WO2017151063A1

WO2017151063A1 - Use of furazano[3,4-b]pyrazine derivatives for chemotherapy

Info

Publication number: WO2017151063A1
Application number: PCT/SG2017/050104
Authority: WO
Inventors: Chit Fang Cheok; Luis Coronel
Original assignee: Agency For Science, Technology And Research
Priority date: 2016-03-03
Filing date: 2017-03-03
Publication date: 2017-09-08

Abstract

The present invention relates to methods of treating cancer with a mitochondrial uncoupler comprising a furazano[3,4 b]pyrazine. The present invention also relates to methods of using the said mitochondrial uncoupler, and the uses thereo to develop target-specific therapeutic agents. In a preferred embodiment, the mitochondrial uncoupler is N5,N6-bis(2 fluorophenyl)-[1,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15). Also provided is a pharmaceutical composition comprising said mitochondrial uncoupler and a second therapeutic agent, wherein the second therapeutic agent preferably also is used in the treatment of cancer.

Description

USE OF FURAZANO[3,4-B]PYRAZINE DERIVATIVES FOR CHEMOTHERAPY

TECHNICAL FIELD

[0001] The present invention generally relates to methods of treating cancer with mitochondrial uncoupler. The present invention also relates to methods of using mitochondrial uncoupler, and the uses thereof to develop target-specific therapeutic agents.

BACKGROUND

[0002] Mitochondrial uncoupling refers to the process whereby protons enter the mitochondrial matrix via a pathway independent of ATP synthase and thereby uncouple nutrient oxidation from ATP production, therefore reducing the efficiency of energy (ATP) production. This process can be pharmacologically induced by small molecule mitochondrial protonophores, which directly shuttle protons across the mitochondrial inner membrane into the matrix. Such small molecules that allow protons to enter the mitochondrial matrix via a pathway independent of ATP synthase are known as "mitochondrial uncouplers".

[0003] BAM 15 is a new synthetic molecule found to have mitochondrial uncoupling function. It has been described as a unique mitochondrial uncoupler that does not depolarize the plasma membrane, unlike conventional mitochondrial uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP). FCCP and DNP are also known to have off-target activity at other (non-mitochondrial) membranes that can result in undesired and unspecific cytotoxicity. While BAM15 has been shown to uncouple the mitochondria, which is expected to result in effects on the metabolic pathway, the effects of BAM 15 on any molecular pathways in cells have not yet been demonstrated to date. In particular, no literature is available on the effect of BAM15 on inhibiting cancer cell proliferation.

[0004] The p53 signaling pathway, which is activated in response to a variety of stress, allows p53 to coordinate transcription programs that contribute to tumor suppression^' The p53 is a gene located on the seventeenth chromosome (17pl3.1). It codes for a protein that regulates the cell cycle and hence functions as a tumor suppressor. One of the genes most commonly mutated in human cancers is p53; in more than 50% of all human cancers p53 gene is mutated. p53-deficient cells include cells that partially or completely lack p53 activity and functions. Yet to date there is no curative therapies for this large cohort of cancers.

[0005] There is a need to provide methods and compositions for treating p53-associated neoplasm and tumor, particularly cancers, that overcome, or at least ameliorate, one or more of the disadvantages described above. There is also a need to provide target-specific therapeutic agents for p53-associated neoplasm and tumor, particularly cancers, to inhibit or prevent the progression of the neoplasm, tumor or cancer, and its recurrence.

SUMMARY

[0006] The present invention is based on the surprising finding BAM15 has increased toxicity for different cancer cell types (as demonstrated in the Examples described herein) and p53 deficiency further sensitizes cells to BAM15. The toxicity of BAM15 for p53- deficient cells and the ability to inhibit cancer cell growth enables its use in the treatment of cancer, which is often characterized by p53 -deficiency and/or unregulated cell growth.

[0007] According to a first aspect, there is provided a method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof a mitochondrial uncoupler, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0008] Advantageously, it was found that a furazano[3,4-b]pyrazine derivative, BAM15, has potent anti-proliferative activity against growth of cancer cells from different tissue origins, such as cells from lung, large intestine, breast, bone, and brain.

[0009] According to a second aspect, there is provided a mitochondrial uncoupler for use in preventing or treating neoplasm or tumor, comprising a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0010] According to a third aspect, there is provided a use of a mitochondrial uncoupler for the manufacture of a medicament for the prevention or treatment of neoplasm or tumor, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0011] According to a fourth aspect, there is provided a pharmaceutical composition comprising a mitochondrial uncoupler and a second therapeutic agent, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0012] Advantageously, BAM15 was shown to increase the cytotoxicity of existing chemotherapy drugs when used in combination.

[0013] According to a fifth aspect, there is provided a method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof the pharmaceutical composition described herein.

[0014] According to a sixth aspect, there is provided a pharmaceutical composition described herein, for use in preventing or treating neoplasm or tumor.

[0015] According to a seventh aspect, there is provided a use of the pharmaceutical composition described herein for the manufacture of a medicament for the prevention or treatment of neoplasm or tumor.

