of antibody specificity (a single Fd fragment can bind up to 10 different light chains without changing antibody specificity), and the Fd fragment retains epitope-binding ability alone.

[0150] Included in the present invention are the complementarity-determining regions (CDRs) of the antibody that directly interact with the epitope of the antigen and the framework regions (FRs) that maintain the tertiary structure of the paratope (see generally Clark, 1986; Roitt, 1991).

[0151] Also included are non-CDR regions of mammalian antibodies, which can be replaced with similar regions of co-specific or heterospecific antibodies while retaining the epitope specificity of the original antibody. Such antibodies are referred to as "chimeric" antibodies, including intact antibody fragments having antigen-binding ability.

[0152] The present invention also provides F(ab’)2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and / or FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric F(ab’)2 fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and / or CDR1 and / or CDR2 and / or light chain CDR3 regions are replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and / or CDR1 and / or CDR2 regions are replaced by homologous human or non-human sequences. The present invention also includes so-called single-chain antibodies.

[0153] The compounds and compositions described herein are administered in therapeutically effective and physiologically acceptable amounts, which are amounts that are physiologically acceptable to the subject and that are necessary or sufficient to achieve the desired beneficial biological effect, in this case the treatment of cancer or the inhibition of angiogenesis. The biologically beneficial effect can be measured, for example, by determining the physiological effect of the treatment after therapeutic administration. The biologically beneficial effect can be the improvement and / or absolute elimination of symptoms resulting from the disorder being treated, or the inhibition of angiogenesis in the disorder being treated, as demonstrated, for example, by a reduction in the number of microvessels (e.g., abnormal microvessels) in imaging.

[0154] A therapeutically effective and physiologically acceptable amount can vary depending on the particular compound or combination of compounds, composition or combination of compositions, and / or therapy employed. It can also vary depending on factors such as the condition being treated (e.g., cancer), the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a compound or combination of compounds of a particular septin target compound without undue experimentation. In combination with the teachings provided herein, by selecting from various compounds and weighting factors such as efficacy, relative bioavailability, patient body weight, severity of adverse side effects, and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned that causes no substantial toxicity but is still fully effective in treating a particular subject.

[0155] In some cases, a dosage below the therapeutic amount of either the compounds or compositions described herein or a second agent, or below the therapeutic amounts of both, is used to treat a subject. For example, when a compound or composition described herein is used together with an anti-cancer agent, the compound or composition and the anti-cancer agent are administered at sub-therapeutic dosages and still produce a desired therapeutic effect. As used herein, a "sub-therapeutic dosage" refers to a dosage that is less than the dosage that would produce a therapeutic result in a subject if administered in the absence of other agents. Thus, a sub-therapeutic dosage of an anti-cancer agent is a dosage that would not produce the same or substantially similar therapeutic results in a subject in the absence of administration of the compounds or compositions described herein. The therapeutic amounts of anti-cancer agents are known in the medical field. These dosages are widely described in references such as Remington’s Pharmaceutical Sciences, 18th ed., 1990, and many other medical references relied upon by medical professionals as guidance for cancer treatment and are well known in the art.

[0156] In the case of the compounds and compositions described herein, a therapeutically effective amount is first determined from in vitro assays such as cell culture assays. A therapeutically effective amount can also be determined in animal experiments. For example, the effective amount of a compound or composition described herein, with or without a second agent, is evaluated using, for example, in vivo assays of tumor regression and / or prevention of tumor formation. Relevant animal models include, for example, assays in which malignant cells are injected into an animal subject, usually at a defined site. Generally, a range of septin target compound doses is administered to the animals. Inhibition of tumor growth after injection of malignant cells indicates the ability to reduce the risk of developing cancer. Inhibition of further growth (or reduction in size) of an existing tumor indicates the ability to treat cancer.

[0157] The applied doses of both agents can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the doses to achieve maximum effectiveness based on the above methods and other methods is within the capabilities of a normal person skilled in the art.

[0158] Preferred target doses of the compounds and compositions described herein typically range from about 0.1 μg to 30,000 mg, more typically from about 1 μg / day to 20,000 mg, even more typically from about 10 μg to 15,000 mg, and most typically from about 100 μg to 10,000 μg. Stated in terms of the subject's body weight, typical dosages range from about 0.1 μg to 200 mg / kg / day, more typically from about 0.5 to 150 mg / kg / day. In some important embodiments, the compound is administered in an amount of about 1 to 100 mg / kg / day. In some other important embodiments, the compound is administered in an amount of 10 to 60 mg / kg / day.

[0159] As used herein, "routine schedule" refers to a predetermined specified period selected for delivering the compounds or compositions described in the present invention. The routine schedule includes periods of the same or different lengths as long as the schedule is pre-determined. The routine schedule can be, for example, administration 2, 3, 4, or 6 times per day, daily administration, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, weekly administration, monthly administration or any set number of days or weeks in between - among which, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, etc., are included. Alternatively, a given routine schedule can include administering daily in the first week, monthly for several subsequent months, and then every 3 months thereafter. Any particular combination is covered by the routine schedule as long as it is pre-determined that the appropriate schedule includes administration on a particular day.

[0160] The compounds or compositions of the present invention are administered in a pharmaceutically acceptable carrier or in the context of a vector or delivery system. Examples of the chemical / physical vectors of the present invention are colloidal dispersions. Colloidal dispersions include oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems including liposomes. A preferred colloidal system of the present invention is liposomes. Liposomes are artificial membrane containers useful as delivery vectors in vivo or in vitro. Large unilamellar vesicles (LUVs) with sizes of 0.2 - 4.0 μm have been shown to be able to encapsulate large macromolecules. RNA, DNA, and intact virions are encapsulated in the aqueous interior and delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., (1981) 6:77).

[0161] The compounds embodying the present invention can be delivered using liposomes. By binding liposomes to specific ligands such as sugars, glycolipids, or proteins, the liposomes can be targeted to specific tissues. Ligands that may be useful for targeting liposomes to cells include, but are not limited to, intact or fragmented molecules that interact with cell-specific receptors, and molecules such as antibodies that interact with cell surface markers of cells. Such ligands can be readily identified by binding assays well known to those skilled in the art. In yet other embodiments, liposomes can target cancer, for example, by binding it to one of the aforementioned immunotherapy antibodies. Further, a vector can bind to a nuclear targeting peptide that directs the vector to the nucleus of a host cell.

[0162] Liposomes used for delivering the compounds or compositions described herein are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™ formed from cationic lipids such as N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and dimethyldioctadecylammonium bromide (DDAB). Methods for making liposomes are well known in the art and are described in many publications. Liposomes are also reviewed by Gregoriadis, G. in Trends in Biotechnology, (1985) 3:235-241.

[0163] In other embodiments, the chemical / physical vectors used for delivering the compounds embodying the present invention include biocompatible microspheres suitable for delivery such as oral or mucosal delivery. Such microspheres are disclosed in Chickering et al., Biotech. And Bioeng., (1996) 52:96-101 and Mathiowitz et al., Nature, (1997) 386:410-414 as well as PCT patent application No. WO97 / 03702.

[0164] Both non-biodegradable and biodegradable polymer matrices that can be used to deliver the compounds or compositions described in the present invention include a biodegradable matrix. Such polymers can be natural or synthetic polymers. The polymers are generally selected on the order of several hours to over a year, based on the period during which release is desired. Usually, release over a period in the range of several hours to 3 months to 12 months is most desirable. The polymer can optionally be in the form of a hydrogel that can absorb up to about 90% of its weight in water and can further optionally be cross-linked with polyvalent ions or other polymers.

[0165] The polymer matrices considered for use in formulating the agents described herein are preferably in the form of microparticles such as microspheres (where the agent is dispersed throughout the solid polymer matrix) or microcapsules (where the agent is contained in the core of a polymeric shell). Other forms of polymer matrices for containing agents that can be utilized to deliver the compounds embodying the present invention include films, coatings, gels, implants, and stents. The size and composition of the polymer matrix device are selected to provide a preferred release profile in the tissue into which the matrix is introduced. The size of the polymer matrix is further selected according to the delivery method used, typically injection into tissue, or administration of a suspension by aerosol to the nasal and / or pulmonary regions. Preferably, when the aerosol route is used, the compounds of the polymer matrix and the septin target compound are included in a surfactant vehicle. The polymer matrix composition can be selected to have both a preferred rate of degradation and to be formed from a bioadhesive material, further enhancing the effectiveness of migration when the matrix is administered to damaged nasal and / or pulmonary surfaces. The matrix composition can also be selected not to degrade, but rather to release by diffusion over a long period of time. In some preferred embodiments, the compounds or compositions described herein are administered to a subject via an implant.

[0166] Particularly interesting bioadhesive polymers include biodegradable hydrogels (such as those described by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587), polyhyaluronic acid, casein, gelatin, gluten, polyanhydrides, polyacrylic acid, alginates, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate)), poly(laurel methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0167] The compositions and methods of the present invention are useful for replacing existing surgical procedures or drug therapies, but in some cases, the present invention is useful for improving the effectiveness of existing therapies for treating such conditions. Thus, combination therapies can be used to treat subjects who are receiving or are scheduled to receive cancer treatment. For example, the agent can be administered to a subject in combination with another anti-proliferative (e.g., anti-cancer) therapy. Suitable anti-cancer therapies include surgical procedures to remove tumor mass, chemotherapy, or local radiation therapy. Other anti-proliferative therapies can be administered before, simultaneously with, or after treatment with the agent of the present invention. There may be a delay of several hours, days, or in some cases weeks between the administration of different treatments, such that the agent can be administered before or after the other treatment. In some embodiments, the compounds or compositions described herein are administered with or without (e.g., before surgery, radiation or chemotherapy) other anti-proliferative treatments, but the timing is not so limited.

