WO2014011120A1 - Endoperoxydes, leur synthèse et leurs utilisations dans le traitement de maladies néoplasiques telles que le cancer - Google Patents

Endoperoxydes, leur synthèse et leurs utilisations dans le traitement de maladies néoplasiques telles que le cancer Download PDF

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WO2014011120A1
WO2014011120A1 PCT/SG2013/000293 SG2013000293W WO2014011120A1 WO 2014011120 A1 WO2014011120 A1 WO 2014011120A1 SG 2013000293 W SG2013000293 W SG 2013000293W WO 2014011120 A1 WO2014011120 A1 WO 2014011120A1
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endoperoxide
aryl
pharmaceutically acceptable
concentration
cells
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PCT/SG2013/000293
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English (en)
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Nguan Soon Tan
Shunsuke CHIBA
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Nanyang Technological University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/02—1,2-Dioxanes; Hydrogenated 1,2-dioxanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Endoperoxide synthesis and uses thereof for the treatment of neoplastic diseases such as cancer.
  • the present invention relates generally to an organic synthetic methodology of endoperoxide compounds.
  • the present invention also relates to endoperoxide compounds that are useful for therapy.
  • Cancer is one of the leading causes of death in humans. Although a variety of drugs against cancer have been developed and techniques are available such as surgery and radiation therapy, there is still a need for alternative and improved methods of treatment of neoplastic diseases.
  • Cancer metastasis is an extremely complex process, which occurs through a series of sequential, interrelated
  • Apoptosis is a determinant factor modulating
  • metastasis efficiency As a barrier to metastases, cells normally undergo apoptosis after they lose contact with
  • anoikis a cell death process termed anoikis.
  • ECM extracellular matrix
  • anoikis resistance a crucial property of metastatic cells that permit malignant tumor cells to survive at crucial steps in the metastasis pathway.
  • Acquisition of anoikis resistance is a pre-requisite for the initial step of metastatic dissemination which requires the detachment of epithelial tumor cells from the ECM, as well as for cancer cells survival while travelling through the lymphatic and circulatory systems into the secondary tissue sites.
  • the signalling mechanisms involved in anoikis resistance are still not completely understood.
  • Tumors exhibit an excessive amount or persistent elevation of reactive oxygen species (ROS, specifically the superoxide anion 0 2 ⁇ ) and utilize a redox-based mechanism to evade death by anoikis.
  • ROS reactive oxygen species
  • a deregulated ROS production leads to cell transformation and aggressive tumor metastasis.
  • Recent findings showed that tumor cells maintained a high 0 2" :H 2 02 ratio to confer resistance to anoikis.
  • therapies that interfere with this ratio, culminating in a relative reduction in 0 2 ⁇ :H 2 0 2 ratio, would sensitize tumor cells to apoptosis by anoikis and severely attenuated metastasis.
  • Much effort has been put into the discovery of novel anti-tumor drugs, however few have specifically exploited this redox-based apoptosis as an anticancer therapeutics strategy.
  • a method of producing an endoperoxide comprising exposing an aryl imine to oxygen in the presence of a metallic catalyst, wherein the aryl imine has formula (I) :
  • Ri and R 2 are independently hydrogen, alkyl or aryl, or Rx and R 2 together with the carbon atom to which they are attached form a ring,
  • aryl said aryl being optionally alkyl, alkoxide, halide or substituted in the remaining positions with alkyl or alkoxide, wherein the -CHRiR 2 and -C(NH)R 3 moieties are attached to carbon atoms on the aryl or heterocycle that are adjacent to each other.
  • the method may additionally comprise the step of reacting a nitrile of formula (II) :
  • the step of reacting the nitrile of formula (II) with the Grignard reagent or organometal reagent and the step of reacting the resulting aryl imine of formula (I) with oxygen may be conducted without isolation of the aryl imine .
  • an endoperoxide or a pharmaceutically acceptable salt thereof, having the following general formula (III)
  • Ri and R 2 are independently hydrogen, alkyl or aryl, or Ri and R 2 together with the carbon atom to which they are attached form a ring,
  • R 3 is alkyl or aryl, said aryl being optionally substituted by alkyl, alkoxide, halide or erhaloalkyl , is an aryl or a heterocycle, optionally substituted in the remaining positions with alkyl or alkoxide, wherein the -CHR ⁇ and -C (NH) R 3 moieties are attached to carbon atoms on the aryl or heterocycle that are adjacent to each other; and
  • R 4 is amino or azido.
  • the method may result in endoperoxide compounds which are extremely stable and possess distinct biological activities useful in redox- based apoptosis anti-cancer therapeutic strategies.
  • the method for - making endoperoxides may be straightforward and may be done from readily available building blocks.
  • a further advantage of the method is that the method for making endoperoxides may be conducted in a concise one-pot manner.
  • the endoperoxide compounds may be able to reduce the 0 2 ⁇ : H 2 0 2 ratio in tumours, thereby inducing redox-based apoptosis.
  • the endoperoxide compounds may have a selective cytotoxic effect, i.e. the compounds may have a cytotoxic effect on cancer cells but little cytotoxic effect on normal cells.
  • alkyl includes within its meaning monovalent (“alkyl”) and divalent (“alkylene”) straight chain .or branched chain saturated aliphatic groups having from 1 to 12 carbon atoms, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.
  • alkyl includes, but is not limited to, methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2 -butyl, isobutyl, tert-butyl, amyl, 1 , 2-dimethylpropyl, 1,1- dimethylpropyl, pentyl, isopentyl, hexyl, 4 -methylpentyl , 1-methylpentyl, 2 -methylpentyl , 3 -methylpentyl, 2,2- dimethylbutyl, 3, 3 -dimethylbutyl , 1 , 2 -dimethylbutyl , 1,3- dimethylbutyl , 1, 2, 2- trimethylpropyl , 1,1,2- trimethylpropyl, 2-ethylpentyl , 3 -ethylpentyl , heptyl, 1- methylhexyl, 2 , 2-dimethylpentyl
  • alcohol includes within its meaning a group that contains one or more hydroxyl moieties.
  • alkoxy or variants such as “alkoxide” as used herein refers to an -O-alkyl radical. Representative examples include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.
  • aryl or variants such as "aromatic group” or “arylene” as used herein refers to monovalent (“aryl”) and divalent (“arylene”) single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • groups include, for example, phenyl, biphenyl, naphthyl, phenanthrenyl , and the like.
  • Aryl groups may be optionally substituted.
  • amino includes an amine group (i.e., - NH 2 ) or a substituted amine group.
  • carbocycle or variants such as “carbocyclic ring” as used herein, includes within its meaning any stable 3, 4, 5, 6, or 7-membered monocyclic or bicyclic or 7, 8, 9, 10, 11, 12, or 13-membered bicyclic or tricyclic, any of which may be saturated, partially unsaturated, or aromatic.
  • carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, [3.3.0] bicyclooctane ,
  • carbocycles are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, ' naphthyl, and indanyl.
  • carbocycle When the term "carbocycle” is used, it is intended to include "aryl”. Carbocycles may be optionally substituted.
  • halogen or variants such as “halide” or “halo” as used herein, includes within its meaning fluorine, chlorine, bromine and iodine.