DEFINITION OF TERMS

[0016] The following words and terms used herein shall have the meaning indicated:

[0017] The term "treatment", or grammatical variants thereof, includes any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Hence, "treatment" includes prophylactic and therapeutic treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e. not worsening) state of condition, disorder or disease; delay or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state; remission (whether partial or total, and whether detectable or undetectable); or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a cellular response that is clinically significant, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. Treatment may entail treatment with a single agent or with a combination (two or more) of agents. Treatment may also entail sensitizing a patient to treatment with a therapeutic agent.

[0018] The term "prevent" or grammatical variants thereof, as used herein, for therapeutic purposes, generally refers to prophylactically interfering with a pathological mechanism that results in the disease or disorder. In the context of the present invention, such a pathological mechanism can be disruption to the p53 pathway, or increase in cell proliferation, or some combination of the two, which result in neoplasm or tumor.

[0019] The term "sensitize" or grammatical variants thereof, as used herein, for therapeutic purposes, generally refers to causing a patient to be susceptible to treatment with a single agent or with a combination (two or more) of agents to thereby allow for more effective treatment of a disease. For example, sensitizing a patient to treatment with an anticancer drug may refer to causing the patient to be susceptible to treatment with, or more effective treatment with, the anti-cancer drug. In another example, sensitizing p53-deficient cells with BAM15 may refer to causing the p53-deficient cells to be susceptible to, or more susceptible to, apoptosis, thereby enabling treatment of, or more effective treatment of, neoplasm or tumor caused by p53-deficient cells.

[0020] The term "neoplasm" as used herein refers to abnormal growth of tissue in its initial stage, which may lead to subsequent formation of a tumor.

[0021] The term "tumor" as used herein refers to a neoplasm that may be malignant (cancerous) or non-malignant (benign).

[0022] The term "mitochondrial uncoupler" as used herein refers to a molecule that is capable of "uncoupling" the mitochondria, for example, by allowing protons to enter the mitochondrial matrix via an ATP-synthase-independent pathway. More specifically, mitochondrial uncoupling is the depolarization of the mitochondrial membrane by the dissipation of the proton gradient that is generated by the NADH-powered pumping of protons from mitochondrial matrix. As the uncoupling occurs through an ATP- synthase independent pathway, uncoupling can lead to inhibition of ATP synthesis (which is dependent on the proton gradient and is mediated through ATP synthase).

[0023] An "agent" is used herein broadly to refer to, for example, a compound, or other means for treatment e.g. radiation treatment or surgery.

[0024] In the context of this invention, the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to a subject, or a surface by any appropriate means. Administration may be via one or more routes, such as via parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. [0025] As used herein, pharmaceutically acceptable salts, esters or other derivatives of the compounds described herein include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to a subject (animals or humans) without substantial toxic effects and that either are pharmaceutically active or are prodrugs.

[0026] As used herein, derivative of a molecule refers to a portion derived from the molecule, or a modified version of the molecule.

[0027] As used herein, the term "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the subject or patient is a primate. In certain embodiments, the subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.

[0028] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

[0029] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0030] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0031] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

DETAILED DISCLOSURE OF THE EMBODIMENT

[0032] Exemplary, non-limiting embodiments of a method of treating neoplasm or tumor, will now be disclosed.

[0033] In a first aspect, there is provided a method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof a mitochondrial uncoupler, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof. Furazano[3,4-b]pyrazine is a com ound of Formula I

Formula I

wherein Rj and R₂ are independently selected from the group consisting of H, halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl and carboxy, each of which is unsubstituted or substituted by one or more groups independently selected from the group consisting of H, halogen, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid side chain, and amino acid.

[0034] The term "halogen" represents chlorine, fluorine, bromine or iodine. The term "halo" represents chloro, fluoro, bromo or iodo.

[0035] The term "hydroxy" is intended to mean the radical -OH.

[0036] The term "acyl" is intended to mean a -C(0)-R radical, wherein R may be selected from, but not limited to, an alkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl group.

[0037] The term "alkyl" refers to a straight- or branched-chain alkyl group having from 1 to 12 carbon atoms in the chain. Exemplary alkyl groups include methyl (Me), ethyl (Et), n- propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl (tBu), pentyl,isopentyl, tert-pentyl, hexyl, isohexyl, and the like.

[0038] The term "alkenyl" refers to a straight-or branched-chain alkenyl group having from 2 to 12 carbon atoms in the chain. Illustrative alkenyl groups include prop-2-enyl, but- 2- enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, and the like.

[0039] The term "alkynyl" refers to a straight- or branched-chain alkynyl group having from 2 to 12 carbon atoms in the chain. Illustrative alkynyl groups include prop-2-ynyl, but- 2-ynyl, but-3-ynyl, 2-methylbut-2-ynyl, hex-2-ynyl, and the like.

[0040] The term "cycloalkyl" as used herein refers to cyclic saturated aliphatic groups and includes within its meaning monovalent ("cycloalkyl"), and divalent ("cycloalkylene"), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 10 carbon atoms, eg, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like.

[0041] The term "heterocyclo" as used herein refers to fully saturated or unsaturated rings of 5 or 6 atoms containing one or two O and S atoms and/or one to four N atoms provided that the total number of hetero atoms in the ring is 4 or less.

[0042] The term "aryl" as used herein refers to a monocyclic, or fused or spiro polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) having from 3 to 12 ring atoms per ring.

