WO2009004496A2 - Bisanthrapyrazoles comme agents anticancer - Google Patents

Bisanthrapyrazoles comme agents anticancer Download PDF

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Publication number
WO2009004496A2
WO2009004496A2 PCT/IB2008/002784 IB2008002784W WO2009004496A2 WO 2009004496 A2 WO2009004496 A2 WO 2009004496A2 IB 2008002784 W IB2008002784 W IB 2008002784W WO 2009004496 A2 WO2009004496 A2 WO 2009004496A2
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group
compound
dna
linker
alkyl
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PCT/IB2008/002784
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WO2009004496A3 (fr
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Brian B. Hasinoff
Lynn J. Guziec
Frank Guziec
Hong Liang
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University Of Manitoba
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/54—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings condensed with carbocyclic rings or ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates generally to the field of cancer treatment. More particularly, it concerns novel compounds useful for chemotherapy, methods of synthesizing these compounds and methods of treatment employing these compounds.
  • novel compounds are bisanthrapyrazoles that are thought to exert their anticancer activity, at least in part, by behaving as DNA intercalators and topoisomerase Il ⁇ inhibitors.
  • the present invention also relates to molecular modeling methods for modeling compounds with DNA and estimating their DNA binding strength.
  • a variety of anti-cancer agents interfere with cancerous activity at the level of
  • chemotherapeutics for example, act as DNA intercalators, while others act as inhibitors or poisons of topoisomerase, an enzyme that acts on the topology of DNA. Certain drugs may act via one or more of these methods.
  • DNA intercalating agents are one of the most widely used classes of cancer chemotherapeutic agents currently employed for the management of human cancers. These agents, which are typically polycyclic, aromatic and planar, stack between base pairs of DNA and induce local structural changes, such as the unwinding of the double helix and lengthening of the DNA strand. These structure modifications lead to functional changes, often the inhibition of transcription and replication processes.
  • DNA intercalators are typically mutagens and are often carcinogenic (e.g., benzopyrene diol epoxide, bisbenzimide, aflatoxin and ethidium bromide).
  • Bisintercalating agents are also known, wherein two parts of the same molecule intercalate DNA. See, e.g., Wakelin, 1986; Phillips et al, 1992; Skorobogaty et al, 1988; and U.S. Patent No. 4,112,217.
  • Anthrapyrazoles are a family of compounds that include certain compounds thought to behave as intercalators (Begleiter et al, 2006; Liang et al, 2006). Because the anthrapyrazoles do not contain a quinone group as do the anthracyclines, they are unable to be reductively activated like doxorubicin and other anthracycline: thus, it is thought that this family of compounds may exhibit less cardiotoxicity, and they were designed with this benefit in mind (Begleiter et al, 2006; Leteurtre et al, 1994; Gogas and Mansi, 1995).
  • anthrapyrazoles examples include teloxantrone, losoxantrone and piroxantrone (Begleiter et al, 2006; Hartley et al, 1998; Showalter et al, 1987). Structure-activity relationship studies have been performed for anthrapyrazole cytotoxicity and DNA binding as well as analyses of their effects on different types of cancer, including breast, prostate, head and neck cancer and leukemia (Gogas and Mansi, 1995; Begleiter et al, 2006; Liang et al, 2006; Ingle et al, 1994; Talbot et al, 1991; Huan et al, 2000). Aza-bioisosteres of anthrapyrazoles have also been studied in clinical trials (Sissi and Palumbo, 2004; Sissi et al, 2004; see also U.S. Patent No. 6,747,039).
  • Piroxantrone shown below, is an anthrapyrazole with broad antitumor activity in vitro (Berg et al, 1993). This synthetic compound, which has undergone clinical trials for anti-tumor activity, intercalates into DNA and inhibits topoisomerase II, thereby inhibiting DNA replication and repair (Ingle et al, 1994). Although less cardiotoxic than doxorubicin, this agent exhibits a narrow spectrum of antineoplastic activity. Losoxantrone, also shown below, is an anti-cancer anthrapyrazole that has also undergone clinical trials for breast cancer treatment (Joshi et al, 2001; Talbot et al, 1991). This synthetic compound is a topoisomerase II inhibitor (Leteurtre et al, 1994).