[0168] The compounds or compositions described herein can also be administered in combination with non-surgical, anti-proliferative (e.g., anti-cancer) drug therapies. In some embodiments, the agent can be administered in combination with an anti-cancer agent such as a cytostatic compound. A cytostatic compound is a compound (e.g., nucleic acid, protein) that inhibits the growth and / or proliferation of cells. In some embodiments, the cytostatic compound is directed to malignant cells of a tumor. In still other embodiments, the cytostatic compound inhibits the growth and / or proliferation of vascular smooth muscle cells or fibroblasts.

[0169] According to the methods of the invention, the compounds or compositions described herein are administered before, simultaneously with, or after other anti-cancer agents. The dosing schedule includes administering different agents alternately. In other embodiments, the combination therapies of the invention are delivered before and during, or during and after, or before and after treatment with other therapies. In some cases, the agent is administered more than 24 hours prior to the administration of other anti-proliferative treatments. In other embodiments, two or more anti-proliferative therapies are administered to the subject. For example, the subject receives the agent of the invention in combination with both surgery and at least one other anti-proliferative compound. Alternatively, the agent is administered in combination with two or more anti-cancer agents.

[0170] The compounds or compositions described herein can be combined with other therapeutic agents, such as adjuvants, to enhance the immune response. The compounds or compositions described herein and the other therapeutic agents can be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously, they can be administered in the same or separate formulations, but are administered simultaneously. The administration of the other therapeutic agents and the compounds or compositions described herein can also be temporally separated, which means that the therapeutic agents are administered at different times, either before or after the administration of the compounds or compositions described herein. The separation in time between the administration of these compounds and agents can be several minutes or more. Other therapeutic agents include, but are not limited to, nucleic acid adjuvants, non-nucleic acid adjuvants, cytokines, non-immunotherapy antibodies, antigens, and the like.

[0171] A nucleic acid adjuvant is an adjuvant that is a nucleic acid. Examples include immunostimulatory nucleic acid molecules such as those containing CpG dinucleotides, as described in U.S. Patent No. 6,194,388 B1, issued February 27, 2001, U.S. Patent No. 6,207,646 B1, issued March 27, 2001, and U.S. Patent No. 6,239,116 B1, issued May 29, 2001.

[0172] A "non-nucleic acid adjuvant" is any molecule or compound other than the immunostimulatory nucleic acids described herein that can stimulate a humoral and / or cellular immune response. Non-nucleic acid adjuvants include, for example, adjuvants that produce a depot effect, immunostimulatory adjuvants, adjuvants that produce a depot effect and stimulate the immune system, and mucosal adjuvants.

[0173] As used herein, an "adjuvant that produces a depot effect" is an adjuvant that slowly releases an antigen such as a cancer antigen present in a cancer vaccine in the body, thereby extending the exposure of immune cells to the antigen. This class of adjuvants includes alum (e.g., aluminum hydroxide, aluminum phosphate), or mineral oil, non-mineral oil, water-in-oil or oil-in-water-in-oil emulsions, such as water-in-oil emulsions like the Montanide adjuvants of the Seppic ISA series (e.g., Montanide ISA 720, AirLiquide, Paris, France), MF-59 (a squalene-in-water emulsion stabilized with Span85 and Tween80, Chiron Corporation, Emeryville, CA), and PROVAX (a water-in-oil emulsion containing a stabilized detergent and micelle former, DEC Pharmaceuticals Corporation, San Diego, Calif.), including, but not limited to, emulsion-based formulations.

[0174] An "immunostimulatory adjuvant" is an adjuvant that causes activation of cells of the immune system. For example, it may cause immune cells to produce and secrete cytokines. This class of adjuvants includes, but is not limited to, saponins purified from the bark of the Q. saponaria tree such as QS21 (a glycolipid that elutes at the 21st peak in an HPLC fraction, Antigenics, Inc., Waltham, Mass.), poly[di(carboxylatophenoxy)phosphazene] (PCPP polymer, Virus Research Institute, USA), monophosphoryl lipid A (MPL, Ribi ImmunoChem Research, Inc., Hamilton, Mont.), derivatives of lipopolysaccharides such as muramyl dipeptide (MDP, Ribi) and threonyl-muramyl dipeptide (t-MDP, Ribi), OM-174 (a glucosamine disaccharide related to lipid A, OM Pharma SA, Meyrin, Switzerland), and Leishmania elongation factor (a purified Leishmania protein, Corixa Corporation, Seattle, Wash.).

[0175] "An adjuvant that produces a depot effect and stimulates the immune system" is a compound with both of the above functions. This class of adjuvants includes ISCOMS (immunostimulating complexes that contain mixed saponins, lipids, and form virus-sized particles with pores that can retain antigens, CSL, Melbourne, Australia), SB-AS2 (SmithKline Beecham adjuvant system #2, an oil-in-water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium), SB-AS4 (SmithKline Beecham adjuvant system #4, containing alum and MPL, SBB, Belgium), nonionic block copolymers that form micelles such as CRL 1005 (these contain straight chains of polyoxypropylene that are sandwiched by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Syntex adjuvant formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer, Syntex Chemicals, Inc., Boulder, Colo.), but is not limited thereto.

Example

[0176] Example 1 Representative Synthesis of the Disclosed Compound UR214-9 Compound UR214-9 was synthesized using the following general procedure.

Chem.

[0177] A solution of an amine (0.01 - 0.1 mol) in dry DMF was added dropwise with a solution of isocyanate (0.01 - 0.1 mol), and then the reaction mixture was heated and stirred at 65 °C overnight. The reaction mixture was cooled to room temperature, and DMF was removed under reduced pressure. A portion of the resulting reaction mixture was purified using preparative thin-layer chromatography with hexane:ethyl acetate or DCM:MeOH as the eluent. The desired band was scraped off, and the compound was detached from the silica gel using MeOH:DCM (90:10). The solvent was evaporated under reduced pressure, and the desired compound was collected as a powder.

[0178] Other compounds of the present application, such as those disclosed or exemplified in the present application (e.g., compounds of formulas (I) - (IV)), are also prepared according to the same synthetic route.

[0179] A representative mass spectrum of UR214 - 9 obtained by this process is presented in FIG. 13.

[0180] Example 2 Development of Potent Septin Inhibitors and Their Cytotoxic Effects in Cancer Cell Lines Introduction Malignant tumors of the ovary and uterus are relatively common and unfortunately very lethal. Uterine cancer, the fourth most common cancer in women, shows approximately 60,000 new cancer diagnoses and 10,000 cancer-related deaths annually in the United States (see Siegel, R.L., Miller, K.D. & Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 67, 7 - 30, 2017). Although less common, ovarian cancer is even more lethal, with a 5-year survival rate of 46.6% (www.cdc.gov / cancer / dataviz, June 2019). Current treatments for these diseases rely on a combination of surgical and medical management. While evolving, the cornerstone of both first-line medical treatments has been the combination of platinum-based drugs and taxane drugs for over a decade.

[0181] Diarylurea derivatives are of great interest in pharmaceutical chemistry and are low-molecular-weight compounds with a wide range of biological activities such as anti-inflammatory, antithrombotic, and antibacterial effects (see Garuti, L., Roberti, M., Bottegoni, G. & Ferraro, M. Diaryl Urea: A Privileged Structure in Anticancer Agents. Curr. Med. Chem. 23, 1528-1548, 2016). Furthermore, diarylurea derivatives such as sorafenib and regorafenib are clinically used in cancer treatment (see Sadeghian-Rizi, S. et al. Synthesis and characterization of some novel diaryl urea derivatives bearing quinoxalindione moiety. Res. Pharm. Sci. 13, 82-92, 2018). Forchlorfenuron (FCF, N-(2-chloro-4-pyridyl)-N’-phenylurea) is a small synthetic urea derivative currently used in agriculture. FCF exhibits strong cytokinin activity and is used worldwide as a plant fertilizer to increase fruit size.Interestingly, FCF has been shown to inhibit the growth, anchorage-independent growth, migration, and invasion of cancer cell lines (see Blum, W. et al. The phytohormone forchlorfenuron decreases viability and proliferation of malignant mesothelioma cells in vitro and in vivo. Oncotarget 10, 6944-6956, 2019, Zhang, N. et al. The requirement of SEPT2 and SEPT7 for migration and invasion in human breast cancer via MEK / ERK activation. Oncotarget 7, 61587-61600, 2016, and Vardi-Oknin, D., Golan, M. & Mabjeesh, N. J. Forchlorfenuron disrupts SEPT9_i1 filaments and inhibits HIF-1. Plos One 8, e73179, 2013). The effects of FCF have been demonstrated in various types of cancer, such as prostate, mesothelioma, lung, colon, breast, ovary, and cervix. FCF has also been found to be effective in mouse models with suppressed tumor growth. At the molecular level, FCF treatment causes the suppression of HIF-1α and HER2, both of which are known to be associated with more malignant cancer phenotypes (see Marcus, E. A. et al. Septin oligomerization regulates persistent expression of ErbB2 / HER2 in gastric cancer cells. Biochem. J. 473, 1703-1718, 2016).

[0182] In this example, a structure-activity relationship study focusing on optimizing the chemical structure of FCF was conducted with the aim of developing a potent septin inhibitor capable of testing the cytotoxic activity against a panel of gynecological cancer cell lines. Also investigated was whether FCF or the derived inhibitor contributes to the secretion of human epididymis protein 4 (HE4), which is associated not only with the progression of ovarian cancer but also with poor patient outcomes such as advanced disease and decreased survival (see James, N.E., Chichester, C. & Ribeiro, J.R. Beyond the Biomarker: Understanding the Diverse Roles of Human Epididymis Protein 4 in the Pathogenesis of Epithelial Ovarian Cancer. Front. Oncol. 8, 124, 2018). The role of the disclosed inhibitor on the expression of cancer cell growth factor receptors was also investigated.

[0183] Methods Cell lines, culture, and reagents. Cells were maintained in either DMEM (SKOV-3, KLE, OVCAR-3, OVCAR8-C5 and IGROV-1) supplemented with 10% fetal bovine serum (or 20% in the case of OVCAR-3), penicillin (100 units / mL), and streptomycin (100 μg / mL) or RPMI-1640 (ECC-1 and HCH-1) at 37 °C with 5% CO2 in a humidified incubator. Antibodies were purchased from Abcam (septin-2, ab179436) and Cell Signaling Technology, ERK (9102), p-ERK (4370), β-actin (3700), α-tubulin (2144), EGFR (4267) and HER2 (4290). FCF was purchased from Abcam. All other chemicals were from Sigma Aldrich.