[0043] The term "heteroaryl" as used herein refers to a monocyclic, or fused or spiro polycyclic, aromatic heterocycle (ring structure having ring atoms selected from carbon atoms as well as nitrogen, oxygen, and sulfur heteroatoms) having from 3 to 12 ring atoms per ring.

[0044] The term "alkoxy" as used herein refers to straight chain or branched alkyloxy groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

[0045] The term "amino" as used herein refers to groups of the form -NR_a¾ wherein R_a and ¾ are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups. In one embodiment, the aryl is optionally substituted with at least one halogen. In one embodiment the halogen comprises fluorine. In one embodiment, the halogen comprises chlorine. In one embodiment, the halogen comprises bromine. In one embodiment, the halogen comprises iodine. Where the aryl is substituted with more than one halogen, the halogen may be independently selected from a group consisting of fluorine, chlorine, bromine, and iodine. In one embodiment, the halogen comprises fluorine and chlorine, fluorine and iodine, fluorine and bromine, chlorine and iodine, chlorine and bromine, or iodine and bromine.

[0046] The term "amide" as used herein refers to the formula -C(0)NRR', wherein R and R' independently can be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group.

[0047] The term "sulfone" as used herein refers to a group -S(0)2R, in which R may be selected from, but not limited to, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0048] The term "sulfoxide" as used herein refers to a group -S(0)R, in which R may be selected from, but not limited to, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

[0049] The term "oxo" as used herein refers to the radical =0,

[0050] The term "oxy," as used herein refers to (-0-).

[0051] The term "nitro" as used herein refers to -N0₂.

[0052] The term "carbonyl," as used herein refers to a -C(O)- group.

[0053] The term "carboxyl" as used herein refers to the radical -C(0)OH.

[0054] The term "amino acid" as used herein is defined as having at least one primary, secondary, tertiary or quaternary amino group, and at least one acid group, wherein the acid group may be a carboxylic, sulfonic, or phosphonic acid, or mixtures thereof. The amino groups may be "alpha", "beta", "gamma" ... to "omega" with respect to the acid group(s). The backbone of the "amino acid" may be substituted with one or more groups selected from halogen, hydroxy, guanido, heterocyclic groups. Thus term "amino acids" also includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparte, glutamine, lysine, arginine and histidine, taurine, betaine, N-methylalanine etc. (L) and (D) forms of amino acids are included in the scope of this disclosure.

[0055] The term "substituted" means that the specified group or moiety bears one or more substituents. The term "unsubstituted" means that the specified group or moiety bears no substituents. [0056] In another aspect, there is provided a mitochondrial uncoupler for use in preventing or treating neoplasm or tumor, comprising a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0057] In another aspect, there is provided a use of a mitochondrial uncoupler for the manufacture of a medicament for the prevention or treatment of neoplasm or tumor, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

[0058] In one embodiment, the mitochondrial uncoupler is selected from the group

consisting of

N5,N6-bis(2-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine BAM15),

N5,N6-bis(2-chlorophenyl)-

[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15A),

N5,N6-bis(2-bromophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15B),

N5,N6-bis(2-iodophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15C),

F F N5,N6-bis(4-chloro-2- fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15D),

difluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15F),

N5,N6-bis(4-chlorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM8) and

N5,N6-bis(4-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM9), and pharmaceutically acceptable salts, esters and derivatives thereof. the mitochondrial uncoupler is

N5,N6-bis(2-fluorophenyl)-[l,2,5]oxadiazolo[3,4- b]pyrazine-5,6-diamine (BAM15), or pharmaceutically acceptable salts, esters or derivatives thereof.

[0060] In one embodiment, the neoplasm or tumor is p53-deficient. In one embodiment, the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer. In one embodiment, the neoplasm or tumor is malignant or cancerous.

[0061] In one embodiment, the cancer is a cancer of the lung, large intestine, breast, bone, and brain. In one embodiment, the cancer of the lung is lung adenocarcinoma. In one embodiment, the cancer of the large intestine is colon carcinoma. In one embodiment, cancer of the bone is osteosarcoma. In one embodiment, the cancer of the brain is glioblastoma. In one embodiment, the cancer of the breast is adenocarcinoma. In one embodiment, the cancer of the breast is ductal carcinoma. In one embodiment, the cancer of the breast is triple negative breast cancer.

[0062] In another aspect, there is provided a pharmaceutical composition comprising a mitochondrial uncoupler and a second therapeutic agent, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof. Advantageously, as described in the examples below, BAM15 was shown to increase the cytotoxicity of existing chemotherapy drugs when used in combination with the chemotherapy drug. [0063] In another aspect, there is provided a method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof the pharmaceutical composition described herein.

[0064] In another aspect, there is provided a pharmaceutical composition described herein, for use in preventing or treating neoplasm or tumor.

[0065] In another aspect, there is provided a use of the pharmaceutical composition described herein for the manufacture of a medicament for the prevention or treatment of neoplasm or tumor.

[0066] In one embodiment, the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis. Advantageously, BAM 15 was shown to be able to increase the cytotoxicity of a second therapeutic agent when used in combination with that second therapeutic agent.