  • anthrapyrazoles While certain anthrapyrazoles have shown less cardiotoxicity than the anthracyclines, piroxantrone has been shown to produce cardiotoxicity at high cumulative doses (Gogas and Mansi, 1995). Further, certain anthrapyrazoles have been shown to cause myelosuppression (Gogas and Mansi, 1995). Thus, preparation of other anthrapyrazole derivatives may offer improved chemotherapeutic effects over known anthrapyrazoles as well as other DNA intercalating compounds.
  • the present invention provides for novel bisanthrapyrazoles as anti-cancer agents.
  • these bisanthrapyrazoles are based on aspects of the structures of losoxantrone and piraxantrone.
  • the present inventors have found that these bisanthrapyrazoles are likely DNA intercalators, some of which surprisingly bind to DNA more tightly than doxorubicin.
  • Certain bisanthrapyrazoles of the present invention are topoisomerase Il ⁇ inhibitors, and have shown in vitro cancer cell growth inhibitory effects.
  • the present invention also involves novel compounds that have utility as anti-tumor and/or chemotherapeutic drugs, methods of synthesizing these compounds and methods of using these compounds to treat patients with cancer.
  • Non-limiting types of cancer that may be treated using the methods and compounds of the present invention include, for example, breast, prostate, head and neck cancer and leukemia.
  • the present invention generally contemplates a compound comprising a first anthrapyrazole operatively linked to a second anthrapyrazole.
  • operatively linked it is meant that the two anthrapyrazoles are joined via any chemical means known in the art, such as covalent, ionic, or dative means, for example.
  • two anthrapyrazoles are joined via an operative linkage comprising, or consisting of, one or more covalent bonds.
  • the first anthrapyrazole is operatively linked to a second anthrapyrazole via a linker.
  • the linker may join the two anthrapyrazoles at any two or more atoms of either anthrapyrazole.
  • a linker may link each Z atom of the first and second anthrapyrazoles to form a bisanthrapyrazole having the core structure of formula (B):
  • each of ring atoms D, E, F, G, H, I, J, K, L, Y, Z, D B , E B , F B , G B , H B , I B , J B , K B , L B , Y B and Z B are each independently carbon or nitrogen; the bond between L and Y and the bond between L B and Y B may each independently be a double or single bond; and R and R B are each independently oxygen or sulfur; and wherein the first and second anthrapyrazoles have the same or different core skeleton.
  • a linker linking the two Z groups (Z and Z B ) comprises at least 7 linear atoms (that is, 7 atoms or higher, including subranges such as 7-10, 7-15, 7-20 linear atoms, etc.), which may be substituted or unsubstituted.
  • the linear atoms may each be any atom known to those of skill in the art.
  • the linear atoms are selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus.
  • the first and second anthrapyrazoles are each independently selected from the compound of formula (I):
  • R 1 -R 7 are each independently hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more Of Y 1 -R 1 , R 1 - R 2 , R 2 -R 3 , R3-R4, R5-R5, R5-R7 or R 7 -Z 1 together forms a cyclic group, or any combination of one or more of these groups; R 8 is either oxygen or sulfur; R 9 is either not present or is a linker; and R 1O is either hydrogen, alkyl, a nucleophile or a leaving group; D, E, F, G, H, I, J, K and L are each independently carbon, -CH or nitrogen; Y 1 is selected from the group consist
  • R 1 -R 1 O may each individually comprise any functional group, as well as H and/or alkyl.
  • R 1 is either H, alkyl, substituted alkyl, or OH.
  • R 4 is hydrogen, halogen, hydroxyl, or substituted alkyl.
  • the substituted alkyl may be, for example, an aminoalkyl.
  • the aminoalkyl group may be, for example, -NH(CH 2 ) 2 N(CH 3 ) 2 .
  • the substituted alkyl may be a haloalkyl.
  • R 5 is hydrogen, halogen, or substituted alkyl.
  • the substituted alkyl may be, for example, aminoalkyl, wherein the aminoalkyl group may be, for example, -NH(CH 2 ) 2 N(CH 3 ) 2 or - NH(CH 2 )3NH 2 .
  • the substituted alkyl may be hydroxyalkylamino, such as - NH(CH 2 ) 3 NH(CH 2 ) 2 OH.
  • the substituted alkyl may be a haloalkyl.
  • R 2 , R 3 , R 6 and R 7 are each hydrogen.
  • R 8 may be oxygen, in certain embodiments.
  • R 9 is not present and R 1O is hydroxyl.