  • heterocycle includes within its meaning a group comprising a covalently closed ring herein at least one atom forming the ring is a carbon atom and at least one atom forming the ring is a heteroatom.
  • Heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms, any of which may be saturated, partially unsaturated, or aromatic . Any number of those atoms may be heteroatoms (i.e., a heterocyclic ring may comprise one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms) .
  • heterocycle e.g., C1-C6 heterocycle
  • the heterocylic ring will have additional heteroatoms in the ring.
  • heterocycles comprising two or more heteroatoms, those two or more heteroatoms may be the same or different from one another.
  • Heterocycles may be optionally substituted. Binding to a heterocycle can be at a heteroatom or via a carbon atom.
  • heterocycles include heterocycloalkyls (where the ring contains fully saturated bonds) and heterocycloalkenyls (where the ring contains one or more unsaturated bonds) such as, but are riot limited to the following:
  • D, E, F, and G independently represent a heteroatom.
  • Each of D, E, F, and G may be the same or different from one another.
  • nitrile refers to the group -CN.
  • perhaloalkyl includes within its meaning an alkyl group in which all hydrogen atoms are replaced by a halo group (e.g., trifluoromethyl , pentafluoroethyl) .
  • ring refers to any covalently closed structure.
  • substituted means the group to which this term refers is substituted with one or more groups other than hydrogen provided that the indicated atom' s normal valency is not exceeded, and that the substitution results in a stable compound.
  • arylalkyl When compounded chemical names, e.g. "arylalkyl” and “arylimine” are used herein, they are understood to have a specific connectivity to the core of the chemical structure.
  • the group listed farthest to the right e.g. alkyl in “arylalkyl”
  • alkyl in “arylalkyl” is the group that is directly connected to the core.
  • an "arylalkyl” group is an alkyl group substituted with an aryl group (e.g. phenylmethyl (i.e., benzyl)) and the alkyl group is attached to the core.
  • An “alkylaryl” group is an aryl group substituted with an alkyl group (e.g., p- methylphenyl (i.e., p-tolyl) ) and the aryl group is attached to the core.
  • the term "grignard reagent” refers to a reagent of structure R 3 MgX wherein R 3 is alkyl or aryl, said alkyl or aryl being optionally substituted.
  • metallic catalyst includes, for example, elemental powders, salts, and organometallic compounds of a metal.
  • Exemplary metallic catalysts are transition metal catalysts.
  • transition metal describes, for example, any metal in Groups III through VII of the periodic table, for example, elements 21 through 30 (scandium through zinc) , 39 through 48 (yttrium through cadmium) , 57 through 80 (lanthanum through mercury), and 89 through 103 (actinium through lawrencium) .
  • Useful metallic catalysts include, for example, copper, iron, gold, silver, cobalt, ruthenium, rhodium, palladium, iridium, platinum, osmium, nickel and zinc catalysts.
  • Useful transition metals include, for example, copper (I) and copper (II) .
  • An example of a transition metal catalyst is copper (II) acetate .
  • organometal reagent refers to a compound with one or more direct bonds between a carbon atom and an electron donor metal.
  • Useful metals of an organometal reagent include, for example, alkali metals or transition metals.
  • alkali metal describes, for example, lithium, sodium, potassium, rubidium, ⁇ cesium.
  • transition metal describes, for example, any metal in Groups III through VII of the periodic table, for example, elements 21 through 30 (sandium through zinc) , 39 through 48 (yttrium through cadmium) , 57 through 80 (lanthanum through mercury) , and 89 through 103 (actinium through lawrencium) .
  • Useful transition metals include, for example, copper, iron, gold, silver, cobalt, ruthenium, rhodium, palladium, iridium, platinum, osmium, nickel and zinc catalysts.
  • Useful organo radicals include, for example, alkyl radicals (such as butyl radical or acetyl radical), . allyl radicals, amino radicals, imido radicals and phosphino radicals.
  • An example of an organometal reagent is a reagent comprising lithium or zinc.
  • compositions according to the present invention are intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like.
  • pharmaceutically acceptable carrier is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated.
  • Supplementary active compounds may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound (s) is calculated to produce the desired therapeutic effect i association with the required pharmaceutical carrier.
  • the compound (s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit .
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • prodrug is intended to include an inactive form of a compound which is transformed in vivo to the active " form.
  • Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.
  • proton donor refers to any chemical compound which is capable of releasing a proton to a proton acceptor.
  • Useful proton donors include, for example, water, alcohol or mixtures thereof.
  • 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 .
  • treatment refers to 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.
  • terapéuticaally effective- amount and “diagnostically effective amount”, . include within their meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect.
  • the exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate "effective ..amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • administering includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means .
  • 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.
  • the method comprises exposing an aryl imine to oxygen in the presence of a metallic catalyst, wherein the aryl imine has formula (I) :
  • Ri and R 2 are independently hydrogen, alkyl or aryl, or Ri and R 2 together with the carbon atom to which they are attached form a ring.
  • Ri may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl , propyl , butyl , pentyl , hexyl , heptyl , octyl , nonyl, decyl, undecyl or dodecyl .
  • R 2 may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • Ri and R 2 may together with the carbon atom to which they are attached form an optionally substituted carbocyclic ring, examples of which include optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl;
  • R 3 is alkyl or aryl, said aryl being optionally substituted by alkyl, alkoxide, halide or perhaloalkyl.
  • R 3 may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl , ethyl , propyl , butyl , pentyl , hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl .
  • R 3 may be optionally substituted aryl, examples of which include phenyl, biphenyl, naphthyl and phenanthrenyl.
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; or CI , F , Br, I; or - CF 3 , - C 2 F 5 , - C 3 F 7 , - C 4 F 9 , -C5F 11 , -C 6 Fi 3 , C 7 F 15 , - C 7 F 17 , - CgFig , - C 9 F2 1 , CioF 23 , -C 1 1F25 or -C 12 F 27 ; and
  • Ring A may be optionally substituted phenyl, naphthyl, phenanthrenyl; or pyrroline, pyrrolidine, imidazoline, imidazolidine , pyrazoline, pyrazolidine , pyrane, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran.
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy.
  • the method may comprise exposing an aryl imine to oxygen in the presence of a metallic catalyst, wherein the aryl imine has formula (I) :
  • Rx and R 2 are independently hydrogen, alkyl or aryl, or Rx and R 2 together with the carbon atom to which they are attached form a ring.
  • R x may be optionally substituted Ci - C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl .
  • R 2 may be optionally substituted C x -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • R x and R 2 may together with the carbon atom to which they are attached form an optionally substituted carbocyclic ring, examples of which include optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl ;
  • R 3 is alkyl or aryl, said aryl being optionally substituted by alkyl, alkoxide, halide or perhaloalkyl .
  • R 3 may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • R 3 may be optionally substituted aryl, examples of which include phenyl, biphenyl , naphthyl and phenanthrenyl .