[0067] In one embodiment, the second therapeutic agent is selected from the group consisting of 5-fluorouracil, 6-mercaptopurine, actinomycin-D, altretamine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, dexamethasone, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, ixabepilone, L-asparaginase, lomustine, mechlorethamine, melphalan, methotrexate, methylprednisolone, mitomycin-C, mitoxantrone, oxalaplatin, paclitaxel, pemetrexed, prednisone, streptozocin, temozolomide, teniposide, thiotepa, topotecan, vinblastine, vincristine and vinorelbine. In one embodiment, the second therapeutic agent is a chemotherapy drug. When used in combination with BAM15, the cytotoxic effect of the chemotherapy drug was shown to increase, resulting in increased cancer cell killing. Suitable chemotherapy drugs for use in combination with BAM15 include, but are not limited to gemcitabine, paclitaxel, doxorubicin and cisplatin.

[0068] The combination treatment may comprise simultaneous or sequential administration of the furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof, and the second therapeutic agent. In one embodiment of sequential administration, the furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof is administered first, followed by the second therapeutic agent. In another embodiment, the furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof is administered after the second therapeutic agent. [0069] The furazano[3,4-b]pyrazine derivative and/or the second therapeutic agent may be administered in the form of a pharmaceutical composition. In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.

[0070] The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.

[0071] Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

[0072] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of the furazano[3,4-b]pyrazine derivative and/or the second therapeutic agent which would be required to treat or prevent the diseases to be treated or prevented. Typically, in therapeutic applications, the treatment would be for the duration of the disease state.

[0073] Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques. [0074] It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0076] Figure 1 shows the inhibition of cell growth by an exemplary furazano[3,4- b]pyrazine derivative, BAM15.

[0077] Figure 1A shows BAM15 exhibited potent antiproliferative activity against each of the cell lines lung adenocarcinoma A549, osteocarcinoma U20S, triple negative breast cancer BT-549, breast adenocarcinoma MCF-7, breast adenocarcinoma MBA-MD-231 and glioblastoma U87MG in vitro, resulting in an estimated IC50 between 7.5 μΜ to 15 μΜ.

[0078] Figure IB shows the accompanying bright field images of colonies that were taken at 5 days following recovery from BAM15. The scale bars represent 400 μπι.

[0079] Figure 2 shows the increased cell killing in combination treatments of BAM15 and a second therapeutic agent. The cell line used for Figures 2A to 2E was HCT116.

[0080] Figure 2A shows the increased cell killing in combination treatment of BAM15 and gemcitabine (at concentrations of 0.01 μΜ, 0.02 μΜ, 0.03μΜ and 0.04μΜ). The data on the table represents the percentage of cell viability. The data on the graph for gemcitabine is based on 0.03μΜ gemcitabine.

[0081] Figure 2B shows the increased cell killing in combination treatment of BAM 15 and paclitaxel (at concentrations of ΙμΜ, 2 μΜ, 3μΜ and 4μΜ). The data on the table represents the percentage of cell viability. The data on the graph for paclitaxel is based on 3μΜ paclitaxel.

[0082] Figure 2C shows the increased cell killing in combination treatment of BAM 15 and doxorubicin (at concentrations of 0.02μΜ, 0.04 μΜ, 0.05μΜ and 0.08μΜ). The data on the table represents the percentage of cell viability. The data on the graph for doxorubicin is based on 0.05μΜ doxorubicin.

[0083] Figure 2D shows the increased cell killing in combination treatment of BAM 15 and cisplatin (at concentrations of 0.25μΜ, 0.5 μΜ, ΙμΜ and 2μΜ). The data on the table represents the percentage of cell viability. The data on the graph for cisplatin is based on 1 μΜ cisplatin.

[0084] Figure 2E shows the accompanying bright field images for the colonies that were taken following treatment with 15μΜ BAM15 alone, 0.04μΜ gemcitabine alone, ΙμΜ cisplatin alone, 0.04μΜ gemcitabine with 15μΜ BAM15 and ΙμΜ cisplatin with 15μΜ BAM15. The scale bars represent 400 μπι.

[0085] Figure 3 shows the inhibition of B AMI 5 on mTOR, autophagy and AMP-activated protein kinase (AMPK) pathways.

[0086] Figure 3A shows the effect of BAM15 on AMPK pathway.

[0087] Figure 3B shows the effect of B AMI 5 on the mTOR and autophagy pathways.

[0088] Figure 4 shows BAM15 sensitizes cancer cells with p53 -deficiency.

[0089] Figures 4A and 4B show the growth of HCT116p53+/+ and HCT116p53-/- cells treated with different concentrations of BAM15.

[0090] Figure 4C shows the selective effects of BAM15 on A549 cells expressing the control NT shRNA and p53 shRNA constructs.

[0091] Figure 5 shows BAM15 is effective in both HCT116p53+/+ and HCT116p53-/- cells.

[0092] Figure 5A shows the oxygen consumption of mitochondria under conditions of BAM15-induced mitochondrial uncoupling, in the presence of oligomycin.

[0093] Figure 5B shows the maximal respiration rate under conditions of BAM15-induced mitochondrial uncoupling

[0094] Figure 6A shows the cell cycle profiles of both HCT116 p53+/+ and HCT116p53 - /- cells.