  • R 9 may be, in certain embodiments, -N(R 11 )(ClH ⁇ -, wherein R 11 may be any functional group, including H and alkyl.
  • R 11 is selected from the group consisting of H, alkyl and substituted alkyl.
  • R 9 is -N(R ⁇ )(CH 2 )2-, R 1 , R 2 , R3, Rs, Re and R 7 is H, R 4 is Cl, R 8 is O, R 10 is hydroxyl and R 11 is CH 3 .
  • R 1O is hydroxyl, azido or amino.
  • the number of linear atoms linking the Z 1 groups of the first and second anthrapyrazoles is 7 or higher (e.g., 7 or more, including subranges such as 7-10, 7-15, 7-20 linear atoms, etc.).
  • the linear atoms may each be any atom known to those of skill in the art.
  • the linear atoms are selected from the group consisting of carbon, nitrogen, oxygen, sulfur and phosphorus.
  • R9 comprises an alkyl group, a substituted alkyl group, a heterocyclyl group, an oxo group, a sulfo group, a thioether group, an aryl group, an amide group, a sulfonamide group, a carbonyl group, a thiocarbonyl group, a secondary amine group, a tertiary amine group, an ester group, a thioester group, a sulfonyl group, or any combination of one or more of these groups.
  • the linker may comprise an alkyl group, an ester, and/or amide, in certain embodiments.
  • Other non- limiting examples of linkers are described herein, and are also known to those of skill in the art.
  • R 1 -R 7 and R ⁇ -Ris are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more OfY 1 -R 1 , R 1 -R 2 , R 2 -R3, R3-R4, R5-R6, R 6 -R 7 , R 7 -Z 1 , Y 2 -R 12 , R 12 -R 13 , Ri 3 -Ri 4 , Ri 4 -Ri 5 , Rie-R ⁇ , R ⁇ -Ri ⁇ or Ri 8 -Z 2 together forms a cyclic group, or any combination of one or more of these groups; Rg and R19 are each independently oxygen or sulfur; D, E, F, F
  • Ri-R 7 and Ri 2 -Ri 8 may each, in certain embodiments, be independently H or halogen.
  • the halogen is chlorine.
  • R 8 and R 19 are each oxygen.
  • D, E, F, G, H, I, J, K, L, M, P, Q, R, T, U, V, W and X are each carbon.
  • Yi, Y 2 , Zi and Z 2 are each carbon.
  • the L-Yi bond and the X-Y 2 bond are each double bonds.
  • the linker of R 2 o may be that of any known to those of skill in the art.
  • the linker is selected from the group consisting of an alkyl group, a substituted alkyl group, a heterocyclyl group, an oxo group, a sulfo group, a thioether group, an aryl group, an amide group, a sulfonamide group, a carbonyl group, a thiocarbonyl group, a secondary amine group, a tertiary amine group, an ester group, a thioester group, a sulfonyl group, or any combination of one or more of these groups.
  • the linker may comprise an alkyl, at least one ester, and/or at least one amide.
  • the compound of formula (II) may be further defined as a prodrug, as defined herein.
  • the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as: In particular embodiments, the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as: In particular embodiments, the compound of formula (II) is further defined as:
  • the compound of formula (II) is further defined as:
  • Another aspect of the present invention contemplates a method of preparing a bisanthrapyrazole comprising preparing a first anthrapyrazole, preparing a second anthrapyrazole, and conjugating the first anthrapyrazole to the second anthrapyrazole either directly or through a linker.
  • the linker may join the two anthrapyrazoles at any two or more atoms of either anthrapyrazole.
  • the linker may be any type of linker described herein and as known to those of skill in the art.
  • the joined atoms of the first and second anthrapyrazoles may be any two atoms as described herein (e.g., the Z atoms of the compounds of core skeleton formula (A), or the Z 1 atoms of the anthrapyrazoles of the compound of formula (I)).
  • one of the two anthrapyrazoles comprises a nucleophile and the other comprises a leaving group, and the two anthrapyrazoles are linked together via reaction of the nucleophile and the atom to which the leaving group is attached.