  • the optional substituents may be straight or branched chain methyl, ethyl-, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; or CI, F, Br, I; or -CF 3 , -C 2 F 5 , - C 3 F 7 , -C 4 F 9 , -C 5 F 1 1, -C 6 F 13 , 7 F1 5 , -C 7 F 17 , -C 8 Fi 9 , -C 9 F2 1 , Ci 0 F 23 , -C 11 F25 or -C 12 F 27 ; and a
  • Ring A may be optionally substituted phenyl, naphthyl , phenanthrenyl ; or pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine , pyrane, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran.
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy,
  • R x and R 2 form an unsubstituted cyclohexyl group and ring A is an unsubstituted phenyl group, then R 3 is not a phenyl group substituted in the 4 -position with a methyl group.
  • the metallic catalyst may include, for example, elemental powders, salts, and organometallic compounds of a metal.
  • the metallic catalyst may be a transition metal catalyst.
  • the transition metal may be any metal in Groups III through VII of the periodic table, for example, elements 21 through 30 (scandium through zinc) , 39 through 48 (yttrium through cadmium), ⁇ 57 through 80 (lanthanum through mercury) , and 89 through 103 (actinium through lawrencium) .
  • the metallic catalyst may include copper, iron, gold, silver, cobalt, ruthenium, rhodium, palladium, iridium, platinum, osmium, nickel or zinc.
  • the metallic catalyst may include copper (I) and copper (II) .
  • the metallic catalyst may be copper (II) acetate.
  • the metallic catalyst may be selected from 5 mol% to 15 mol% based on the nitrile, about 7 mol% to 13 mol% based on the nitrile, or about 9 mol% to 11 mol% based on the nitrile.
  • the metallic catalyst may be 10 mol% based on the nitrile.
  • the method may additionally comprise the step of reacting a nitrile of formula (II) :
  • X may represent a fluorine, chlorine, bromine or iodine atom.
  • M may represent an alkali metal or transition metal. When M represents an alkali metal, it may be lithium, sodium, potassium, rubidium, cesium.
  • M represents a transition metal
  • it may be any metal in Groups III throug VII of the periodic table, for example, elements 21 through 30 (scandium through zinc) , 39 through 48 (yttrium through cadmium) , 57 through 80 (lanthanum through mercury) , and 89 through 103 (actinium through lawrencium), for example, copper, iron, gold, silver, cobalt, ruthenium, rhodium, palladium, iridium, platinum, osmium, nickel or zinc.
  • M may represent lithium or zinc.
  • the step of reacting the nitrile of formula (II) with the Grignard reagent or organometal reagent and the step of reacting the resulting aryl imine of formula (I) with oxygen may be conducted without isolation of the aryl imine.
  • the method may additionally comprise adding a proton donor following reaction of the nitrile with the Grignard reagent or organometal reagent .
  • the proton donor may be selected from the group consisting of water, an alcohol, and mixtures thereof.
  • the alcohol may be CH 3 OH, C 2 H 5 OH, C 3 H 7 OH, C 4 H 9 OH, C 5 H 1:L OH, C 16 H 33 OH, C 2 H 4 (OH) 2 , C 3 H 6 (OH) 2, C 3 H 5 (OH) 3 , C 4 H 6 (OH) 4 , C 5 H 7 (OH) 5 , C 6 H 8 (OH) 6 , C 7 H 9 (OH) 7 , C 3 H 5 OH, Ci 0 H 17 OH, C 3 H 3 OH, C S H 6 (OH) 6 or Ci 0 H 19 OH .
  • the reaction with the Grignard reagent or organometal reagent may be conducted at a temperature of about 40°C to about 80°C, about 40°C to about 80°C, about 50°C to about 70°C, about 55°C to about 65°C.
  • the temperature may be about 60 °C.
  • the method may additionally comprise the step of converting the amino moiety of the endoperoxide to the corresponding azido moiety in the presence of an azide reagent.
  • the azide reagent may be sodium azide, lithium azide, and tetra-n-butylammonium or trimethylsilyl azide (TMSN 3 ) .
  • Intramolecular nucleophilic attack of the terminal oxygen of V to the iminyl carbon result in the formation of benzo [d] [1 , 2] dioxin-1-amine 1 with regeneration of Cu(II) species that maintains the catalytic turnover.
  • Ri and R 2 are independently hydrogen, alkyl or aryl, or Ri and R 2 together with the carbon atom to which they are attached form a ring.
  • R may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl .
  • R 2 may be optionally substituted C 1 -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • R x and R 2 may together with the carbon atom to which they are attached form an optionally substituted carbocyclic ring, examples of which include optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl ;
  • R 3 is alkyl or aryl, said aryl being optionally substituted by alkyl, alkoxide, halide or perhaloalkyl
  • R 3 may be , optionally substituted C x -C 12 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • R 3 may be optionally substituted aryl, examples of which include phenyl, biphenyl, naphthyl and phenanthrenyl .
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; or CI , F , Br, I; or - CF 3 , - C 2 F 5 , - C 3 F 7 , - C 4 F 9 , -C 5 F 1 1, - C6F 13 , C 7 F 15 , - C 7 F1 7 , - C 8 Fi 9 , - C 9 F 21 , CioF -C 11 F 25 or _ Ci 2 F 27 ; is an aryl
  • Ring A is optionally substituted phenyl, naphthyl, phenanthrenyl ; or pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyrane, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran.
  • the optional substituents may be straight or branched chain methyl, ethyl, ; propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; and
  • R 4 is amino or azido.
  • R 4 may be -NH 2 , an alkylamino group, a dialkylamino group, an arylamino group, a diarylamino group, an alkylarylamino group; or -N 3 .
  • Ri and R 2 are independently hydrogen, alkyl or aryl, or Ri and R 2 together with the carbon atom to which they are attached form a ring.
  • Ri may be optionally substituted Cx - Ci 2 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl .
  • R 2 may be optionally substituted Ci - Ci 2 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl.
  • Ri and R 2 may together with the carbon atom to which they are attached form an optionally substituted carbocyclic ring, examples of which include optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl ;
  • R 3 is alkyl or aryl, said aryl being optionally substituted by alkyl, alkoxide, halide or perhaloalkyl .
  • R 3 may be optionally substituted Ci-Ci 2 alkyl, examples of which include optionally substituted straight chain or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl or dodecyl .
  • R 3 may be optionally substituted aryl, examples of which include phenyl, biphenyl, naphthyl and phenanthrenyl .
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; or CI, F, Br, I; or -CF 3 , -C 2 F 5 , - C 3 F 7 , - C 4 F 9 , - 5F 11 , -CeFn , C 7 F 1 5 , - C 7 F1 7 , -CsFig, - 9 F2 1 , C10F23, - 11F25 or -C12F27 ;
  • Ring A is optionally substituted phenyl, naphthyl, phenanthrenyl; or pyrroline, pyrrolidine, imidazoline, imidazolidine , pyrazoline, pyrazolidine , pyrane, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran.
  • the optional substituents may be straight or branched chain methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; or straight or branched chain methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy; and
  • R is amino or azido.
  • R 4 may be -NH 2 , an alkylamino group, a dialkylamino group, an arylamino group, a diarylamino group, an alkylarylamino group; or -N 3 ,
  • R 3 is not a phenyl group substituted in the -position with a methyl group .