[0095] Figure 6B shows the dot plots from the flow cytometry analysis for HCT116 cells treated with B AMI 5.

DETAILED DESCRIPTION OF THE DRAWINGS EXAMPLES

[0096] Non-limiting examples of the invention, including the best mode, and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Methods: Cell lines

[0097] The cell lines used are HCT116p53+/+, HCT116p53-/-, U-2 OS (ATCC^® HTB- 96™), MCF7 (ATCC^® HTB-22™), MDA-MB-231 (ATCC^® HTB-26™), A549 (ATCC^® CCL-185™), HCT 116 (ATCC^® CCL247™), U-87 MG (ATCC^® HTB-14™), and BT549 (ATCC^® HTB 122™).

Drug inhibition assay

[0098] Cells were plated on 6-well dish at a density of 50000 cells per well. Cells were treated with the indicated concentrations for 48 hours. BAM15 was purchased from TimTec LCC (Catalog No: ST056388). Drug-containing media was removed and replaced with drug free media for 8 days. Cell colonies were stained using crystal violet (0.5% w/v in 50% methanol). 0.1% DMSO-treated cells were used as non-drug treated control in each cell line. In all cell lines, at concentrations above 20μΜ, 100% colony growth inhibition was observed.

Drug cytotoxicity assay

[0099] Cells were seeded at a density of 5000 cells/well and incubated overnight at 37°C prior to drug treatment. Drugs were diluted with cell culture media and added to each well at the indicated concentrations. Cells were treated with BAM15 for 48 hours before allowing cells to recover for 5 days in drug free media. Cell viability was measured using WST1 proliferation assay (Roche). Absorbance was measured using a microplate reader. Cell viability of drug treated cells was displayed as a percentage of control DMSO treated cells. The final concentration of DMSO was no more than 0.1% (v/v). The results were from two independent experimental values.

Cell viability assay (WST1 proliferation assay) [00100] The cell proliferation assay was performed using the Cell Proliferation Reagent WST-1 (Roche Catalogue No.: 11644807001) according to the manufacture's protocol.

In vitro mitochondrial functional assay

[00101] The Seahorse XF Cell Mito Stress Test Kit was used according to the manufacturer's protocol. FCCP provided in the kit was replaced with BAM15 to show that BAM 15 is also uncoupling the mitochondria similar to the known mitochondria uncoupler FCCP. Flow cytometry analysis

[00102] 50000 cells were seeded on a single well in a 6 well dish. The next day, cells were treated with 20μΜ or 30μΜ of BAM15. After 48 hours, the cells were harvested and fixed in 70% ethanol, followed by incubation in PBS containing O. lmg/ml RNaseA and O. lmg/ml Propidium Iodide (PI) stain. Flow cytometry was performed using BD LSR II Flow Cytometer (fluorescence-activated cell sorting) system.

Results and Discussion:

BAM15 inhibits cancer cells growth

[00103] To determine if BAM15 is active against cancer cell growth, in vitro assays were performed by treating cancer cell lines with serial dilutions of BAM15 using the "Drug inhibition assay" in the Methods section above. The cancer cell lines cell lines tested were colon carcinoma lung adenocarcinoma A549, osteocarcinoma U-2 OS, triple negative breast cancer or ductal carcinoma BT549, breast adenocarcinoma MCF7, breast adenocarcinoma MB A-MD-231 and glioblastoma U-87 MG (described above in Paragraph [0098]).

[00104] BAM 15 was shown to exhibit potent anti-proliferative against each of these cell lines in vitro, resulting in an estimated IC50 between 7.5 μΜ and 20 μΜ (Figure 1A and IB). Therefore, BAM 15 is active against cancer cells growth and was shown to inhibit the growth of cancer cell lines of different tissue origins.

BAM15 increases the cytotoxicity of existing chemotherapy drugs [00105] To determine if BAM15 could increase the cytotoxicity of commonly used chemotherapeutic drug when used in combination, cytotoxicity assays were performed by treating the cells with a combination of BAM 15 and a chemotherapy drug for 48 hours as described in the "Drug cytotoxity assay" in the Methods section above. The viability of the cells was determined after a period of recovery in drug-free growth media. The chemotherapy drugs tested were gemcitabine, paclitaxel, doxorubicin, and cisplatin. The control treatment was 0.1% dimethyl sulfoxide (DMSO).

(i) Increased cell killing in combination treatment of BAM15 and gemcitabine (Figure 2A and 2E)

[00106] Using cell viability assays (i.e. the WST1 proliferation assay as described in the Methods section above) the inventors found that BAM 15 resulted in the inhibition of HCT116 at an estimated IC50 of 20-25μΜ.

[00107] Dilutions of gemcitabine were tested on HCT116 cells. When gemcitabine alone was added at Ο.ΟΙμΜ to 0.04μΜ, it reduced cell viability by l l%-44%. When the same concentrations of gemcitabine were used in combination with 10μΜ-20μΜ of BAM15, further increase in cytotoxicity of cells was observed, resulting in reduction of cell viability by 67-99%. This indicates that the combination of gemcitabine with BAM 15 resulted in at least additive cell killing.