  • Also contemplated by the present invention is a method of preparing a compound of formula (II) comprising: preparing a first anthrapyrazole of formula (I):
  • R 1 -R 7 are each independently hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more Of Y 1 -R 1 , R 1 - R 2 , R 2 -R 3 , R3-R4, R5-R6, R6-R7 or R 7 -Z 1 together forms a cyclic group, or any combination of one or more of these groups; Rs is either oxygen or sulfur; R 9 is either not present or is a linker; and R 1O is either hydrogen, alkyl, a nucleophile or a leaving group; D, E, F, G, H, I, J, K and L are each independently carbon, -CH or nitrogen; Y 1 is selected from the group consisting
  • R 12 -R 18 are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more of Y 2 -R 12 , R 12 -R 13 , R13-R14, R14-R15, R16-R17, R17-R18 or R 18 -Z 2 together forms a cyclic group, or any combination of one or more of these groups;
  • R 49 is either oxygen or sulfur;
  • R 21 is either not present or is a linker; and
  • R 22 is either H, alkyl, a nucleophile or a leaving group;
  • M, P, Q, R, T, U V, W and X are each independently selected from the group consisting of carbon
  • the conjugation may take place via any method known to those of skill in the art, and may take place between two or more atoms on either anthrapyrazole.
  • the conjugation step comprises conjugating said first anthrapyrazole to said second anthrapyrazole through a linker that joins the Z 1 and Z 2 positions of each anthrapyrazole.
  • the linker comprises an alkyl group, a substituted alkyl group, a heterocyclyl group, an oxo group, a sulfo group, a thioether group, an aryl group, an amide group, a sulfonamide group, a carbonyl group, a thiocarbonyl group, a secondary amine group, a tertiary amine group, an ester group, a thioester group, a sulfonyl group, or any combination of one or more of these groups.
  • the linker may comprise an alkyl group, at least one ester, and/or at least one amide.
  • R 1 O and R 22 are selected from the group consisting of a nucleophile and a leaving group, wherein R 1 O ⁇ R 22 .
  • Another general aspect of the present invention contemplates inhibiting the catalytic decatenation activity of topoisomerase Il ⁇ , comprising administering to a cell an effective amount of a bisanthrapyrazole.
  • Another general aspect of the present invention contemplates a method of treating a patient with cancer, comprising administering to the patient a therapeutically effective amount of a bisanthrapyrazole.
  • Yet another general aspect of the present invention contemplates a therapeutic kit comprising, in suitable container means, a pharmaceutically acceptable composition comprising a bisanthrapyrazole.
  • the present invention pertains also to molecular modeling methods that may show how a compound binds to DNA as well as provide estimates regarding the binding strengths of various compounds. These methods may be used with any compound known in the art, including anthrapyrazoles, bisanthrapyrazoles and any other putative or known DNA intercalator.
  • the present invention also generally contemplates a method of estimating the binding strength of a compound to DNA using molecular modeling comprising modeling the compound docked into DNA and obtaining a GOLDScore.
  • the method may, in certain embodiments, comprise the steps of:
  • the compound is a DNA intercalator or a putative DNA intercalator.
  • the compound may be, in certain embodiments, an anthrapyrazole or a bisanthrapyrazole.
  • the compound may be a compound of formula (I) or (II), as discussed herein, for example.
  • alkyl refers to a straight, branched or cyclic carbon- carbon or hydrocarbon chain, optionally including alkene or alkyne bonding, containing 1-20 carbons.
  • Lower alkyl refers to alkyl radicals comprising 1-5 carbons. In any embodiment wherein “alkyl” radicals may be employed, “lower alkyl” radicals may be employed, in certain embodiments. Non-limiting examples of lower alkyls include methyl, ethyl, propyl, cyclopropyl, butyl and isopropyl.
  • Substituted alkyl refers to an alkyl radical substituted with at least one atom known to those of skill in the art.
  • one or more substituents may be selected from the group consisting of hydrogen, halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy, acyl, aryl, acetyl, carbonyl, cyano, heterocyclyl, amido, aminocarbonyl, amino, -NH-alkyl, -N(alkyl) 2 , -NH-(substituted alkyl), -N-(substituted alkyl) 2 , -NH-aryl, -N(aryl) 2 , trialkylsilyloxy, acyloxy, acylamino, bis-acylamino, ester, NO, NO 2 and sulfo (e.g., thioether, thioester, thiocarbonyl, sulfonamido, sulfonyl) and any combination thereof.
  • oxo e.g., ether
  • a substituted alkyl is an aminoalkyl radical, such as -NH(CH 2 ) 2 N(CH 3 ) 2 or -NH(CH 2 ) 3 NH 2 .