[0184] Synthesis of derivatives. The derivatives of FCF were synthesized by coupling variously substituted aryl isocyanates with differently substituted 4-aminopyridine in a molar ratio of (0.1:0.1) in dry DMF at 65 °C overnight. The reaction was monitored using a thin layer chromatography plate with DCM-MeOH or pure ethyl acetate as the eluent. The spots were monitored in a UV chamber. After completion of the reaction, the reaction mixture was poured into a wet ice mixture, triturated, and then the separated solid was filtered under vacuum. The product was washed with hexane followed by diethyl ether and dried under vacuum. The compounds were characterized by spectrophotometry.

[0185] Quantitative real-time PCR. The indicated cell lines were transfected with siRNA targeting septin-2 or non-targeting control siRNA (Santa Cruz Biotechnology: sc-37007 or sc-40936) using Lipofectamine 3000 (Invitrogen). Total RNA was lysed and isolated using Tri reagent and Direct-zol kit (Zymo Research). Reverse transcription was performed using the iScript cDNA synthesis kit (BioRad) according to the manufacturer's recommendations for both. Quantitative real-time PCR was performed using a QuantStudio 12 K Flex real-time PCR system (ABI) and a Taqman gene expression assay (ABI) consisting of the following FAM-labeled probes: SEPT2 (Hs01565417_m1), WFDC2 (Hs00899484_m1), TBP (Hs00427620_m1) or B2M (Hs00187842_m1) (used as a reference for normalization).

[0186] Cell viability, proliferation and apoptosis assays. Cell viability, proliferation, and cell apoptosis were measured using the MTS (Promega), BrdU cell proliferation (Cell Signaling Technology), and Caspase-Glo 3 / 7 apoptosis detection (Promega) assays according to the respective manufacturer's recommendations with appropriate modifications.

[0187] Enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Cells were maintained overnight in the above medium. The medium was removed and replaced with fresh complete medium containing either vehicle (DMSO) or FCF / disclosed inhibitor under the indicated conditions. After treatment, cell lysates and supernatants were collected and subjected to the HE4 assay (Human HE4 Quantikine ELISA Kit, R&D Systems). The amount of HE4 in the supernatant was normalized to the protein concentration of the corresponding cell lysate. The level of HER2 was determined by Human Total ErbB2 / Her2 DuoSet IC ELISA (R&D systems). Western blot analysis was performed using a previously published protocol (see Kim, K.K. et al. Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1alpha in cancer cells. Sci. Rep. 5, 14296, 2015).

[0188] Data acquisition and statistical analysis. Gene expression profiling of septins comparing microdissected normal ovarian stroma (N = 8) with tumor stroma samples from high-grade serous ovarian cancer patients (N = 31) was obtained from the Mixed Ovarian Cancer (CAF)-Wong-77-u133p2 dataset by the ’R2: Genomics Analysis and Visualization Platform (http: / / r2 / am / nl). Mean values of SD were plotted and compared using Student's t-test (two-sided, unpaired t-test for unequal variances, significance for p < 0.01). Prognostic evaluation of septin-2 or septin in endometrial cancer among various cancers was performed using TCGA data obtained through the Human Protein Atlas (http: / / www.proteinatlas.org) (see Uhlen, M. et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248-1250, 2010). Survival curves between high and low populations of septins and their statistical significance were analyzed using GraphPad Prism software (Mantel-Cox test).

[0189] Results Treatment with FCF reduced the viability of endometrial and ovarian cancer cell lines. The effect of FCF on cell viability was first tested in a panel of gynecological cancer cell lines using endometrial (ECC-1 and KLE) and ovarian (HCH-1, OVCAR-3, and SKOV-3) cancer cell lines. FCF treatment reduced the viability of these cells in a time- and dose-dependent manner (Figure 1). FCF showed significant cytotoxic activity against the panel of cell lines, although such inhibition was achieved only at relatively high concentrations (above 100 μM).

[0190] Structural modification of FCF. For the purpose of generating very potent derivatives, systematic structural modifications of the FCF scaffold were carried out (Table 1). The substituents on the phenyl moiety were first varied while keeping 2-chloropyridine constant (except for UR214-5 and UR214-6). The substituents were changed to read the effect of the modification on the anti-proliferative activity of the molecule. As described in Figure 2A, substitutions such as benzyloxy (UR214-2), pyrimidinyl (UR214-4) and pyrrolyl (UR214-3) were not tolerated. However, the CF3S substitution (UR214-1) on the phenyl ring of FCF generated very potent cytotoxic activity. The importance of 2-chloropyridine was demonstrated by replacing 2-chloropyridine with 2-chloropyrimidine while leaving the CF3S-Ph moiety (UR214-6) intact, and this change resulted in a lack of activity. UR214-1 emerged as the most potent molecule against cancer cells, followed by UR214-7 (Figure 2A). UR214-1 was not further modified, but UR214-7 was selected for further optimization to enhance its efficacy.

Table 1

[0191] The effects of the synthesized derivatives on cell viability and proliferation. The structure of UR214-7 was further refined to develop molecules from UR214-8 to UR214-11. The dose-dependent effects of these compounds (UR214-7~UR214-11) on the viability of tumor cells were tested using the MTS assay (Figure 2E). Treatment with compounds UR214-7~UR214-11 exerted a potent anti-viability effect on cancer cells of the ovary (SKOV-3, OVCAR-3, and HCH-1) and endometrium (ECC-1 and KLE). To produce the most active compound (UR214-9) in the series, a chloride was placed at the 6th carbon of pyridine. UR214-9 showed strong activity across most cell lines, except for HCH-1 in which UR214-7 had excellent potency for unclear reasons (Figure 2E). In most cases, the halide substituents of pyridine were well tolerated. Surprisingly, the substitution of 2-chloro to 2-bromo was tolerated, so UR214-11 did not lose its activity and an opportunity for further modification was opened. Next, the BrdU assay was used to measure the anti-proliferative activities of UR214-7 and UR214-9 against ECC-1 cells in the low-dose range. In this assay, FCF was used as a positive control. As shown in Figure 2B, 48-hour treatment with UR214-9 suppressed viability and proliferation at approximately 33 μM and 4 μM, respectively. UR214-7 did not change the viability of tumor cells at less than 11 μM but reduced proliferation (Figure 2B, right). In both assays, FCF lacked activity. Next, the ability of UR214-7 and UR214-9 to induce apoptosis in ECC-1 cells was analyzed. As shown in Figure 2D, treatment with both UR214-7 and UR214-9 caused apoptosis, but the latter was much more effective. The effects of UR214-7 and UR214-9 treatments were time- and dose-dependent (Figure 2C).

[0192] The derivative selectively inhibits HER2 expression. FCF has been shown to downregulate HER2, a growth factor fixed to the cell membrane that is involved in the invasiveness of several types of cancer. Therefore, it was investigated whether the newly generated derivative affects HER2 expression in cancer cell lines. Treatment with UR214-7 and UR214-9 did not affect EGFR expression. However, treatment with UR214-7 and UR214-9 resulted in inhibition of HER2 expression in both ECC-1 and HCH-1 cells, as was the case with FCF at significantly higher doses (Figure 3A). Previous studies suggest that the effect of FCF on HER2 may be achieved by disruption of septins, and that knockdown of septin-2 decreases HER2 expression. Therefore, septin-2 was knocked down and its effect on HER2 levels was determined. Knockdown of septin-2 resulted in downregulation of HER2 (Figure 3B). Inhibition of septin-2 also decreased the viability of cancer cell lines (Figure 3C).

[0193] The derivative reduces HE4 secretion. HE4 is highly upregulated in patients with ovarian and endometrial cancers (see Moore, R.G. et al. The use of multiple novel tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol. Oncol. 108, 402 - 408, 2008, and Moore, R.G. et al. Serum HE4 levels are less frequently elevated than CA125 in women with benign gynecologic disorders. Am. J. Obstet. Gynecol. 206, 351 e351 - 358, 2012), and is a small secreted glycoprotein associated with the malignant phenotype of cancer. Therefore, it was determined whether FCF or its potent analogs could affect HE4 secretion. As shown in Figure 4A, FCF treatment (300 μM for 7 hours) resulted in a decrease in HE4 secretion by OVCAR8 - C5, which stably overexpresses HE4, along with a slight increase in intracellular levels of HE4. This effect was found to be dose - dependent in both ECC - 1 and HCH - 1 cells (Figure 4B). Similar inhibition of HE4 secretion was achieved by UR214 - 1 and UR214 - 7 at much lower concentrations compared to FCF (<10 - 27 - fold, Figure 4C). FCF treatment was found to moderately suppress HE4 mRNA (Figure 4D). Disruption of septin - 2 by small interfering RNA also resulted in a reduction in HE4 expression (Figure 4E).