(ii) Increased cell killing in combination treatment of BAM15 and paclitaxel (Figure 2B)

[00108] Dilutions of paclitaxel were tested on HCT116 cells. When paclitaxel alone was added at ΙμΜ to 4μΜ, it reduced cell viability by 10%-72%. When the same concentrations of paclitaxel were used in combination with 10μΜ-20μΜ of BAM15, further increase in cytotoxicity of cells was observed, resulting in reduction of cell viability by 54-85%. This indicates that the combination of gemcitabine with BAM15 resulted in at least additive cell killing.

(iii) Increased cell killing in combination treatment of BAM15 and doxorubicin (Figure 2C)

[00109] Dilutions of doxorubicin were tested on HCT116 cells. When doxorubicin alone was added at 0.02μΜ to 0.08μΜ, it reduced cell viability by 10%-74%. When the same concentrations of doxorubicin were used in combination with 10μΜ-20μΜ of BAM15, further increase in cytotoxicity of cells was observed, resulting in reduction of cell viability by 53-91 . This indicates that the combination of doxorubicin with BAM 15 resulted in at least additive cell killing.

(iv) Increased cell killing in combination treatment of BAM15 and cisplatin (Figure 2D and 2E)

[00110] Dilutions of cisplatin were tested on HCT116 cells. When cisplatin alone was added at 0.25μΜ to 2μΜ, it reduced cell viability by 14%-64%. When the same concentrations of cisplatin were used in combination with 10μΜ-20μΜ of BAM15, further increase in cytotoxicity of cells was observed, resulting in reduction of cell viability by 60- 96%. This indicates that the combination of cisplatin with BAM15 resulted in at least additive cell killing. BAM15 inhibits mTOR, autophagy and AMP-activated protein kinase (AMPK) pathways

[00111] Uncoupling of the mitochondria by BAM 15 is expected to have important consequences on the metabolic pathway. However, the effects of BAM 15 on any molecular pathways in cells have not yet been determined. To determine the molecular pathways in which BAM15 acts on, cell lysates treated with BAM15 were probed using specific biomarkers for mTOR, AMPK and autophagy (see Figure 3)

[00112] The inventors describe for the first time that BAM15 leads to partial degradation of AMPK and ACC proteins but enhanced phosphorylation of AMPK substrate, ACC (as indicated using an antibody to phosphor- ACC). The inventors also show activation of the AMPK pathway, as indicated by the increase in phosphorylation of the AMPK and acetyl- CoA carboxylase (ACC, which is a substrate of AMPK) (see Figure 3A). In this case, activation of AMPK is being investigated widely as a strategy for treatment of cancers. There are activators of AMPK that are being identified for cancer therapy. The inventor's observation that even though AMPK and its substrate ACC appear to be degraded upon BAM15 treatment, on higher exposure of the western blots, it seems that the ACC and AMPK are highly phosphorylated upon BAM 15 treatment which corresponds to increase activation of the AMPK signaling pathway (see Figure 3A). Therefore, activation of AMPK by BAM15 may represent one potential mechanism in which BAM15 kills cancer cells.

[00113] The inventors also describe for the first time that BAM15 leads to inhibition of mTOR pathway, as indicated by the decrease in phosphorylation of mTOR substrate p70S6K and the decrease in phosphorylation of 4EBP1 (see Figure 3B). mTOR inhibitors have been designed to inhibit either mTORCl or mTORC2 or both, and have been shown to have tumor responses in clinical trials against various tumor types. Dysregulation of mTOR signaling is apparent in human cancers, and may be the reason for increased sensitivity to these mTOR inhibitors. Furthermore, over-activation of mTOR signaling can contribute to the initiation and development of tumors. Therefore inhibition of mTOR can curb the growth of tumors and it has also been previously shown that mTOR inhibition leads to autophagy and induced apotosis. Therefore, the inhibition of mTOR by BAM15 may represent another potential mechanism in which BAM15 kills cancer cells.

[00114] Furthermore, the inventors showed a clear induction of autophagy, as indicated by the increase in LC3BII band (see Figure 3B) which suggests that BAM15 leads to autophagic induction in cells.

BAM15 sensitizes cancer cells with p53-deficiency [00115] One of the genes most commonly mutated in human cancers is p53; in more than 50% of all human cancers p53 gene is mutated. Yet to date there is no curative therapies for this large cohort of cancers. The inventors have shown that BAM 15 has anti-tumor potential as discussed above. To assess if B AMI 5 will also preferentially sensitize cells harboring loss of p53, a pair of isogenic cell lines HCT116p53+/+ and HCT116p537- was analyzed.

[00116] HCT116p53+/+ (50000 cells) and HCT116p53 -/- (50000 cells) were grown in 6 well plate. On the next day, these cells were either treated with DMSO (<0.1% DMSO) or treated with the indicated concentrations of BAM15 (25μΜ, 30μΜ, 35μΜ, 40μΜ, or 45μΜ, as shown in Figure 4A). After 48 hours of drug incubation, the media was replaced with drug-free media and the cells were allowed to recover. After 6 days of recovery, the surviving colonies were stained using crystal violet for visualization.