  • the subsituted alkyl is an hydroxyalkylamino radical, such as -NH(CH 2 ) 3 NH(CH 2 ) 2 OH.
  • a substituted alkyl is a haloalkyl.
  • a substituted alkyl is a hydroxyalkyl.
  • a substituted alkyl is a alkoxyalkyl.
  • cycloalkyl refers to carbocycle alkyl radicals of three or more atoms.
  • a "substituted cycloalkyl” is a cycloalkyl group, the ring atoms of which comprise one or more functional group as substituents.
  • a “heterocyclyl” refers to a cycloalkyl radical, the ring atoms of which are substituted with at least one heteroatom (e.g., S, O, or N).
  • a “substituted heterocycloalkyl” is a heterocycloalkyl group, the ring atoms of which comprise one or more functional group as substituents.
  • Substituents may be selected, in some embodiments, from the group consisting of hydrogen, alkyl, halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy, acyl, aryl, acetyl, carbonyl, cyano, amido, aminocarbonyl, amino, -NH-alkyl, -N(alkyl) 2 , -NH- (substituted alkyl), -N-(substituted alkyl) 2 , -NH-aryl, -N(aryl) 2 , trialkylsilyloxy, acyloxy, acylamino, bis-acylamino, ester, NO, NO 2 and sulfo (e.g., thioether, thioester, thiocarbonyl, sulfonamido, sulfonyl) and any combination thereof.
  • oxo e.g., ether
  • aryl refers to a carbo cyclic aromatic group, including but not limited to those selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, and anthracenyl; or a heterocyclic aromatic group, including but not limited to those selected from the group consisting of furyl, furanyl, thienyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trithianyl, indolizinyl, indolyl, isoindolyl
  • Aryl groups may independently contain one or more functional groups as substituents ("substituted aryl").
  • substituents may be selected from the group consisting of hydrogen, alkyl, halogen, oxo (e.g., ether), hydroxy, alkoxy, silyloxy, acyl, aryl, acetyl, carbonyl, cyano, heterocyclyl, amido, aminocarbonyl, amino, -NH-alkyl, -N(alkyl) 2 , -NH-(substituted alkyl), -N-(substituted alkyl) 2 , -NH-aryl, -N(aryl) 2 , trialkylsilyloxy, acyloxy, acylamino, bis-acylamino, ester, NO, NO 2 and sulfo (e.g., thioether, thioester, thiocarbonyl, s
  • halogen refers to fluoro, chloro, bromo or iodo.
  • amino alone or in combination, is used interchangeably with “amine” and refers to a primary amine, secondary amine or tertiary amine — that is, derivatives of ammonia (NH 3 ), in which one (primary), two
  • cyclic group refers to a cycloalkyl group, a substituted cycloalkyl group, a heterocyclyl group, a substituted heterocyclyl group, an aryl group, a substituted aryl group, or any combination thereof.
  • nucleophile or “nucleophilic” generally refers to atoms bearing lone pairs of electrons. Such terms are well known in the art and include -NH 2 , thiolate, carbanion and hydroxyl.
  • leaving group generally refers to a group readily displaceable by a nucleophile, such as an amine, an alcohol, or a thiol nucleophile.
  • Such leaving groups are well known and include carboxylates, N- hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides), triflates, tosylates, mesylates, alkoxy, thioalkoxy and the like.
  • the term "functional group” generally refers to how persons of skill in the art classify chemically reactive groups.
  • functional groups include hydroxyl, amine, sulfhydryl, amide, carboxyls, carbonyls, etc. While hydrogen and unsubstituted alkyl groups are not typically considered functional groups, as used herein hydrogen and/or unsubstituted alkyl groups may, in certain embodiments, be considered functional groups. In certain embodiments, hydrogen and/or unsubstituted alkyl groups are explicitly not considered functional groups.
  • protecting group refers to a moiety attached to a functional group to prevent an otherwise unwanted reaction of that functional group. Protecting groups are well-known to those of skill in the art. Non-limiting exemplary protecting groups fall into categories such as hydroxy protecting groups, amino protecting groups, sulfhydryl protecting groups and carbonyl protecting groups. Such protecting groups may be found in Greene and Wuts, 1999 (incorporated herein by reference in its entirety).
  • a "linker” is any diradical species that may covalently join one anthrapyzrazole to another anthrapyrazole such that the linker does not chemically react with either anthrapyrazole.