  • the endoperoxide compounds, or pharmaceutically acceptable salts thereof, may be selected from the group consisting of:
  • the endoperoxide compounds, or. pharmaceutically acceptable salts thereof may be selected from the group consisting of:
  • composition comprising at least one compound of formula (III), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • composition comprising at least one compound selected from the group consisting of:
  • composition comprising (i) 4-p-tolyl-4H- spiro [benzo [d] [1,2] dioxine-1, 1 ' -cyclohexan] -4-amine, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • an endoperoxide of formula (III) or a pharmaceutically acceptable salt thereof, for use in therapy.
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 /M.
  • an endoperoxide selected from the group consisting of:
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than . 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of ⁇ '500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than . 20% at a concentration of 500 ⁇ , greater than 25% at ' a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide of formula (III) for use as a cytotoxic agent .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide selected from the group consisting of:
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide of formula (III) for use in the treatment of cancer .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide of formula (III) or a pharmaceutically acceptable salt thereof , in the manufacture of a medicament for the treatment of cancer.
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide selected from the group consisting of:
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 MM, greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the method includes the step of administering a therapeutically effective amount of an endoperoxide of formula (III) , or a pharmaceutically acceptable salt thereof .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater. than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • an endoperoxide selected from the group consisting of:
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the endoperoxide may have an apoptotic index of greater than 5% at a concentration of 500 ⁇ , greater than 10% at a concentration of 500 ⁇ , greater than 15% at a concentration of 500 ⁇ , greater than 20% at a concentration of 500 ⁇ , greater than 25% at a concentration of 500 ⁇ , greater than 30% at a concentration of 500 ⁇ , greater than 35% at a concentration of 500 ⁇ , greater than 40% at a concentration of 500 ⁇ .
  • the cancer to be treated may be (A) lung cancer (e.g., lung adenocarcinoma and non small cell lung cancer), (B) pancreatic cancers (e.g., pancreatic carcinoma such as, for example, exocrine pancreatic carcinoma), (C) colon cancers (e.g., colorectal carcinomas, such as, for example, colon adenocarcinoma and colon adenoma), (D) myeloid leukemias (for example, acute myelogenous leukemia (AIVIL) , CIVIL, and CIVIML) , (E) thyroid cancer, (F) myelodysplastic syndrome (IVIDS) , (G) bladder carcinoma, (H) epidermal carcinoma, (I) melanoma, (J) breast cancer, (K) prostate cancer, (L) head and neck cancers (e.g., squamous cell cancer of the head and 5 neck) , (M.) ovarian cancer, (N)
  • Certain compounds may exist in different isomeric (e.g., enantiomers, diastereoisomers, atropisomers) forms.
  • the invention contemplates all such isomers both in pure form and in admixture, including racemic mixtures. Enol forms are also included.
  • All stereoisomers (for example, geometric isomers, optical isomers and the like) of the present compounds including those of the salts, solvates and prodrugs of the compounds as well as the salts and solvates of the prod rugs), such as those which may exist due to asymmetric carbons on various substituents , including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are contemplated within the scope of this invention.
  • Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
  • the chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.
  • the use of the terms “salt”, “solvate” “prodrug” and the like, is intended to equally apply to the salt, solvate and prodrug of enantiomers, stereoisomers, rotamers, tautomers, racemates or prodrugs of the inventive compounds .
  • Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization.
  • Enantiomers can be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers.
  • an appropriate optically active compound e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride
  • some of the compounds of Formula (I) may be atropisomers (e.g., substituted biaryls) and are considered as part of this invention.
  • Enantiomers can also be separated by use of chiral HPLC column.
  • the compounds of formula (III) form salts that are also within the scope of this invention.
  • Reference to a compound of formula (III) herein is understood to include reference to salts thereof, unless otherwise indicated.
  • the term "salt(s)”, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases.
  • zwitterions inner salts may be formed and are included within the term "salt(s)" as used herein.
  • Salts of the compounds of the formula (III) may be formed, for example, by reacting a compound of formula 1.0 with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
  • Exemplary acid addition salts include acetates, adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates , bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates , cyclopentanepropionates, digluconates , dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates , glycerophosphates, hemisulfates , heptanoates, hexanoates, hydrochlorides, hydrobromides , hydroiodides , 2 - hydroxyethanesulfonates, lactates, maleates, methanesulfonates, methyl sulfates, 2 - naphthalenesulfonates, nicotinates, nitrates, oxalates, pam
  • Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts, zinc salts, salts with organic bases (for example, organic amines) such as benzathines, diethylamine, dicyclohexylamines , hydrabamines (formed with N,N- bis (dehydroabietyl) ethylenediamine) , N-methyl-O- glucamines, N-methyl-O-glucamides, t-butyl amines, piperazine, phenylcyclohexylamine, choline, tromethamine , and salts with amino acids such as arginine, lysine and the like.
  • organic bases for example, organic amines
  • organic bases for example, organic amines
  • benzathines diethylamine, dicyclohexylamines , hydrabamines (
  • Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides) , dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates) , long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides) , aralkyl halides (e.g. benzyl and phenethyl bromides), and others.
  • lower alkyl halides e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates
  • prodrugs form prodrugs that are also within the scope of this invention.
  • prodrug denotes a compound that is a drug precursor which, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of formula (III) or a salt and/ or solvate thereof.
  • a compound of formula (III) or a pharmaceutically acceptable salt, hydrate or solvate of the compound, contains a carboxylic acid, alcohol or amine functional group
  • a prodrug can be formed by the replacement of the hydrogen atom in the carboxylic acid, alcohol or amine functional group.
  • the present invention also embraces isotopically- labelled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as 2 H, 3 H, "C, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 35 S, 18 F, 36 CI, and 123 I, respectively.
  • Certain isotopically- labelled compounds of formula (III) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., 3 H) and carbon- 14 (i.e., 14 C) isotopes are particularly preferred for their ease of preparation and detectability . Certain isotopically- labelled compounds of Formula (III) can be useful for medical imaging purposes.
  • those labeled with positron- emitting isotopes like 1:L C or 18 F can " be useful for application in Positron Emission Tomography (PET) and those labeled with gamma ray emitting isotopes like 1 3 I can be useful for application in Single photon emission computed tomography (SPECT) .
  • substitution with heavier isotopes such as deuterium (i.e., 2 H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances.
  • Isotopically labeled compounds of Formula (III) in 15 particular those containing isotopes with longer half lives (Tl/2 >1 day), can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.
  • compound (s) of the invention When used for the treatment or prevention of microbial infection, compound (s) of the invention may be administered alone. Alternatively, the compounds may be administered as a pharmaceutical, veterinarial , agricultural, or industrial formulation which comprises at least one compound. The compound (s) may also be present as suitable salts, including pharmaceutically acceptable salts.
  • the compounds may be used in combination with other known treatments or antimicrobial agents, including antifungal treatments, antibiotics, disinfectants, etc.
  • Combinations of active agents, including compounds of the invention, may be synergistic.
  • salt By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art .
  • suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, asparate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate , citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2 -hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate,
  • alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine , trimethylamine , triethylamine , ethylamine, triethanolamine and the like.
  • Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration.
  • the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound.
  • the compound may also be administered parenterally or intraperitoneally .
  • Dispersions of the compounds according to the Invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of ' sterile injectable solutions or dispersions.
  • the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi .
  • the compound (s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier.
  • the " compound (s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet.
  • the compound (s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • such compositions and preparations may contain at least 1% by weight of active compound.
  • the percentage of the compound(s) of formula (I) and/or (II) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit.
  • the amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the carrier may be an orally administrable carrier.
  • Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.
  • delayed release formulations are also included in the scope of this invention.
  • prodrug is an inactive form of a compound which is transformed in vivo to the active form.
  • Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by including various antibacterial and/or anti-fungal agents.
  • Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol , phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above .
  • Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum gragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules can be coated with shellac, sugar or both.
  • a syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the analogue can be incorporated into sustained-release preparations and formulations.
  • the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
  • compositions according to the invention may be carried out .
  • One skilled in the art would be able, by routine experimentation, to determine effective, nontoxic dosage levels of the compound and/or " composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.
  • an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; suitably, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight.
  • an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight .
  • an effective dosage may be up to about 500mg/m 2 .
  • an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , about 25 to about 350mg/m 2 , about 25 to about 300mg/m 2 , about 25 to about 250mg/m 2 , about 50 to about 250mg/m 2 , and about 75 to about 150mg/m 2 .
  • Figure 1 shows the synthesis and structure of 13 representative endoperoxide compounds of the present invention.
  • Figure la shows the synthetic conditions and yields of 13 representative endoperoxide compounds according to the present invention.
  • Figure lb shows the structures of 13 representative endoperoxide compounds.
  • Figure 2 shows the _i.a_yi-tro_an-t ⁇ cance-r—e-f-f-e-e-fe-s—e-f- representative endoperoxide compounds according to the present invention.
  • the apoptotic index of attached MDA- MB-231 cells treated with 12 individual representative compounds under the indicated concentrations for 0.5h is shown. There were three replicates in each experimental and control group and each experiment was independently performed three times. The concentration 0 indicates vehicle control. Error bars, S.E.M. Comparison was performed against cognate vehicle controls. n.s. represents not significant. * p ⁇ 0.05.
  • Figure 3 also shows the in vitro anti-cancer effects of representative endoperoxide compounds according to the present invention.
  • Figure 3a shows the percentage of Annexin V positive (apoptotic) cells in adhered MDA-MB-231 and MCF10A cells treated with indicated representative endoperoxide compounds under indicated concentrations for 0.5 h. See Figures 8 to 11 (discussed below) for detailed FACS. Experiments were independently performed three times with at least three replicates of each sample. The concentration 0 indicates vehicle control. Error bars: S.E.M. Comparison was performed against cognate vehicle controls. *P ⁇ 0.05, **P ⁇ 0.01; ***P ⁇ 0.001.
  • Figure 3b shows the percentage of Annexin V positive (apoptotic) cells in suspended MDA-MB-231 and MCF10A cells treated with indicated representative endoperoxide compounds under indicated concentrations for 0.5 h. See Figures 8 to 11 (discussed below) for detailed FACS. Experiments were independently performed three times with at least three replicates of each sample. The concentration 0 indicates vehicle control. Error bars: S.E.M. Comparison was performed against cognate vehicle controls. *P ⁇ 0.05, **P ⁇ 0.01; ***P ⁇ 0.001.
  • Figure 4 shows that a representative endoperoxide compound 4 -Me targets Nox4 to cause apoptosis of MDA-MB- 231 cells.
  • Figure 4a shows a flowchart of the strategy to identify cellular interacting partners of -Me. 4 -Me with an alkyne group (40-alkyne) was used as a bait.
  • Figure 4b shows the immunodetection of Nox 4 from immunoprecipitates of 40-alkyne conjugated to biotin- azide.
  • 40-alkyne-azide-biotin was affinity pulldown using streptavidin Sepharose beads.
  • Figure 4c shows a representative sensogram of three independent experiments showing binding profiles between immobilized- full -length (FL) Nox4 and indicated concentrations of 4 -Me. The sensogram was corrected against a reference flow cells with no immobilized proteins .
  • Figure 4d shows a representative sensogram of three independent experiments showing binding profiles between immobilized-FL Nox4 and 500 nM of 4 -Me, 3-Cl, 4- CF3, and H. The sensogram was corrected against a reference flow cells with no immobilized proteins.
  • Figure 4e shows a representative sensogram of three independent experiments showing binding profiles between either immobilized-FL Nox4, DFAD/ NADPH or DHEME with 4 -Me. The sensogram was corrected against a reference flow cells with no immobilized proteins.
  • Figure 4f shows relative mRNA and protein levels of Nox4 in various human cell lines.
  • HaCaT is a non- tumorigenic skin line
  • A-5RT3 is a metastatic skin line
  • MKN28 is a gastric carcinoma line.
  • the mRNA data (means+S. D.) are from two independent qPCR experiments performed in triplicates.
  • the ribosomal protein 18S gene serves as a reference housekeeping gene and b- tubulin serves as a loading and transfer control in immunoblot . ***P ⁇ 0.001.
  • Figure 4g shows percentage of suspension- induced apoptotic (Annexin V positive) cells treated with indicated concentrations of 4 -Me for 0.5 h as analyzed by FACS (5000 events) . Vehicle- treated cells served as control. The sum of Annexin V + /PI " (early apoptosis) and Annexin V + /PI + (late apoptosis) cells were considered apoptotic. Results are representative of three independent experiments. See Figures 13D and 14A (discussed below) for FACS ' data. Error bars: S.E.M. Comparison was performed against cognate vehicle controls, n.s. indicates not significant; ***P ⁇ 0.001. [0136] Figure 5 shows that endoperoxide compounds of the present invention regulate ROS levels in cancer cells.
  • Figure 5a shows general ROS levels (expressed as fold change) measured by CM-H2DCFDA in MCF10A.
  • the cells were suspended in the presence of 4 representative compounds under indicated concentrations for 0.5h. Each experiment was independently performed three times with triplicates. The concentration 0 indicates vehicle control and its ROS value was set as 1. Error bars, s.e.m. Comparison was performed against cognate vehicle controls, n.s. represents not significant. *p ⁇ 0.05.
  • Figure 5b shows general ROS levels (expressed as fold change) measured by CM-H2DCFDA in MDA-MB-231.
  • the cells were suspended in the presence of 4 representative compounds under indicated concentrations for 0.5h. Each experiment was independently performed three times with triplicates. The concentration 0 indicates vehicle control and its ROS value was set as 1. Error bars, s.e.m. Comparison was performed against cognate vehicle controls, n.s. represents not significant. *p ⁇ 0.05.
  • Figure 6 also shows that endoperoxide compounds of the present invention regulate ROS levels in cancer cells.
  • concentration 0 indicates vehicle control and its ROS value was set as 1.
  • Figure 6a shows general ROS levels (expressed as fold change) in MCF10A and MDA-MB-231 cells suspended in the presence of the four representative endoperoxide compounds at indicated concentrations for 0.5 h as measured by CM-H2DCFDA.