[00117] Figure 4A showed a dose dependent effect of BAM15 in HCT116 parental p53+/+ cells. Interestingly, HCT116 p53 7- showed an increased sensitivity to BAM15, as indicated by the decrease in colonies at all tested concentrations of BAM15. This data suggest that p53- deficient cells exhibit increased sensitivity to BAM15 and further suggest that BAM15 could be advantageously useful as a pharmacologic tool to more selectively target cells with loss of p53 functions. The experiment was repeated multiple times and the increased sensitivity of p53-deficient cells to BAM15 is indeed reproducible, as shown in another representative experiment (Figure 4B).

[00118] To further illustrate the selective effects of BAM15 on HCT116 p53-/- cells, the inventors used a WST1 cell viability proliferation assay to compare the growth of HCT116p53+/+ and HCT116p53 -/- cells after BAM 15 treatment. The cells were seeded at a density of 5000 cells/well and incubated overnight at 37°C prior to drug treatment. Drugs were diluted with cell culture media and added to each well at the indicated concentrations. Cells were treated with BAM15 for 48 hours before allowing cells to recover for 5 days in drug free media. Figure 4C shows the selective effects of BAM 15 on A549 cells expressing the p53 shRNA construct as compared to the cells expressing NT shRNA construct, indicating that cells deficient in p53 show lower viability than control cells when treated with BAM15.

[00119] To show that BAM15 is indeed effective in both HCT116p53+/+ and HCT116p53 -/- cells and that the differential effects on the reduction of cell viability is not due to trivial reasons such as the drug uptake (in other words, higher viability of HCT116p53+/+ cells is due to BAM15 not being taken in by the HCT116p53+/+ cells), the inventors made use of the biochemical property of the drug in inducing mitochondrial uncoupling, and tested if BAM 15 induces equivalent mitochondrial uncoupling in both HCT116p53+/+ and HCT116p53 7- cells. The in vitro mitochondrial functional assay was performed as described in the Methods section above to study the effects of BAM 15 on mitochondrial uncoupling. Figure 5 A shows that BAM 15 led to a dramatic spike in oxygen consumption, in the presence of oligomycin, which is a hallmark of mitochondrial uncoupling. Indeed BAM 15 resulted in mitochondrial uncoupling in both HCT116p53+/+ and HCT116p53 7- cells and to the same extent in both cell lines.

[00120] Using the data from in vitro mitochondrial functional assay, the extent of maximal respiration was measured and presented in Figure 5B, which shows that both HCT116p53+/+ and HCT116p53 7- cells exhibited equivalent maximal respiration under conditions of BAM15-induced mitochondrial uncoupling. [00121] To determine if cell cycle profiles of both HCT116 p53+/+ and HCT116p53 7- cells are similar before and after treatment with BAM15, the cells were harvested and flow cytometry was performed using propidium iodide staining as described in "Flow cytometry analysis" of the Methods section above. The FACs data was gated to show single cell population. Figure 6 A shows that BAM 15 induced a pronounced G2 arrest in HCT116 p53+/+. Similarly in the HCT116p53 7- cells, the inventors observed a pronounced G2 arrest. Therefore the differential sensitivity in p537- cells cannot be explained by a simple Gl arrest which could have protected cells against any possible BAM15 induced cytotoxicity in S or G2 phase of the cell cycle. The data also suggest that BAM15-induced damage is likely independent of specific cell cycle phases.

[00122] In an ungated population, the inventors observed that BAM15 induced massive polyploidy in both HCT116 p53+/+ and HCT116p53 7- (Figure 6B). In HCT116 p53+/+ cells, the inventors observed an arrest in both G2/M phase and in the tetraploid G2/M phase. In HCT116p53 7- cells, the inventors observed also a significant population in G2/M phase and in the tetraploid G2/M phase. However, in addition, the inventors observed that cells were not fully arrested in G2/M phase as indicated by the presence of tetraploid S phase cells (with a PI staining in between G2/M and tetraploid G2/M). This suggests that HCT116p53 7- could continue to undergo mitotic slippage giving rise to more tetraploid G2/M cells and this could provide an additional mechanism that explains in part the sensitization of HCT116p53 - /- cells to BAM15. In other words, the sensitization of HCT116p53 7- cells to BAM15 may be a result of HCT116p53 7- cells continuing to undergo mitotic slippage thus accumulating DNA damage and chromosomal instability that can eventually kill the HCT116p53 7- cells.

Claims

A method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof a mitochondrial uncoupler, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

The method of claim 1, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

The method of claim 1 or 2, wherein the neoplasm or tumor is malignant or cancerous.

The method of any one of claims 1 to 3, wherein the neoplasm or tumor is p53- deficient.

The method of any one of claims 1 to 4, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.

The method of any one of claims 1 to 5, wherein the mitochondrial uncoupler is

N5,N6-bis(2-fluorophenyl)-

[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15),

N5,N6-bis(2-chlorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15A),

N5,N6-bis(2-iodophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15C), SI N

F F N5,N6-bis(4-chloro-2- fluoro hen l -[l,2,5]oxadiazolo[3,4-b] razine-5,6-diamine BAM15D),

difluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15F),

N5,N6-bis(4-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM9),

and pharmaceutically acceptable salts, esters and derivatives thereof.

7. the mitochondrial uncoupler is

N6-bis(2-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15),

or pharmaceutically acceptable salts, esters or derivatives thereof.