  • Non- limiting examples include (-CH 2 -) p , wherein p is 1-10 (that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or higher; - (CH 2 )2N(CH3)(CH2)2OC(O)(CH2)nC(O)O(CH2)2N(CH3)(CH 2 )2-, wherein n is 1-5 or higher (that is 1, 2, 3, 4, 5 or higher), - (CH 2 )2N(CH3)(CH2)2NHC(O)(CH2) n C(O)NH(CH2)2N(CH3)(CH 2 )2-, wherein n is 1-5 or higher, -(CH2)2N(CH 3 )(CH2)2OC(S)(CH2)nC(S)O(CH2)
  • Compounds as described herein may contain one or more asymmetric centers and thus can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. All possible stereoisomers of the all the compounds described herein, unless otherwise noted, are contemplated as being within the scope of the present invention.
  • the chiral centers of the compounds of the present invention can have the S- or the R-configuration, as defined by the IUPAC 1974 Recommendations. The present invention is meant to comprehend all such isomeric forms of the compounds of the invention.
  • the claimed invention is also intended to encompass salts of any of the synthesized compounds of the present invention.
  • salt(s) as used herein, is understood as being acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • Zwitterions are understood as being included within the term “salt(s)” as used herein, as are quaternary ammonium salts such as alkylammonium salts.
  • Nontoxic, pharmaceutically acceptable salts are preferred as described below, although other salts may be useful, as for example in isolation or purification steps.
  • Non-limiting examples of acid addition salts include but are not limited to acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyan
  • Non- limiting examples of basic salts include but are not limited to ammonium salts; alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts comprising organic bases such as amines (e.g., dicyclohexylamine, alkylamines such as t-butylamine and t- amylamine, substituted alkylamines, aryl-alkylamines such as benzylamine, dialkylamines, substituted dialkylamines such as N-methyl glucamine, trialkylamines, and substituted trialkylamines); and salts comprising amino acids such as arginine, lysine and so forth.
  • amines e.g., dicyclohexylamine, alkylamines such as t-butylamine and t- amylamine, substituted alkylamines, aryl-alkylamines such as benzylamine
  • the basic nitrogen-containing groups may be quaternized 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, myrtistyl and stearyl chlorides, bromides and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides) and others known in the art.
  • lower alkyl halides e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates e.g., dimethyl, diethyl, dibutyl, and dia
  • an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer.
  • Anti-cancer agents are well-known in the art and include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reo viral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.
  • chemotherapy agents such as DNA intercalators
  • radiotherapy agents radiotherapy agents
  • a surgical procedure a surgical procedure
  • immune therapy agents immunotherapy
  • genetic therapy agents gene therapy
  • reo viral therapy hormonal therapy
  • other biological agents biotherapy
  • alternative therapies include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reo viral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.
  • an effective amount means adequate to accomplish a desired, expected, or intended result.
  • Treatment and “treating” as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a subject e.g., a mammal, such as a human
  • a treatment comprising administration of one or more bisanthrapyrazoles of the present invention.
  • therapeutic benefit refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • a therapeutically effective amount of a bisanthrapyrazole of the present invention may be administered to a subject having a cancerous tumor, such that the tumor shrinks. It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • FIG. 1 Bisanthrapyrazole ID shown bound in the minor groove of a 6 base- pair piece of DNA.
  • the two doxorubicin molecules were removed from the Protein Data Bank Ida9.pb x-ray structure of the doxorubicin-DNA complex and ID was docked into the DNA with the genetic algorithm docking program GOLD.
  • FIG. 2. ⁇ T m for DNA binding as a function of the number of CH 2 linker groups in bisanthrapyrazoles 1A-1E.
  • FIGS. 3 A, 3B. 3 A GOLD docking score as a function of the number of CH 2 linker groups in certain bisanthrapyrazoles.
  • 3B GoldScore does not predict DNA ⁇ T m .
  • FIG. 4 DNA ⁇ T m , cytotoxicity and topoisomerase Il ⁇ inhibitor effects of bisanthrapyrazoles 1A-1E.
  • FIG. 5 K562 cytotoxicity is not correlated with DNA ⁇ T m .
  • FIG. 6 Structures of the anthrapyrazoles, mitoxantrone and doxorubicin.
  • the core structure used for alignment of the anthrapyrazoles for the 3D-QSAR CoMFA and CoMSIA analyses is shown in bold.