[0194] Overexpression of septin is associated with increased cancer-related mortality. In this study, knockdown of septin 2 decreased the expression of HER2 and HE4 and reduced the survival rate of cancer cell lines. Furthermore, septin is associated with the malignant cancer phenotype (see Dolat, L. et al. Septins promote stress fiber-mediated maturation of focal adhesions and renal epithelial motility. J. Cell Biol. 207, 225-235, 2014, Mizutani, Y. et al. Possible role of a septin, SEPT1, in spreading in squamous cell carcinoma DJM-1 cells. Biol. Chem. 394, 281-290, 2013, Jiang, H. et al. MicroRNA-127-3p promotes glioblastoma cell migration and invasion by targeting the tumor-suppressor gene SEPT7. Oncol. Rep. 31, 2261-2269, 2014, Froidevaux-Klipfel, L. et al. Septin cooperation with tubulin polyglutamylation contributes to cancer cell adaptation to taxanes. Oncotarget 6, 36063-36080, 2015, and Gonzalez, M.E., Makarova, O., Peterson, E.A., Privette, L.M. & Petty, E.M. Up-regulation of SEPT9_v1 stabilizes c-Jun-N-terminal kinase and contributes to its pro-proliferative activity in mammary epithelial cells. Cell Signal. 21, 477-487, 2009). Therefore, it was investigated whether septin expression is associated with the prognosis of patients with endometrial cancer and ovarian cancer.Kaplan–Meier survival analysis was performed on endometrial cancer stratified by the expression of septin-2, -3, and -7. These septins are of particular interest because they were used in the binding studies of FCF (see Angelis, D., Karasmanis, E.P., Bai, X. & Spiliotis, E.T. In silico docking of forchlorfenuron (FCF) to septins suggests that FCF interferes with GTP binding. Plos One 9, e96390, 2014). Analysis of The Cancer Genome Atlas (TCGA) (Figure 5A) has shown that overexpression of both septin-2 and septin-3 correlates with increased mortality in endometrial cancer. In endometrial cancer, the relative expression of septin-2 was the highest, followed by septin-7. The expression of septin-3 was found to be very low (Figure 5B). The correlation between the expression of septin-2 and increased mortality was also seen in cancers of the kidney, lung, liver, and pancreas, but not in ovarian cancer. Cancer-associated fibroblasts (CAFs) are found in the stroma that surrounds and supports cancer cells. The septin network is known to support CAFs in creating a tumor-promoting microenvironment (see Calvo, F. et al. Cdc42EP3 / BORG2 and Septin Network Enables Mechano-transduction and the Emergence of Cancer-Associated Fibroblasts. Cell Rep. 13, 2699–2714, 2015). Therefore, data profiling septin expression in ovarian cancer-associated stroma and normal ovarian stroma were also analyzed (Wong-77-MAS5.0-u133p2). In cancer-associated stroma, the expression of septin-2 and septin-9 was significantly upregulated, while septin-6 and septin-7 did not change (Figure 5C).

[0195] Discussion In this example, it was found that the cell viability of ovarian and endometrial cancer cell lines was reduced in the range of 100 - 300 μM by treatment with FCF, which is pharmacologically undesirable. Through efforts to optimize the structure of FCF, very potent analogs, UR214-1, UR214-7, and UR214-9, were generated. Treatment with these analogs blocked the growth of multiple endometrial and ovarian cancer cells at significantly lower doses than FCF. UR214-9 was found to inhibit cancer cell growth in the range of 4 - 5 μM. Further structure optimization may lead to nanomolar disruptors suitable for further animal and potentially human testing.

[0196] Septins are a class of cytoskeletal proteins associated with the malignant phenotypes of cancer. Furthermore, septins are increasingly associated with cancer gene expression, such as the EGFR, HER2, HIF-1α / angiogenesis axis (see Diesenberg, K., Beerbaum, M., Fink, U., Schmieder, P. & Krauss, M. SEPT9 negatively regulates ubiquitin-dependent downregulation of EGFR. J. Cell Sci. 128, 397-407, 2015, Angelis, D. & Spiliotis, E.T. Septin Mutations in Human Cancers. Front. Cell Dev. Biol. 4, 122, 2016, and Amir, S., Wang, R., Simons, J.W. & Mabjeesh, N.J. SEPT9_v1 up-regulates hypoxia-inducible factor 1 by preventing its RACK1-mediated degradation. J. Biol. Chem. 284, 11142-11151, 2009). Therefore, it is not surprising that high septin-2 levels are associated with reduced patient survival in several cancer types, including endometrial cancer. Furthermore, ovarian tumor stroma has been found to be highly enriched in septin-2 and -9, and further investigation of the roles of these two structural proteins in ovarian cancer is warranted.

[0197] HE4 is a secreted glycoprotein encoded by WFDC2. It has been found to be upregulated in ovarian and endometrial cancers (see Moore, R.G. et al. Utility of a novel serum tumor biomarker HE4 in patients with endometrioid adenocarcinoma of the uterus. Gynecol. Oncol. 110, 196 - 201, 2008), and has been shown to increase cancer cell proliferation, migration, invasion, metastasis, and chemotherapy resistance. The present results show that treatment with UR214 - 1 and UR214 - 7 results in a reduction of HE4 secretion. Therefore, these FCF analogs may provide a particular therapeutic benefit in gynecological cancers.

[0198] FCF is the only small molecule septin inhibitor known to date. Since septins are not found in terrestrial plants (see Yamazaki, T. et al. Localization and evolution of septins in algae. Plant J. 74, 605 - 614, 2013), it seems reasonable that FCF, which has a major impact on plant growth (see Kopecny, D. et al. Phenyl - and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase / dehydrogenase: a structural study. Biochim. 92, 1052 - 1062, 2010), is involved in cell processes that do not contain septins. Indeed, the non - septin effects of FCF have been described (see Heasley, L. R., Garcia, G. III & McMurray, M. A. Off - target effects of the septin drug forchlorfenuron on nonplant eukaryotes. Eukaryot. Cell 13, 1411 - 1420, 2014). Nevertheless, FCF has also been shown to inhibit septin dynamics and interfere with the assembly of septin - based structures (see Hu, Q., Nelson, W. J. & Spiliotis, E. T. Forchlorfenuron alters mammalian septin assembly, organization, and dynamics. J. Biol. Chem. 283, 29563 - 29571, 2008). In silico studies suggest that FCF may interact with the nucleotide - binding pocket of septins. In this example, treatment with FCF analogs down - regulated both HER2 and HE4, mimicking the effect of septin - 2 knockdown.

[0199] Example 3. Septin disruption controls tumor growth and enhances the efficacy of trastuzumab (Herceptin (trademark)) Septins are a family of GTP-binding cytoskeletal proteins involved in cytokinesis, cell migration, chromosome dynamics, and protein secretion. Septins heterooligomerize to form scaffold filaments, bundles, and rings within cells. Furthermore, septins are important cytoskeletal components that regulate the functions of tubulin and actin. Changes in septin protein expression have been observed in pancreatic, kidney, lung, colorectal, skin, brain, endometrial, ovarian, breast, and other malignancies. Aberrant septin expression has also been associated with neurodegenerative / neuromuscular diseases, blood disorders, infertility, and developmental disorders. It is unclear whether abnormal enrichment of individual septin family members is sufficient to enhance tumorigenesis or whether specific hetero-oligomeric assemblies may be involved. Drugs targeting septins remain poorly understood, mainly because the oligomeric structural makeup of septins poses difficult challenges in the design of therapeutic approaches.

[0200] In this example, the effect of individual septins on the survival of patients with pancreatic cancer, breast cancer, lung cancer, kidney cancer, liver cancer, or melanoma was investigated. To examine the effect of septins on survival, the transcription data and tools published in the Human Protein Atlas (HPA) and R2: Genomics Analysis and Visualization Platform (https: / / hgserver1.amc.nl / cgi-bin / r2 / main.cgi) were used. UR214-9, a potent septin modulator, has been described, which disrupts the structural organization of septin-2 and septin-9, and β-actin, and controls cancer cell proliferation and tumor growth. Molecular docking techniques were used to investigate how UR214-9 and its analogs interact with the elements of the GDP-binding domain and the known FCF-binding pocket. Transcriptome analysis of breast and pancreatic cancer cells treated with UR214-9 was performed to identify how gene expression is affected by UR214-9 and thereby characterize its off-target tendencies. In summary, this example presents UR214-9 as a potent septin filament modulator and shows that the disruption of septin structures in pancreatic, ovarian, and breast cancer cells by UR214-9 can be an effective therapeutic strategy.

[0201] Method Cell lines, cell culture, and reagents. PANC-1, BXPC-3, and CAPAN-1, SKOV-3, MCF7, MDA-MD-231 cells were obtained from ATCC and maintained in DMEM, RPMI-1640, and IMDM supplemented with 10% fetal bovine serum, penicillin (100 units / mL), and streptomycin (100 μg / mL) in a humidified incubator containing 5% CO2 at 37 °C. JIMT-1 cells were purchased from AddexBio Inc, USA (Catalog number: C0006005) and maintained in DMEM supplemented with 10% FBS and antibiotics. Septin-2 (Catalog number: HPA018481), Septin-7 (Catalog number: HPA029524), Septin-9 (Catalog number: HPA029524) antibodies were purchased from Sigma Aldrich Inc. DyLight 488 (Catalog number: DI-1488, rabbit), Dylight594 (Catalog number: DI-2594, mouse) were purchased from Vector Laboratories Inc. Phylozin-TRITC was purchased from ECM Biosciences (Catalog number: PF7551). HER2 (Cell Signaling Technology, Catalog number: 4290), pSTAT-3 (Catalog number: 9145p), STAT-3 (Catalog number: 4904), and GAPDH antibodies (Catalog number: 2118s) were purchased from Cell Signaling Technology Inc., USA and used at the manufacturer-recommended dilutions.

[0202] Synthesis of derivatives. UR214-9 was synthesized by coupling aryl isocyanate and 2,6-dichloro-4-aminopyridine in a molar ratio of (0.1:0.1) in dry DMF at 65 °C overnight under an argon-flash atmosphere. The reaction was monitored using a thin-layer chromatography plate with DCM-MeOH or pure ethyl acetate as the eluent. The spots were monitored in a UV chamber. After completion of the reaction, the reaction mixture was poured into a wet ice mixture, triturated, and the separated solid was filtered under vacuum. The product was washed with hexane and subsequently with diethyl ether and dried under vacuum. The compound was characterized by mass spectrometry.