  • Figure 6b shows H 2 0 2 levels in MDA-MB-231 cells treated like in Figure 6a as determined by Amplex Red and 2-methyl-6- (4 -methoxyphenyl) -3, 7-dihydroimidazo [1 , 2-a] pyrazin-3-one hydrochloride (MCLA) assays, respectively.
  • MCLA pyrazin-3-one hydrochloride
  • Figure 6c shows 0 2 ⁇ levels in MDA-MB-231 cells treated like in Figure 6a as determined by Amplex Red and 2 -methyl- 6- (4 -methoxyphenyl) -3 , 7-dihydroimidazo [1 , 2-a] pyrazin-3-one hydrochloride (MCLA) assays, respectively.
  • MCLA pyrazin-3-one hydrochloride
  • Figure 6d shows the representative EPR spectra of DEPMPO- superoxide spin adduct from MDA-MB-231 cells suspended in the presence of -Me.
  • the EPR spectra represent the averaged signals of 10 scans.
  • Figure 6e shows the EPR signal intensity (expressed as percentage of control) at 3480 G from MDA- MB-231 cells in Figure 6d. Tiron-treated measurements serve as negative signal controls.
  • Figure 6f shows arbitrary relative 0 2 " :H 2 0 2 ratios calculated based on H 2 0 2 and 0 2 ⁇ values from Figures 6b and c .
  • Figure 7 shows that representative endoperoxide compounds of the present invention upregulate OH levels in cancer cells.
  • concentration 0 indicates vehicle control and its ⁇ 0 ⁇ value was set as 1.
  • Figure 7a shows levels of OH (expressed as fold change) in MDA-MB-231 cells suspended in the presence of the four representative endoperoxides at indicated concentrations for 0.5 h measured using TA.
  • Figure 7b shows representative EPR spectra of BMPO-OH spin adduct from MDA-MB- 231 suspended in the presence 4 -Me.
  • spectrometer parameters were: modulation frequency, 100 kHz; scan range, 100 G; field set, 350 G; microwave power, 10 m ; modulation amplitude, 1 G; time constant, 5 ms; and scan time 5.14 s.
  • the EPR spectra represent the averaged signals of 10 scans. Dimethylthiourea was used as negative controls for •OH measurements.
  • Figure 7c shows the EPR signal intensity expressed as percentage of control at 3410 G from MDA-MB-231 cells in Figure 7b.
  • Figure 8 shows endoperoxides of the present invention selectively causing cancer cells to undergo apoptosis.
  • Adherent MDA-MB-231 cells induced by treatment with indicated amino endoperoxide compounds under indicated concentrations for 0.5 h.
  • Figure 9 shows endoperoxides of the present invention selectively causing cancer cells to undergo apoptosis.
  • Adherent and suspension MCF10A cells induced by treatment with indicated amino endoperoxide compounds under 500 ⁇ concentrations for 0.5 h;
  • Figure 10 shows endoperoxides of the present invention selectively causing cancer cells to undergo apoptosis.
  • Figure 11 shows endoperoxides of the present invention selectively causing cancer cells to undergo apoptosis.
  • Figure 12 shows the percentage of Annexin V positive (apoptotic) cells calculated based on Figure 11. Error bars: S.E.M. ***p ⁇ 0.001. Vehicle-treated cells served as cognate controls for comparison. The sum of Annexin V + /PI " (early apoptosis) and Annexin V + /PI + (late apoptosis) cells were considered apoptotic. Values (bold) denote apoptotic cells (%) . Results are representative of three independent experiments. All experiments were performed three or four times with consistent results.
  • Figure 13 shows that a representative compound according to the present invention, 4—Me, targets Nox4 to cause cell death.
  • Figure 13a shows the amino acid sequence of full length human Nox4 protein. Peptide sequence underlined by various blue color lines shows LC-MS/MS results, indicating Nox4 as a potential target of 4 -Me.
  • Figure 13b shows that cDNA encoding for the various truncated Nox4 were constructed using site mutagenesis and subcloning into mammalian expression vector. Recombinant proteins were purified via nickel - affinity chromatography from lysate of transfeeted HEK293T cells. Representative Coomassie-stained gel and immunblot using anti-histidine tag antibody were shown. Lysate of untransfected (U) and transfected (T) cells served as negative and positive controls, respectively. Solutions with increasing concentrations of imidazole (1 st , 2 nd and 3rd ) were used to wash and eluted the bound proteins.
  • Figure 13c shows the relative mRNA and protein levels of Nox4 in MDA-MB-231 control and MDA-MB-231 Nox4 siRNA cells.
  • the mRNA data (means ⁇ SD) are from two independent qPCR experiments performed in triplicates.
  • the ribosomal protein 18S gene serves as a reference gene in qPCR and ⁇ - tubulin serves as a loading and transfer control in immunoblot.
  • Figure 13d shows FACS graphs on Annexin V and propidium iodide (Pi) staining assays done on various cells induced by treatment with 4 -Me under indicated concentrations for 0.5 h as analysed by FACS (5000 events) . Vehicle- treated cells served as cognate controls for comparison.
  • Annexin V + /PI * (early apoptosis) and Annexin V + /PI + (late apoptosis) cells were considered apoptotic. Values (bold) denote apoptotic cells (%) . Results are representative of three independent experiments. All experiments were performed three or four times with consistent results.
  • Figure 13e shows a representative sensogram of three independent experiments showing binding profile between immobilized-C0X2 and 500 nM of 4-Me.
  • Anti-COX2 antibody served as positive control. Sensograms were corrected against a reference flow . cells with no immobilised proteins.
  • Figure 14 shows that a representative compound according to the present invention, 4-Me, causes Nox4 overexpressed HEK 293T cells to undergo apoptosis.
  • Figure 14a shows percentage of Annexin V positive stained .A- 5RT3 that of 0.5 h treatment of 500 ⁇ 4-Me as analysed by FACS (5000 events) . Vehicle-treated cells served as control. The sum of Annexin V + /PI " (early apoptosis) and Annexin V + /PI + (late apoptosis) cells were considered apoptotic. Values (bold) denote apoptotic cells (%) . Results are representative of three independent experiments.
  • Figure 14b shows the immunoblot of indicated antibodies on total cell lysates of control and Nox4- transfected HEK 293T cells. Immunoblot data are from three independent experiments performed in duplicate. B- tubulin serves as a loading and transfer control.
  • Figure 14c shows percentage of Annexin V positive stained Nox4- transfected HE.K 293T cells (C) that of 0.5 h treatment of 500 ⁇ 4 -Me as analysed by FACS (5000 events). Vehicle- treated cells served as control. The sum of Annexin V + /PI " (early apoptosis) and Annexin V + /PI +
  • Figure 15 shows that a representative compound according to the present invention, 4 -Me, induces apoptosis and DNA damage on breast cancer cells but has no effect on their cell cycle.
  • Figure 15a shows Immunoblots of DNA damage and apoptotic markers (cleaved PARP and cleaved caspase-3, respectively) on cell lysate of vehicle and indicated concentrations of 4-Me treated MDA-MB-231 cells. Immunoblot data are from three independent experiments performed in duplicate, ⁇ - tubulin serves as a loading and transfer control.