8. A mitochondrial uncoupler for use in preventing or treating neoplasm or tumor, comprising a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

9. The mitochondrial uncoupler of claim 8, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

10. The mitochondrial uncoupler of claim 8 or 9 , wherein the neoplasm or tumor is malignant or cancerous.

11. The mitochondrial uncoupler of any one of claims 8 to 10, wherein the neoplasm or tumor is p53-deficient.

12. The mitochondrial uncoupler of any one of claims 8 to 11, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.

13. The mitochondrial uncoupler of any one of claims 8 to 12, comprising a compound

N5,N6-bis(2-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15),

N5,N6-bis(2-iodophenyl)- l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15C),

N5,N6-bis(4-chloro-2- fluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15D),

difluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15F),

N5,N6-bis(4-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM9),

and pharmaceutically acceptable salts, esters and derivatives thereof.

14. claims 8 to 12, comprising

[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15), or pharmaceutically acceptable salts, esters or derivatives thereof.

15. Use of a mitochondrial uncoupler for the manufacture of a medicament for the

prevention or treatment of neoplasm or tumor, wherein the mitochondrial uncoupler comprises a furazano[3,4-b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

16. The use of claim 15, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

17. The use of claim 15 or 16, wherein the neoplasm or tumor is malignant or cancerous.

18. The use of any one of claims 15 to 17, wherein the neoplasm or tumor is p53- deficient.

19. The use of any one of claims 15 to 18, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.

20. The use of any one of claims 15 to 19, wherein the mitochondrial uncoupler is

selected from the group consisting of

N5,N6-bis(2-chlorophenyl)-

[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15A),

N5,N6-bis(2-bromophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15B), N5,N6-bis(2-iodophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15C),

difluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15F),

N5,N6-bis(4-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM9),

and pharmaceutically acceptable salts, esters and derivatives thereof.

21. The method of an one of claims 1 to 1 wherein the mitochondrial uncoupler is

N5,N6-bis(2-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15), or pharmaceutically acceptable salts, esters or derivatives thereof.

22. A pharmaceutical composition comprising a mitochondrial uncoupler and a second therapeutic agent, wherein the mitochondrial uncoupler comprises a furazano[3,4- b]pyrazine, or pharmaceutically acceptable salts, esters or derivatives thereof.

23. The pharmaceutical composition of claim 22, wherein the mitochondrial uncoupler is selected from the group consisting of

N5,N6-bis(2-iodophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15C),

difluorophenyl)-[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15F),

N5,N6-bis(4-fluorophenyl)- [l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM9),

and pharmaceutically acceptable salts, esters and derivatives thereof.

24. , wherein the mitochondrial uncoupler is

N5,N6-bis(2-fluorophenyl)-

[l,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine (BAM15),

or pharmaceutically acceptable salts, esters or derivatives thereof.

25. The pharmaceutical composition of any one of claims 22 to 24, wherein the second therapeutic agent is selected from the group consisting of 5-fluorouracil, 6- mercaptopurine, actinomycin-D, altretamine, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, dexamethasone, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, floxuridine, fludarabine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, ixabepilone, L-asparaginase, lomustine, mechlorethamine, melphalan, methotrexate, methylprednisolone, mitomycin-C, mitoxantrone, oxalaplatin, paclitaxel, pemetrexed, prednisone, streptozocin, temozolomide, teniposide, thiotepa, topotecan, vinblastine, vincristine and vinorelbine,

26. A method of preventing or treating neoplasm or tumor, comprising administering to a subject in need thereof the pharmaceutical composition of any one of claims 22 to 25.

27. The method of claim 26, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

28. The method of claim 26 or 27, wherein the neoplasm or tumor is malignant or

cancerous.

29. The method of any one of claims 26 to28, wherein the neoplasm or tumor is p53- deficient.

30. The method of any one of claims 26 to 29, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.

31. The pharmaceutical composition of any one of claims 22 to 25, for use in preventing or treating neoplasm or tumor.

32. The pharmaceutical composition of claim 31, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

33. The pharmaceutical composition of claim 31 or 32, wherein the neoplasm or tumor is malignant or cancerous.

34. The pharmaceutical composition of any one of claims 31 to 33, wherein the neoplasm or tumor is p53 -deficient.

35. The pharmaceutical composition of any one of claims 31 to 34, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.

36. Use of the pharmaceutical composition of any one of claims 22 to 25 for the

manufacture of a medicament for the prevention or treatment of neoplasm or tumor.

37. The use of claim 36, wherein the preventing or treating comprises sensitizing the neoplasm or tumor to apoptosis.

38. The use of claim 36 or 37, wherein the neoplasm or tumor is malignant or cancerous.

39. The use of any one of claims 36 to 38, wherein the neoplasm or tumor is p53- deficient.

40. The use of any one of claims 36 to 39, wherein the neoplasm or tumor is selected from the group consisting of adenoma, fibroma, hemangioma, lipoma, meningioma, myoma, brain tumor, carcinoid tumor, leukemia, lymphoma, myeloma, skin cancer, oral cancer, breast cancer, brain cancer, colorectal cancer, colon cancer, rectal cancer, lung cancer, ovarian cancer, renal cancer, prostate cancer, liver cancer and cervical cancer.