[0203] Molecular docking. Docking experiments to investigate the potential binding modes of 9 and related compounds were carried out using Molsoft's ICM software package (v. 3.8-7). The molecules are quite small and somewhat symmetric, consisting of a central urea group flanked by two lipophilic substituted aromatic rings. Since compounds 8, 9, and 10 are the most active ones, it was assumed that they might share a similar binding mode. Therefore, compounds FCF, UR214-8, -9, and -10 were docked to the nucleotide binding site of PDB ID 2QNR, which is the highest quality structure of the septin-2 dimer complex available to date (see Angelis, D., et al., In silico docking of forchlorfenuron (FCF) to septins suggests that FCF interferes with GTP binding. PLoS One. 9, e96390, 2014). Preparation of the receptor (based on the GDP binding site of chain A) and construction of the ligand were carried out within ICM using standard settings. The ICM scores of each compound and its pose were calculated and compared to FCF. Compounds were docked with the "Dock Table" function with an effort of 2.0 and a setting of 20 poses per compound. Visual inspection of the docking poses revealed two sets of low-energy poses ("Set A" and "Set B") that stand out, and the highly active compounds can adopt similar conformations.

[0204] Cell viability and cell cycle analysis. The cell viability of PANC-1, BXPC-3, and CAPAN-1 pancreatic cancer cells treated with UR214-9 was measured using the Cell Titre96R Aqueous One Solution Cell Proliferation Assay (Promega Corp., catalog number: G3580) according to the previously published procedure. The Live / Dead dye kit (Invitrogen Coro., catalog number: L34975) was used to estimate the number of live and dead cells in PANC-1 and BXPC-3 pancreatic cancer cells treated with UR214-9 or vehicle. Briefly, cells were treated with vehicle or UR214-9 (3 μM) for 72 h. Cells were harvested by trypsinization and fixed and permeabilized using a fixation-permeabilization reagent (prepared by diluting the concentrate with the diluent at a ratio of 1:3) (Biogem Inc., diluent: catalog number 92160-00-160 and concentrate catalog number: 2550-00-50), and stained with the Live / Dead dye for 1 h. Cells were centrifuged at 1000 rpm for 5 min, the pellet was washed, and spun down 3 times with DPBS. Cells were analyzed on a 305 flow cytometer, and the relative populations of live and dead cells were calculated by inputting the same number of cells into both the vehicle and control groups.

[0205] For cell cycle analysis, BXPC-3 and PANC-1 and JIMT-1 cells (100,000 / well) were seeded in 6-well dishes overnight and allowed to adhere overnight. The medium was replaced with fresh complete medium supplemented with DMSO or UR214-9 (100 nM and 3 μM), and the cells were incubated for 72 hours. The medium containing the drug was removed, the cells were washed twice with PBS, and gently trypsinized. The cells were collected in 15 mL tubes, complete DMEM medium was added to block trypsin, and the cells were centrifuged. The supernatant was removed, and the cells were gently treated with 70% cold EtOH for 30 minutes. The fixed cells were centrifuged, and the resulting pellet was collected in a flow cytometry tube and stained with a pre-formulated PI / RNase solution (Cell Signaling Technology, catalog number: 4087s) for 30 minutes. The PI content was analyzed using a flow cytometer. The data was processed using Flowjo software.

[0206] Expression of cell cycle proteins. Changes in proteins after drug treatment were investigated using the Cell Cycle Antibody Array (FullMoon BioSystems Inc, catalog number: ACC:058), a high-throughput ELISA-based antibody array designed for qualitative / semi-quantitative protein expression profiling. PANC-1 cells were lysed in a buffer containing protease and phosphatase inhibitors (Cell Signaling, catalog number: 9803S). The total protein content was quantified by Bradford assay, and equal amounts of protein were analyzed twice using an array containing 4 - 6 spots for each of 60 probes according to the manufacturer's instructions (ACC058, Cell Cycle Antibody Array, Full Moon Biosystems, Sunnyvale, CA). After background correction, the average signal intensity was measured using the imaging service of FullMoon Inc. Protein expression in both the naive and both treatment groups of 328 was normalized to the GAPDH signal.

[0207] Confocal analysis of septin disarrangement. To determine the effect of UR214-9 treatment on septin-2 structure in cells, PANC-1 or JIMT-1 cells were seeded onto glass slides and allowed to adhere overnight. The medium was replaced with complete DMEM medium supplemented with DMSO or UR214-9 (1 μM and 70 nM), and the cells were incubated for 48 h. The medium was replaced again with fresh complete medium, and the cells were fixed with neutral buffered formalin at 40°C for 15 min. The medium was removed, and the cells were washed repeatedly with PBST (5 x 5 mL). The cells were stained overnight at 40°C with septin-2 antibody (Sigma Aldrich, catalog number: HPA018481) in PSB. The medium was removed again, and the cells were washed with 2 x 5 mL PBST. The cells were stained with fluorescent-conjugated secondary antibody for 1 h in the dark. The slides were washed repeatedly with 7 x 5 mL PBST in the dark, mounted with mounting medium containing DAPI (Vector labs), and covered with a glass slide. The slides were stored at 40°C in the dark until analysis. Confocal images were acquired and processed basically as previously published (see Moore, R.G., Hill, E.K., Horan, T., Yano, N., Kim, K., MacLaughlan, S., Lambert-Messerlian, G., Tseng, Y.D., Padbury, J.F., Miller, M.C., Lange, T.S., Singh, R.K. Sci Rep. 4, 3574, 2014). Pancreatic tumor microarrays (US Biomax, catalog number T142a) were deparaffinized, processed, stained overnight with septin-2 antibody (Sigma Aldrich, catalog number: HPA018481), washed with PBST, and incubated for 1 h with secondary (FITC) matching the source. The slides were washed with PBST (5 x 10 mL) for 5 min each. DAPI containing mounting medium was applied and covered with a glass slide. Confocal images were acquired with a Nikon C1si confocal microscope (Nikon Inc. Mellville, NY.) using diode lasers 402, 488, and 561. Serial optical sections were obtained with EZ-C1 computer software (Nikon Inc. Mellville, NY).Z-series sections were collected at 0.3 μm with a 40x PlanApo lens and ScanZoom 2, or at 0.25 μm with a 60x Plan Apo objective and ScanZoom 2. Deconvolution measurements were performed using Elements (Nikon Inc. Mellville, NY) computer software. Outlining and analysis of five cells per field were performed.

[0208] Xenograft studies to evaluate the antitumor response of UR214-9. 1 million PANC-1 (HER2+, n = 12), JIMT1 (number of animals = 10), and SKOV-3 (number of animals = 10) cells in Matrigel: medium (1:1) were transplanted into the left flank of 356 NSG mice, respectively. The mice were randomized, identified by ear punch, and subdivided into vehicle and treatment groups when tumors were found to be palpable. Both JIMT1 and SKOV-3 formed invasive tumors within 1 week and were treated with vehicle or UR214-9 (25 mg / kg, IP, 7 days a week). PANC-1 formed slow-growing tumors, and treatment was initiated when the tumor length exceeded 5 mm. The group of SKOV-3 cells was also treated with trastuzumab or trastuzumab + UR214-9. The vehicle formulation was: 40% hydroxypropyl-beta-cyclodextrin [Acros Organics] and Soltrol HS15 (Sigma) in sterile water. 25 mg / kg equivalent of UR214-9 (1 μL in DMSO = 200 μg) was dissolved in 600 μL of PBS + 400 μL of vehicle and vortexed to obtain a clear suspension. Tumor volume and animal body weight were measured manually with a digital caliper on a weekly or bi-weekly routine. Tumor volume was calculated using the formula 1 / 2 (LxW^2) (where L is the longest diameter and W is the widest width). Statistical differences between the vehicle and treatment groups were analyzed by GraphPrism-8 software using one-way ANOVA. P < 0.05 was considered significant. After the treatment period, the mice were euthanized, the tumors were excised, weighed, and frozen in liquid nitrogen. A portion of the tumors from the control and treatment groups was fixed in neutral buffered formaldehyde and paraffin embedded. Tissue slides with a thickness of 5 μM were prepared for histochemistry.

[0209] mRNA sequencing. Total RNA concentration was measured using a NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE), and RNA quality was evaluated using an Agilent Bioanalyzer (Agilent, Santa Clara, CA) (see Bolger, A.M., Lohse, M., Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30, 2114 - 2120, 2014). The TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used to construct next-generation sequencing libraries according to the manufacturer's protocol. Briefly, mRNA was purified from 200 ng of total RNA using oligo dT magnetic beads and fragmented. First-strand cDNA synthesis was performed by random hexamer priming, followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. Next, end repair and 3' adenylation were performed on the double-stranded cDNA. Illumina adapters were ligated to both ends of the cDNA, purified by gel electrophoresis, and amplified with PCR primers specific to the adapter sequences to generate cDNA amplicons with a size of approximately 200 - 500 bp. The amplified library was hybridized to an Illumina single-end flow cell and amplified using cBot (Illumina, San Diego, CA). Using an Illumina NextSeq550, 75-nt single-end reads were generated for each sample (see Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., Gingeras, T.R. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 29, 15 - 21, 2012).

[0210] Analysis of full transcriptome data. Raw reads generated from the NovaSeq6000 sequencer were demultiplexed using bcl2fastq version 2.19.0. Quality filtering and adapter removal were performed using Trimmomatic-0.36 with the following parameters: "TRAILING:13 LEADING:13 ILLUMINACLIP:adapters.fasta:2:30:10 SLIDINGWINDOW:4:20 MINLEN:35". Next, the processed / cleaned reads were mapped to the Homo sapiens reference sequence (GRCh38, hg38) using STAR-2.6.0c with the following parameters specified: "--twopassMode Basic --runMode alignReads --genomeDir ${GENOME} --readFilesIn ${SAMPLE} --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif - outFilterIntronMotifs RemoveNoncanonical". Gene counts were derived using the subread-1.6.1 package (featureCounts) with the following parameters specified: "-s 2 -t exon -g gene_name". Differential expression analysis and data normalization were performed using DESeq2-1.16.1 with an adjusted p-value threshold of 0.05 within the R-3.4.1 environment.The heatmap was created using the pheatmap R package (see R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. (2016). URL https: / / www.R-project.org / , Love, M. I., Huber, W., Anders, S. “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology 15, 550 (2014), and 39. Liao, Y., Smyth, G. K., Shi, W. featureCounts: an efficient general-purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923 - 930, 2014).