  • Figure 15b shows FACS graphs on cell cycle analysis using PI staining on suspension MDA-MB-231 cells under various treatments for 0.5 h. Percentage of various cell cycle phases in each sample conditions are shown. No significant change was observed between the various treatments . Results are representative of three independent experiments with triplicates.
  • Figure 15c shows percentage of Annexin V positive (apoptotic) cells calculated based on (A). Error bars: s.e.m. ***p ⁇ 0.001.
  • Figure 15d shows FACS graphs on Annexin V and propidium iodide (PI) staining assays done on suspension MDA-MB-231 cells induced by treatment either with 4-Me alone or in a combination of 4-Me and NAC under indicated concentrations for 0.5 h as analyzed by FACS (5000 events) .
  • the sum of Annexin V + /PI " (early apoptosis) and Annexin V + /PI + (late apoptosis) cells were considered apoptotic. Values (bold) denote apoptotic cells (%) . Results are representative of three independent experiments performed three or four times with consistent results.
  • Figure 16 shows the suppression of Nox4 abolishes the -Me effect on cellular ROS modulation.
  • Figure .16a shows representative FACS-derived histograms showing general ROS levels in suspension MDA- MB-231 control and MDA-MB-231 Nox4 siR A cells in the presence of indicated concentrations -Me for 0.5 h as measured by CM-H2DCFDA.
  • Figure 16b shows relative fold change in general ROS level (mean DCF fluorescence intensities) as calculated from result of Figure 16a.
  • Figure 16c shows H 2 0 2 levels in suspension MDA-MB- 231 control and MDA-MB- 231 Nox4 siRNA cells in the presence of 4 -Me under indicated concentrations for 0.5 h as determined by Amplex Red and MCLA assays , respectively.
  • Figure 16d shows 0 2 levels in suspension MDA-MB-231 control and MDA-MB- 231 Nox4 siRNA cells in the presence of 4 -Me under indicated concentrations for 0.5 h as determined by Amplex Red and MCLA assays, respectively.
  • Figure 16e shows arbitrary relative 0 2" : H 2 0 2 ratios calculated based on H 2 0 2 and 02 - values from figures 16b and c.
  • Figure 16f shows levels of ⁇ 0 ⁇ (expressed as fold change) in suspension MDA-MB-231 control and MDA-MB-231 Nox4 siRNA cells in the presence of 4 -Me under indicated concentrations for 0.5 h measured using terephthalic acid.
  • the ⁇ 0 ⁇ quencher dimethylthiourea (DMTU) was used as the negative control .
  • DMTU dimethylthiourea
  • Each experiment was independently performed two to three times with triplicates.
  • the value of cognate vehicle controls was set as 1. Error bars, s.e.m. n.s. represents not significant, * p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 17 shows that high dosages of 4 -Me shows little drug-related toxicity on the liver, kidney and heart .
  • Figure 17a shows Haematoxylin and eosin staining of liver, kidney and heart sections from mice treated with 51.5 pmol/kg and 103 ⁇ /kg of 4-Me-treated mice.
  • Figure 17b shows immunblot analysis of caspase 3 in indicated tissues.
  • Antibodies against caspase 3 detected both the full-length and cleaved forms.
  • Figure 18 shows that 4 -Me treatment suppresses MDA-MB-231 orthotopic tumor growth.
  • Figure 18a shows orthotopic tumor volumes induced in nude mice by MDA-MB-231 cells.
  • Cells (2 x 10 "6 ) were inoculated into the fourth mammary fat pad of each mouse .
  • Figure 18b shows representative pictures (top panels) of vehicle control and 4-Me-treated nude mice (day 36) in Figure la. Higher magnifications of tumor pictures are shown in the smaller rectangles. Bottom panels show enlarged (indicating metastasis) and normal sized lymph nodes in vehicle and 4-Me-treated mice, respectively .
  • Figure 18c shows Haematoxylin and eosin and immunofluorescence staining of tumor sections from vehicle- and 4-Me-treated nude mice. Proliferating (Ki67) and apoptotic (TUNEL) cells were identified using the indicated antibodies or assay. Sections were counterstained with DAPI (blue) . Images were acquired in one z-plane using a LSM710 confocal laser scanning microscope and ZEN 2008 software (Carl Zeiss, Oberkochen, Germany) . Scale bars represent 100 mm. All experiments were performed using tumor biopsies harvested , from mice described in Figure 18a at day 36.
  • Figure 18d shows immunoblots of proliferation (cyclin Dl and PCNA) , and apoptosis (cleaved PARP and cleaved caspase-3) markers on tumor biopsies of vehicle and 4 -Me- treated nude mice.
  • Immunoblot data are from three independent experiments performed in duplicate, b- Tubulin serves as a loading and transfer control. All experiments were performed using tumor biopsies harvested from mice described in Figure 18a at day 36.
  • Endoperoxides selectively cause cancer cell apoptosis in vitro.
  • Amino endoperoxides targets Nox4 to cause cancer cell death.
  • cancer cell lines expressing elevated level of Nox4 would be more susceptible to the anticancer action of these endoperoxides when compared with low Nox4 -expressing cell lines.
  • Real-time qPCR and immunoblot analyses revealed that non-tumorigenie human keratinocyte HaCaT and metastatic squamous cell carcinoma A-5RT3 express very low (negligible) levels of Nox4 mRNA (Figure 4f) .
  • 4-Me did not induce apoptosis in A-5RT3 ( Figure 4g and Figure 14a) .
  • Amino endoperoxides modulate ROS levels in cancer cells.
  • Nox4 -dependent ROS production is important for the survival of many cancer types .
  • Nox4 has been reported to predominantly produce H 2 0 2 more than 0 2 ⁇ .
  • the 4 representative endoperoxides, 4- Me, 4-CF3, 3 -CI and H mediated their anti-cancer effects by regulating the ROS levels.
  • Treatment of normal breast cells MCF-IOA with the 4 representative endoperoxides showed little effect on the ROS level (Figure 6a).
  • 0.5 h treatment of breast cancer cells MDA-MB-231 significantly increased the ROS levels in a dose-dependent manner ( Figure 6a) .
  • H 2 0 2 we measured the level of H 2 0 2 in cancer cells in the presence of a specific catalase inhibitor, 3-amino-l, 2, 4,- triazole.
  • the H 2 0 2 levels were greatly increased in all of the 4 representative endoperoxide-treated MDA-MB-231 cells ( ⁇ 3.5-fold increase with 500 ⁇ of 4-Me; Figure 6b) .
  • Redox-based anti -cancer action of amino endoperoxide (4- Me) impairs orthotopic tumor growth.
  • mice were well tolerated without obvious signs of drug-related toxicity throughout the course of this study, even at 16-32 mg/kg dosages (equivalent to 51.5 x 10 "3 ⁇ /kg) as evidenced by undetectable necrotic tissue damage and expression of active cleaved caspase-3 (Figure 17) .
  • immunofluoresence ( Figure 18c) and immunoblot (Figure 18d) analyses of the tumor biopsies indicated significantly reduced cell proliferation and enhanced cell apoptosis in 4-Me treated mice tumors as compared to vehicle treated mice tumors. Together, these observations clearly support the anti -metastatic cancer effect of amino endoperoxides.