[0211] Data acquisition and statistical analysis. Prognostic evaluation of septin-2, -7, and -9 in panels of various cancers was performed using the Human Protein Atlas tool. Alternatively, the R2 Genome.org tool was used to determine the impact of septin enrichment on survival outlook. A p-value of less than 0.05 was considered significant. The relative tumor sizes of the naive and treated groups were calculated using GraphPrism 8 with a one-way analysis of variance (anova) setting. A p-value of less than 0.05 was considered significant.

[0212] Results Septin enrichment correlates with a decrease in the survival rate of cancer patients. Publicly accessible microarray databases of pancreatic and ovarian cancer patients deposited in the R2: Genomics Analysis and Visualization Platform (https: / / hgserver1.amc.nl / cgi-bin / r2 / main.cgi) were analyzed. Septin-2 mRNA was enriched in malignant pancreas compared to normal pancreas (Figure 6A, p = 1.3e-4). Similarly, ovarian cancer epithelium showed significantly enriched expression of Septin-2 compared to normal stroma (Figure 6B, left, p = 1.2e-7). Microdissected stroma of malignant ovarian stroma also showed an increase in the expression of Septin-2 mRNA compared to normal stroma (Figure 6B, right, p = 1.21e-4). Similarly, the tumor epithelial component of malignant breast showed an increase in Septin-2 mRNA enrichment compared to normal stroma (Figure 6C, left, p = 0.49e-3). Furthermore, an increase in the invasive area of breast tumors led to an increase in Septin-2 enrichment (Figure 6C, right, p = 8.9e-3). The Kaplan-Meier survival rate of pancreatic cancer patients grouped by the degree of Septin-2 expression (from microarray data available in https: / / hgserver1.amc.nl / cgi-bin / r2 / main.cgi20 and Human Protein Atlas21) indicates that Septin-2 mRNA enrichment significantly (p = 0.0011) correlates with an increase in mortality (Figure 6D, left). Similarly, the enrichment of Septin-7 and -9 correlates with an increase in the mortality of pancreatic cancer patients. Septin-2 enrichment is also an adverse factor for patients with breast cancer (Figure 6D, middle, p = 3.9e-3) and ovarian cancer (Figure 6D, right, p = 0.011). Analysis of the survival outlook based on other septins indicates that the enrichment of Septin-7 is adverse for patients diagnosed with malignant breast tumors (p = 0.0079).

[0213] UR214-9 causes the destruction of septin-2 intracellularly. The chemical structure of UR214-9 is shown in Figure 7A. UR214-9 was obtained by optimization based on the structure-activity relationship of FCF. By incorporating a fluorine atom group into the phenyl ring and installing a chlorine atom at C-6 of the pyridine ring, UR214-9 became a potent disruptor of the septin filament structure. Using a high-resolution (60x2) confocal microscope, the effects of DMSO, FCF (positive control), and UR214-9 on the structural arrangement of septin-2, 6, 7, and -9 were measured in a panel of BXPC-3, CAPAN-1, Panc-1 (pancreas) and JIMT-1 (breast) and SKOV-3 ovarian cancer cells. FCF appears to strengthen the septin-2 filaments in BXPC-3 cells (Figure 7B, lower left), but the septin-2 needles in PANC-1 were disrupted and translocated at the cell surface after UR214-9 treatment (1 μM) (Figure 7C, lower left). Similarly, the septin-2 needles in JIMT-1 cells after drug treatment showed structural disruption and rearrangement around the nucleus (Figure 7C, lower right). Next, using a confocal microscope, the responses of other septin family members in PANC-1 cells when treated with UR214-9 were investigated. Septin-7 showed a reduction in expression, while septin-9 showed disruption of the filament structure. Septin-4 and -6 did not show a clear filament structure but instead showed punctate staining, which was reduced in the treatment group compared to the DMSO-treated control or the drug effect was not conclusive (data not shown). Similarly, JIMT-1 breast cancer cells treated with UR214-9 showed strong structural disruption and rearrangement of septin-2 around the nucleus. Since JIMT-1 cells did not show a clear septin-7 structure, the effect of UR214-9 on septin-7 remained ambiguous. However, confocal microscopy of PANC-1 and JIMT-1 cells treated with UR214-9 showed clear disruption of the septin-9 filament structure. Whether UR214-9 treatment changes the expression of septin family proteins was investigated by immunoblotting the whole cell lysates of PANC-1, MDA-MB-231, JIMT-1, and MCF-7 cancer cells.Immunoblots were probed with validated septin-2, 6, 7, and -9 antibodies. In PANC-1 cells, the expression of septin-9 was interestingly completely inhibited, while the expressions of septin-2, -6, and -7 were unaffected (Figure 7D). Similarly, Western blot analysis of MDA-MB-231, JIMT-1, and MCF-7 cells showed that UR214-9 did not change the protein expression levels of septin-2, 6, and -9 family proteins, despite their filament structures being overwhelmingly disrupted (Figure 7E). The septin destruction phenomenon in cancer cells was further verified using SKOV-3 ovarian cancer cells, which showed complete disruption of septin-2 filaments, where septin-2 appeared to be re-localized to the cell surface after drug exposure when treated with UR214-9 (1 μM, 48 hours) (Figure 7F). Further examination of 125 septin-6, 7, and -9 structures in drug-treated SKOV-3 cells showed reorganization of septin-9 (Figure 7G, bottom). Septin-6 was non-filamentous and was found to decrease upon treatment with UR214-9 (Figure 7G, top). The change in septin-7 expression was not clear due to the non-filamentous and diffuse / punctate expression of UR214-9 (Figure 7G, middle).

[0214] UR214-9 causes disruption of actin filaments in pancreatic and breast cancer cells. Septins have previously been shown to control the function of actin (see Schmidt, K., Nichols, BJ. Functional interdependence between septin and actin cytoskeleton. BMC Cell Biol. 12, 43, 2004). Confocal microscopy of PANC-1 and JIMT1 cells treated with UR214-9 showed disruption of actin filaments when treated with a dose of 1 μM for 48 hours (Figure 8). Representative structural disorganization of actin filaments needles in both PANC-1 and JIMT1 cells (Figure 8). Regions of interest are shown within the white boxes indicated.

[0215] In silico docking shows the major interactions of UR214-9 with septin-2. Docking experiments were performed to investigate the potential binding modes of UR214-9 and related compounds (including FCF). UR214-9 and its analogs are small in size, similar in structure and symmetry, and they are composed of a central urea group flanked by two lipophilic substituted aromatic rings. Compounds UR214-8, 9, and 10 are the most active compounds described herein, and taking this into account, it was hypothesized that they could share a similar binding mode. Thus, compounds FCF, UR214-8, UR214-9, and UR214-10 were docked into the nucleotide binding site of PDB ID 2QNR, the highest quality structure of the available septin-2 dimer complex. Visual inspection of the docking poses identified two sets of low energy poses ( "Set A, up and down" and "Set B, up and down") in which all highly active compounds could adopt a similar conformation.

[0216] The two sets are similar to each other in that the three major parts of the molecule (the central urea part, the pyridine, and the phenyl ring) are in approximately the same region and the pyridine ring is present instead of guanine with GDP (Figures 9A-9D). In Set A, the pyridine nitrogen atom is seen instead of the guanine carbonyl oxygen atom and forms a hydrogen bond with the backbone of G241 of chain A at 152. The ICM scores for Set A were found to be -8.85 for compound UR214-8, -8.59 for compound UR214-9, -10.4 for compound UR214-10, and -7.21 for FCF, indicating that the binding energy of the synthesized analogs is stronger than that of the parent FCF. Set B appears similar to the previously reported docking pose of FCF with the same structural template obtained with Autodock software. In Figures 9C and 9D, the identity of the amino acid residues that interact with the atoms of UR214-9 or its analogs is shown.

[0217] UR214-9 reduces the viability of cancer cells and arrests the progression of the cell cycle. Treatment with UR214-9 reduced the viability of human pancreatic cancer cells (BXPC-3 and PANC-1) during 72 hours of treatment (Figure 9A). PANC-1 and BXPC-3 cells treated with UR214-9 showed a large population of non-viable cells based on staining with a Live-Dead cell kit and flow cytometry 72 hours after treatment. (Figures 9B and -9C). Considering the role of septins in the cell cycle process, the effect of UR214-9 on the cell cycle progression of PANC-1 and BXPC-3 pancreatic cancer cells at a non-cytotoxic concentration of 100 nM was analyzed. Treatment with a 100 nM dose of UR214-9 caused a mild S-phase arrest in PANC-1, but BXPC-3 cells showed no change in cell cycle distribution at the non-toxic dose. Increasing the dose to 3 μM concentration of UR214-9 caused an overwhelming arrest in the G1 phase of BXPC-3 cells (about 95% compared to 21%), while PANC-1 cells showed a complete arrest in the sub-G1 / G0 phase. Similarly, JIMT-1 cells treated with an increased dose of UR214-9 (3 μM) showed an arrest in the G1 phase and a significant increase in the accumulation in the G0 phase.

[0218] Analysis of cell cycle protein expression. Using a spotted antibody array, multiple cell cycle-related proteins expressed in drug-treated or naive PANC-1 cells were studied simultaneously. By measuring the relative photon count, Cullin-3, glycogen synthase kinase-3 (GSK-3b), p19ARF, 14.3.3.Pan, APC11, APC2, ATM, C-able, CD14A phosphatase, CDC25C, CDC34, CDC37, CDC47, CDC7, CDH1, CDK1 and CDK-3 were shown to be the most expressed and affected proteins with a fold change >2.0 considered significant in treated versus naive PANC-1. β-actin showed the most prominent expression but the expression level did not change after treatment. On the other hand, Cullin-3 showed the most prominent upregulation in treated versus naive PANC-1 cells. Cullin-3, a member of the Cullin-based ubiquitin ligase family, interacts with proteins containing the Hrt1 and BTB domains. The resulting complex functions as a Cullin3-based E3 ligase, ubiquitinating and degrading specific substrates (see Pintard, L., Willems, A., Peter, M. Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family. EMBO J. 23, 1681-7, 2004), which indicates the role of septins in the suppression of ubiquitination and subsequent degradation via Cullin-3.