  • MDA-MB-231 human breast adenocarcinoma
  • HEK 293T human embryonic kidney cells
  • HaCaT human keratinocyte
  • A-5RT3 human metastatic squamous cell carcinoma
  • MCF10A cells normal human mammary epithelial cells
  • DMEM/F12 fetal calf serum
  • hydrocortisone 0.5 g/ml
  • cholera toxin 100 ng/ml
  • insulin 10 ⁇ g/ml
  • EGF 20 ng/ml
  • MKN28 human gastric carcinoma cells were maintained in RPMI1640 supplemented 10% FBS. All cells were cultured at a 37 °C, 5% C0 2 and 75% humidified incubator. All the amino endoperoxides and derivatives treatment experiments were performed in cognate serum- free medium.
  • Annexin V/propidium iodide (PI) staining coupled with flow cytometry (BD Accuri C6) were employed.
  • Adhered cells were exposed to each of the amino endoperoxides and derivatives at different concentrations for 30 min, harvested by trypsinization, washed with PBS and subjected to Annexin V/PI staining for FACS analysis.
  • PI polypropidium iodide
  • FACS analysis for anoikis assay, cells were subjected to suspension by seeding cells onto poly-HEMA coated plate and treated with endoperoxides as described above before FACS analysis. All experiments were performed in triplicates. Vehicle controls were performed accordingly.
  • Annexin V + /PI and Annexin V + /PI + cells were considered apoptotic.
  • ROS Reactive oxygen species
  • ROS measurement was performed as previously described with modifications. Briefly, cells were incubated with 10 ⁇ CM-H2DCFDA (Life Technologies) for 30 min at 37 °C. Next, cells were treated with indicated concentrations of endoperoxides and derivatives for 30min as described above for FACS analysis. Vehicle controls were performed accordingly. Data were analyzed with Flowjo 7.6. All experiments were performed in triplicates. Measurement of 0 2 ⁇ : H 2 0 2 and ⁇ levels.
  • EPR measurement ⁇ and 0 2 ⁇ were performed as previously described with modifications. Briefly, direct trapping 0 2 ⁇ and -OH in aqueous media was performed using the spin trap DEPMPO and BMPO, which form relatively stable 0 2 ⁇ and ⁇ adducts, respectively. EPR spectra were recorded at room temperature with a Bruker D-200 ER spectrometer, operating at X-band with a TM 110 cavity with a quartz flat cell.
  • the EPR parameters were set at 100 KHz, X-band microwave frequency, 9.5 GHz; microwave power, 20 m ; modulation amplitude, 1 G; time constant, 160 s ; scan time, 50 s ; and receiver gain, 5 x 10 s .
  • EPR signal amplitude at 3480 G represent the pure lines corresponding to the 0 2 ⁇ adduct.
  • spectrometer parameters were: modulation frequency, 100 KHz ; scan range, 100 G; field set, 350 G; microwave power, 10 mW; modulation amplitude, 1 G; time constant, 5 ms; scan time 5.14s.
  • EPR signal amplitude at 3410 G represent the pure lines corresponding to the ⁇ adduct.
  • Tiron and dimethylthiourea (DMTU) were used as negative controls for 0 2 ⁇ and ⁇ measurements, respectively.
  • the EPR spectra represent the averaged signals of 10 scans. All experiments were performed in triplicates.
  • Mice were sacrificed at the end of the experiment (day 36) , and tumors were harvested for further analyses .
  • 5x (16 mg/kg) and lOx (32 mg/kg) more -Me were used.
  • the liver, heart and kidney were harvested at end of experiment and analysed for necrosis and cellular apoptosis. All animals were maintained in pathogen- free conditions.
  • Apoptotic tumor cells were detected using the TUNEL assay according to the manufacturer' s protocol (Roche) .
  • the section was pretreated with DNase I.
  • the slides were mounted with antifade reagent (ProLong Gold; Invitrogen) with DAPI . Images were acquired in one zplane using a LSM710 confocal laser scanning microscope and ZEN 2008 software (Carl Zeiss) . Transfection in HEK 293T cells
  • Mammalian expression plasmids containing the cDNA sequences encoding human fulllength Nox4 ( T) , the heme (AFAD/NADPH) or NADPH ( ⁇ ) region with Nterminal histidine tag were transfected into HEK 293T cells using FuGENE ® HD (Promega) following manufacturer' s protocol. An empty plasmid vector was used as control. Transfected cells were lysed in lysis buffer (300 mM NaCl, 50 mM Tris.HCl, pH8.0, 0.1% Triton-X) .
  • Recombinant WT, AFAD/NADPH and ⁇ Nox4 proteins were affinity purified using Ni-NTA and verified using anti-His antibody (Santa Cruz Biotechnology) . Purified recombinant proteins were dialyzed against 100 mM NaCl, 20 mM HEPES, pH 8.0 before surface plasmon resonance analysis. Nox -expressing HEK293 cells were also used for amino endoperoxide treatment assay.
  • the primer sequences were: Nox4 sense (5' GCT GAC GTT GCA TGT TTC AG 3' ); Nox4 antisense (5' CGGGAG GGT GGG TAT CTA A 3' ) ; 18S sense (5' GTA ACC CGT TGA ACC CCA TT 3' ) ; 18S antisense (5' CCA TCC AAT CGG TAG TAG CG 3' ) ⁇
  • the method may be straightforward and can be made from readily available building blocks.
  • the method may be conducted in a concise one-pot manner.
  • the method may provide endoperoxide compounds which are extremely stable and possess distinct biological activities useful in redox-based apoptosis anti-cancer therapeutic strategies.
  • the endoperoxide compounds may reduce the 0 2 ⁇ : H 2 0 2 ratio in tumours, thereby inducing redox-based apoptosis.
  • the endoperoxide compounds may have a selective cytotoxic effect, i.e. the compounds of the present invention have a cytotoxic effect on cancer cells but little cytotoxic effect on normal cells.

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Abstract

La présente invention concerne généralement des procédés et des méthodes pour la synthèse de composés endoperoxydes. Plus spécifiquement, l'invention concerne une méthode pour préparer des composés endoperoxydes à partir d'une arylimine de départ de formule (I) en présence d'un catalyseur métallique et d'oxygène. La présente invention concerne également des composés endoperoxydes de formule (III) et leurs sels pharmaceutiquement acceptables, utiles en thérapie. Tous les substituants sont définis ici. Des méthodes pour le traitement du cancer utilisant les composés de formule (III) sont également décrites.
PCT/SG2013/000293 2012-07-13 2013-07-12 Endoperoxydes, leur synthèse et leurs utilisations dans le traitement de maladies néoplasiques telles que le cancer WO2014011120A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE RN accession no. 276042-20-6 *
ZHANG, L. ET AL.: "Copper-Catalyzed Benzylic C-H Oxygenation under an Oxygen Atmosphere via N-H Imines as an Intramolecular Directing Group", ORGANIC LETTERS, vol. 13, no. 7, 2011, pages 1622 - 1625 *

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