[0219] The UR214-9 treatment delays the growth of HER2+ xenograft tumors. Septin-2 regulates HER2 expression in gastric cancer cells (see Kumar, V., Abbas, A., Aster, J. Robbins basic pathology. Philadelphia: Elsevier / Saunders. 2013; p. 697. ISBN 9781437717815). HER2 is overexpressed in various malignancies (see Buza, N., Roque, D.M., Santin, A.D. “HER2 / neu in Endometrial Cancer: A Promising Therapeutic Target With Diagnostic Challenges”. Archives of Pathology & Laboratory Medicine. 138, 343-50, 2014, and Ruschoff, J., Hanna, W., Bilous, M., Hofmann, M., Osamura, R.Y., Penault-Llorca, F., van de Vijver, M,, Viale, G. “HER2 testing in gastric cancer: a practical approach”. Modern Pathology. 25: 637-50, 2012) and is known to promote tumorigenesis, progression, metastasis, and chemotherapy resistance (see Ruiz-Saenz, A., Dreyer, C., Campbell, M.R., Steri, V., Gulizia, N., Moasser, M.M. HER2 Amplification in Tumors Activates PI3K / Akt Signaling Independent of HER3. Cancer Res. 78, 3645-3658, 2018).Septins have been shown to protect and stabilize the HER2 receptor at the plasma membrane of tumor cells, thereby perpetuating the organized tumorigenesis of HER2 (see Marcus, E.A., Tokhtaeva, E., Turdikulova, S., Capri, J., Whitelegge, J.P., Scott, D.R., Sachs, G., Berditchevski, F., Vagin, O. Septin oligomerization regulates persistent expression of ErbB2 / HER2 in gastric cancer cells. Biochem J. 473, 1703 - 18, 2016). It was hypothesized that targeting septin - 2 might potentially emerge as a new approach to control the organized tumorigenesis of HER2. The MTS assay showed that treatment with UR214 - 9 reduced the growth of BXPC - 3 and PANC - 1 pancreatic cancer cells in a dose - dependent manner up to 48 hours of drug exposure (Figure 10A). Treatment with UR214 - 9 [3 μM] analyzed the entire population of BXPC - 3 cells with a Live - dead kit (Invitrogen Inc), and 29.4% dead cells were generated during 48 - hour drug exposure. Similarly, PANC - 1 cells showed a population of more than 38% dead cells when treated with UR214 - 9 [3 μM]. Next, the effect of UR214 - 9 treatment on pancreatic cancer xenograft tumor growth in vivo was determined. Mice xenografted with HER2+ PANC - 1 cells showed significantly delayed growth (p < 0.0001) (Figure 10D). The antitumor effect of UR214 - 9 was further evaluated against HER2 - positive xenografts derived from JIMT1 (breast cancer) cells. In addition to the increased cell death of JIMT - 1 cells by UR214 - 9 exposure in vitro (Figure 10E), JIMT1 xenograft tumors treated with UR214 - 9 resulted in significant growth control based on both measurements of tumor volume and weight (Figure 10G) (Figure 10F).

[0220] UR214-9 inhibits HER2 expression and blocks STAT-3 phosphorylation. Immunoblot analysis of whole cell lysates of pancreatic cancer cell line PANC-1 (HER2+) showed a dose-dependent decrease in HER2 expression in PANC-1 cells treated with UR214-9 for 72 hours (Figure 11A, top). Phosphorylation of STAT3 is a downstream readout of HER2 activation33, and thus, UR214-9 treatment also reduced phosphorylated STAT-3 in PANC-1 cells (Figure 11A, bottom). Similarly, UR214-9 treatment reduced HER2 expression in a panel of MDA-MB-231, JIMT-1, and MCF-7 breast cancer cells (Figure 11B) and reduced phosphorylation of STAT-3 in each cell line (Figure 11C). We recently showed that septin-2 is highly overexpressed in ovarian cancer (see James, N.E., Cantillo, E., Yano, N., Chichester, C.O., DiSilvestro, .PA., Hovanesian, V., Rao, R.S.P., Kim, K.K., Moore, R.G., Ahsan, N., Ribeiro, J.R. Septin-2 is overexpressed in epithelial ovarian cancer and mediates proliferation via regulation of cellular metabolic proteins. Oncotarget. 10, 2959-2972, 2019).Similar to the JIMT-1 and PANC-1 cell lines, the SKOV-3, platinum-resistant ovarian cancer cell line is characterized by HER2 amplification (see DeFazio-Eli, L., Strommen, K., Dao-Pick, T., Parry, G., Goodman, L., Winslow, J. Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action. Breast Cancer Res. 13(2), R44, 2011, and English, D.P., Roque, D.M., Santin, A.D. HER2 expression beyond breast cancer: therapeutic implications for gynecologic malignancies. Mol. Diagn. Ther. 17, 85-99, 2013). Therefore, xenografts derived from the SKOV-3 cell line were used to verify the antitumor effect of UR214-9 against HER2-amplified xenograft tumors. To further confirm the results of the combination of UR214-9 and trastuzumab, mice were further treated with trastuzumab alone or in combination with UR214-9. As shown in Figure 11D, both UR214-9 and trastuzumab controlled tumor growth. This combination clearly controlled tumor growth significantly more than either drug alone. The true advantage of the combination of UR214-9 and trastuzumab became apparent when treatment was stopped and the tumors were allowed to grow. As shown in Figure 11D, the tumor sizes in the UR214-9 and trastuzumab groups reached the mean size of the control when treatment was stopped, but the combination maintained greater control over tumor growth (combination p<0.0001**** vs. vehicle p=0.0004*** and 0.0001*** for UR214-9 and trastuzumab).When the weight of the excised tumors was measured, the combination group showed the presence of smaller tumors, while both the UR214-9 group and the trastuzumab group generated tumors that were consistent with the mean size seen in the vehicle group (Figure 11E).

[0221] Global transcriptome analysis revealed that UR-214-9 was selective. RNA-Seq was performed in the JIMT-1 and Panc-1 cell lines in three treatment groups (10 nM afatinib, 1 μM UR214-9, and DMSO) consisting of four replicates each. Samples were sequenced with an average depth of 58 million reads, and more than 90% of the read data for each sample was uniquely aligned to the human reference genome (hg38) after adapter and quality trimming. Differentially expressed genes were determined by comparing drug treatment to the control group (adjusted p-value < 0.05). There were 1236 (713 up and 523 down) dysregulated genes between afatinib treatment and control (Figures 12A and 12C). Using the ENRICHR web tool, it was confirmed that the upregulated genes (ALPP, TRIM29, CYP1A1) were associated with extracellular matrix organization and cadherin binding, and the downregulated genes (EGR1, DUSP6, HMGA2, etc.) were associated with purine metabolism and ribosome biogenesis. In contrast, only 11 (7 up and 4 down) genes were called dysregulated between UR214-9 treatment and control (Figures 12B and 12D). For the Panc-1 cell line, only two genes (COL13A1 and PRSS22) were determined to be significantly differentially expressed with afatinib treatment compared to the control group, and no genes were called differentially expressed between UR214-9 and control.

[0222] Discussion The associations between septins and malignant tumors are being continuously revealed, and it has become important to identify septin-targeted therapeutic agents that inhibit the abnormal functions of septins in cancer cells. Starting from FCF, which essentially enhances septin-240, UR214-9, a small molecule that degrades septin-2 and -9 filament assembly in cancer cells without killing the cells or changing the intracellular septin protein levels, was developed. Disrupting the oligomeric septin filament structure via UR214-9 treatment may affect cytokinesis and be key to controlling cancer cell proliferation. Destruction of septin filaments by UR214-9 not only reduced the proliferation of pancreatic cancer cells (and breast, ovarian endometrial, lung, and kidney cancers) in vitro, but also showed reduced tumor growth in xenograft tumors of breast, ovarian, pancreatic, and lung malignancies treated with UR214-9. Interestingly, combined use with trastuzumab enabled more potent control of the growth of HER2-positive SKOV-3 xenografts (Figure 11D).

[0223] Enhancement of the antitumor effect of trastuzumab by combination treatment with UR214-9 in a HER2-positive ovarian cancer xenograft model is probably due to the association between septin-2 and HER2. Septin-2 has been shown to maintain HER2 signaling in cancer cells. Septins protect and stabilize the HER2 receptor in the plasma membrane of tumor cells, perpetuating oncogenic signaling and tumorigenesis organized by HER2. Targeting septins is expected to improve the survival rate of HER2-positive breast, pancreatic, and other malignancies such as ovarian and lung cancers. Overexpression of HER2 leads to invasive breast malignancies and a decrease in patient survival rate (see Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A., McGuire, W.L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2 / neu oncogene. Science 235:177-182, 1987). The current repertoire of treatment methods for HER2+ malignancies is insufficient. More than 60% of HER2+ breast cancer patients do not respond to trastuzumab treatment, and resistance to treatment develops rapidly in virtually all patients (see Pohlmann, P.R., Mayer, I.A., Mernaugh, R. Resistance to Trastuzumab in Breast Cancer. Clin Cancer Res. 15:7479-7491, 2009).Furthermore, since trastuzumab cannot block the secreted (truncated) HER2 that penetrates solid breast tumors and promotes resistance and metastasis, its usefulness in completely and continuously controlling the growth of HER2-organized breast tumors is limited (see Hayes, D.F., Yamauchi, H., Broadwater, G., Cirrincione, C.T., Rodrigue, S.P., Berry, D.A., Younger, J., Panasci, L.L., Millard, F., Duggan, D.B., Norton, L., Henderson, I.C.; Cancer and Leukemia Group B. Circulating HER-2 / erbB-2 / c-neu (HER-2) extracellular domain as a prognostic factor in patients with metastatic breast cancer: Cancer and Leukemia Group B Study 8662. Clin. Cancer Res. 7, 2703-11, 2001). Similarly, the treatment or prevention of brain metastases in HER2+ breast cancer patients is difficult, especially at stages following trastuzumab treatment (Belkacemi, Y., Hanna, N.E., Besnard, C., Majdoul, S., Gligorov, J. Local and Regional Breast Cancer Recurrences: Salvage Therapy Options in the New Era of Molecular Subtypes. Front Oncol. 8, 112, 2018).Approximately two-thirds of patients develop brain metastases despite extracranial disease control or response to trastuzumab (see Clayton, A.J., Danson, S., Jolly, S., Ryder, W.D., Burt, P.A., Stewart, A.L., Wilkinson, P.M., Welch, R.S., Magee, B., Wilson, G., Howell, A., Wardley, A.M. Incidence of cerebral metastases in patients treated with trastuzumab for metastatic breast cancer. Br J Cancer 91, 639-643, 2004). Since trastuzumab does not penetrate the central nervous system, the brain may function as a sanctuary site (see Morris PG, McArthur HL, Hudis CA. Therapeutic options for metastatic breast cancer. Expert Opin Pharmacother. 10, 967-981, 2009). Blood-brain barrier (BBB) penetrants are necessary to more appropriately control brain metastases in patients with HER2+ cancer. UR214-9 has structural attributes of a small polar surface area that facilitate passage through the BBB (calculation for Ur214-9 = 53.49 vs <90 required for BBB passage).

[0224] Therefore, UR214-9 may improve the outcome of patients with brain metastases from HER2+ cancer.

[0225] The relevance of septin signaling is not fully understood. To determine the relevance of septin and the perturbation-induced signaling by UR214-9 treatment, global rna-seq analysis of breast and pancreatic cancer cells treated with UR214-9 and, as a comparison, afatinib, a HER2-targeted therapy, was performed. As shown in FIGS. 12A-12D, afatinib treatment clearly had the greatest impact on the transcriptional profile of PANC-1 cells, while treatment with DMSO and UR214-9 had little effect on the transcriptome. The absence of differentially expressed genes between UR214-9 treatment and the control suggests that the mechanism of action of UR214-9 is non-transcriptional and that treatment with UR214-9 does not induce an overall transcriptional response.

[0226] In summary, this example shows that abnormal expression of septins indicates poor prognosis in cancer patients. UR214-9 is the first prototype of a small molecule that can induce the destruction of septin-2 and -9 filaments, which are pharmacological and cytoskeletal responses of cells not previously described, and can control cancer cell proliferation and tumor growth. Furthermore, an important pharmacological feature of UR214-9 is the advantage of limited off-target involvement. As shown in Figures 12A - 12D, compared to afatinib, an EGFR-targeted therapy that affects the gene expression of over 1200 genes in JIMT-1 breast cancer cells, treatment with UR214-9 at a 100-fold higher dose significantly affected less than 20 genes. UR214-9 is a close structural analog of FCF, but UR214-9 is pharmacologically very different from FCF. FCF has been shown to enhance septin 2, while UR214-9 disassembles the filamentous aggregates of septin-2 and septin-9. The ICM score calculated by molecular docking showed that the binding affinity of UR214-9 to the septin-2:septin-2 dimer complex is higher than that of FCF. Except for FCF, which is clinically inappropriate due to its weak pharmacological effects, off-target effects, and functions related to the enhancement of septin-2 filaments, UR214-9 may be the only septin modulator described so far that can disassemble the septin structural array at nanomolar concentrations (70 nM - 1 μM). Considering the preliminary anti-tumor responses in xenograft models of breast, pancreatic, ovarian, and lung cancers (data not shown) and its ability to significantly enhance the response to trastuzumab in HER2-expressing xenograft tumors, it is clear that disassembling septins is an effective and clinically promising approach to prevent tumor growth, but it is necessary to optimize the dosage, delivery formulation, and dosing frequency, and to identify synergistic or at least additive combination agents to achieve more complete control beyond tumor growth. Based on the promising results of combination with trastuzumab, the results of the combination of UR214-9 with paclitaxel and trastuzumab in a breast cancer model are currently being evaluated to enhance the clinical utility of UR214-9.

[0227] The present invention has been described in conjunction with its detailed description, but the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0228] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. The Genbank and NCBI submissions indicated by the accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference.

[0229] The present invention has been shown and described with particular reference to its preferred embodiments, but it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as encompassed by the appended claims. The invention described in the original claims of this application is appended below. [1] Structure of formula (I):

Chemical formula

Chem.

Chem.

Chem.

Chem.

[10] R 1 and each of R 2 is independently F, Cl, Br, I, or C 1 -C 6 alkyl, wherein said C 1 -C 6 The compound according to any one of [1] and [3] to [9], wherein the alkyl optionally contains one, two, three or more substitutions by F, Cl, Br, I, or phenyl.

[11] R 12 The compound according to any one of [7] to

[10] , wherein it is an ortho substituent.

[12] Each R 12 is F, Cl, Br, I, or C 1 -C 6 alkyl, and the C 1 -C 6 alkyl optionally contains one, two, three or more substitutions by F, Cl, Br, or I, and is the compound according to any one of [7] to

[11] .

[13] Each R 11 is independently C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl or 5- to 10-membered heteroaryl, and the C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl or 5- to 10-membered heteroaryl optionally contains one, two, three or more substitutions by F, Cl, Br, I, (C=O)-(C 1 -C 6 alkyl), (C=O)-O(C 1 -C 6 alkyl), (C=O)-NH(C 1 -C 6 alkyl), or (C=O)-N(C 1 -C 6 alkyl) 2 , and is the compound according to any one of [1] and [3] to

[12] .

[14] i) R 2 is not phenoxy, or ii) R 3 is not imidazolyl or pyrimidinyl, and is the compound according to any one of [1] and [3] to [5].

[15] Structure:

Chem.

Chem.

[16] Structure:

Chem.

[14] .

[17] Structure:

Chem.

[16] .

[18] A pharmaceutical composition comprising the compound according to any one of [1] to

[17] , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[19] The pharmaceutical composition according to

[18] , further comprising one or more additional therapeutic agents.

[20] The pharmaceutical composition according to

[19] , wherein the additional therapeutic agent is a chemotherapeutic agent, or an antibody, or a nucleic acid molecule, or a therapeutic cell.

[21] A method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject a therapeutically effective amount of the compound according to any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to

[18] .

[22] The method according to

[21] , wherein the cancer is pancreatic cancer, breast cancer, lung cancer (e.g., small cell and non-small cell lung cancer), kidney (renal) cancer, liver cancer, ovarian cancer, endometrial cancer, basal cell cancer, biliary tract cancer, bladder cancer, bone cancer, brain and / or CNS cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, fibroma, head and neck cancer, stomach cancer, intraepithelial neoplasia, laryngeal cancer, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), lymphoma (e.g., Hodgkin and non-Hodgkin lymphoma), melanoma, oral cancer (e.g., lip, tongue, mouth, and pharyngeal cancer), prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, or any combination thereof.

[23] The method according to

[21] or

[22] , further comprising administering one or more additional therapeutic agents.

[24] The method according to

[23] , wherein the one or more additional therapeutic agents comprise a chemotherapeutic agent.

[25] The method according to

[24] , wherein the chemotherapeutic agent comprises a taxane or paclitaxel.

[26] The method according to

[23] , wherein the one or more additional therapeutic agents comprise an antibody.

[27] The method according to

[26] , wherein the antibody comprises an anti-HER2 antibody.

[28] The method according to

[27] , wherein the anti-HER2 antibody comprises trastuzumab.

Claims

1. A compound selected from the following (UR214-4), (UR214-7), (UR214-8), (UR214-9), (UR214-10), and (UR214-11) 【Chemical 1】 or a pharmaceutically acceptable salt thereof.

2. The compound according to Claim 1, which is the following compound (UR214-9) [Chemical Formula 2] or a pharmaceutically acceptable salt thereof.

3. The compound according to Claim 1, or a pharmaceutically acceptable salt thereof, selected from the following (UR214-7), (UR214-8), (UR214-9), (UR214-10), and (UR214-11). 【Chemical Formula 3】

4. The compound according to Claim 1, or a pharmaceutically acceptable salt thereof, selected from the following (UR214-7) and (UR214-9). 【Chemical Formula 4】

5. A pharmaceutical composition comprising the compound according to any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to Claim 5, further comprising one or more additional therapeutic agents.

7. The pharmaceutical composition according to Claim 6, wherein the additional therapeutic agent is a chemotherapeutic agent, or an antibody, or a nucleic acid molecule, or a therapeutic cell.

8. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of Claims 1 to 4, or a pharmaceutically acceptable salt thereof, for treating cancer in a subject in need of cancer treatment.

9. The cancer is selected from the group consisting of pancreatic cancer, breast cancer, lung cancer (e.g., small cell and non-small cell lung cancer), kidney (renal) cancer, liver cancer, ovarian cancer, endometrial cancer, basal cell cancer, biliary tract cancer, bladder cancer, bone cancer, brain and / or CNS cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, fibroma, head and neck cancer, stomach cancer, intraepithelial neoplasia, laryngeal cancer, leukemia (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, etc.), lymphoma (e.g., Hodgkin and non-Hodgkin lymphoma), melanoma, oral cancer (e.g., lip, tongue, mouth, and pharyngeal cancer), prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, and any combination thereof. The pharmaceutical composition according to Claim 8.

10. The pharmaceutical composition according to claim 8 or 9, which is administered in combination with one or more additional therapeutic agents.

11. The pharmaceutical composition according to claim 10, wherein the one or more additional therapeutic agents are chemotherapeutic agents.

12. The pharmaceutical composition according to claim 11, wherein the chemotherapeutic agent is a taxane or paclitaxel.

13. The pharmaceutical composition according to claim 10, wherein the one or more additional therapeutic agents are antibodies.

14. The pharmaceutical composition according to claim 13, wherein the antibody is an anti-HER2 antibody.

15. The pharmaceutical composition according to claim 14, wherein the anti-HER2 antibody is trastuzumab.

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