  • FIG. 7 QSAR correlations of growth inhibition of K562 cells by the anthrapyrazoles and mitoxantrone.
  • the anthrapyrazoles are identified by the numbers inside the symbols and losoxantrone, piroxantrone and mitoxantrone are identified as L, P, and M, respectively.
  • FIG. 8 Effect of anthrapyrazoles on the topoisomerase Il ⁇ -mediated cleavage of supercoiled pBR322 DNA.
  • This fluorescent image of the ethidium bromide-stained gel shows that topoisomerase Il ⁇ relaxed supercoiled pBR322 plasmid DNA (SC) to relaxed DNA (RLX).
  • SC supercoiled pBR322 plasmid DNA
  • RLX relaxed DNA
  • Topoisomerase Il ⁇ was present in the reaction mixture for all other lanes.
  • etoposide treatment produced linear DNA (LIN) and inhibited the relaxation of supercoiled pBR322 DNA.
  • a small amount of nicked circular (NC) is normally present in the pBR322 DNA.
  • AP-I, AP-2, AP-6, AP-IO, AP-I l and AP-12 are identified as having produced linear DNA above control levels (lane 2).
  • the binding of some of the fluorescent anthrapyrazoles (AP-3, AP-4, AP-8 and AP-9) to DNA obscured the band where the linear DNA would be expected to be found and also caused a mobility shift due to their binding.
  • Topo Il ⁇ is topoisomerase Il ⁇ .
  • FIG. 9 Docking of the protonated anthrapyrazoles into DNA, their aligned docked structures and CoMSIA contour plots.
  • FIG. 9A The highest scoring structure of the most potent anthrapyrazole AP-IO (ball-and-stick structure) is shown docked into DNA (stick structure). The complex forms both base stacking and H- bonding interactions (green dotted lines).
  • the side chains of AP-IO formed 3 hydrogen bonds (1 from the terminal OH and 2 others from the secondary and tertiary nitrogens) with DNA.
  • the hydroxyl side chain of AP-IO was positioned in the minor groove, and the dimethyl amino side chain was in the major groove.
  • the H-atoms are not shown for clarity.
  • the DNA structure is 1DA9 from the Protein Data Bank and is a doxorubicin/DNA x-ray structure in which two doxorubicin molecules are bound to a 6-bp piece of DNA. Only the first 3 base pairs of the DNA are shown in the figure for clarity. One doxorubicin was removed and AP-IO was docked into its place with the genetic algorithm docking program GOLD.
  • FIG. 9B The structures of all the anthrapyrazoles and mitoxantrone that were docked into DNA and aligned to the core structure as shown in Figure 1 (bold bonds) are shown superimposed.
  • FIG. 9B The structures of all the anthrapyrazoles and mitoxantrone that were docked into DNA and aligned to the core structure as shown in Figure 1 (bold bonds) are shown superimposed.
  • FIG. 9B The structures of all the anthrapyrazoles and mitoxantrone that were docked into DNA and aligned to the core structure as shown in Figure 1
  • FIG. 9C CoMSIA stddev*coeff contour hydrogen bond donor plots for the K562 growth inhibition data are shown for AP-IO, the most potent anthrapyrazole.
  • the green contours indicate regions where hydrogen bond donors increase activity, and the red contours indicate regions where hydrogen bond donors decrease activity.
  • the green contours were located near the nitrogens of the side chains indicating the importance of these groups.
  • FIG. 9D CoMSIA stddev*coeff contour electrostatic plots for the K562 growth inhibition data are shown for AP-IO, the most potent anthrapyrazole.
  • the green contours indicate regions where electrostatic interactions increase activity and the red contours indicate regions where electrostatic interactions decrease activity.
  • the green contours were located near the protonated nitrogens of the side chains, indicating the importance of protonated nitrogens in the activity of the anthrapyrazoles.
  • FIG. 10 QSAR correlations of /C50 values for growth inhibition of K562 cells by the anthrapyrazoles and mitoxantrone with energy terms obtained by docking the anthrapyrazoles into a DNA x-ray structure.
  • FIG. 11 CoMSIA predictions for the effect of the anthrapyrazoles and mitoxantrone on /C50 for growth inhibition of K562 cells (FIG. HA) and the increase in the DNA melt temperature ⁇ T m (FIG. HB).
  • the straight lines are the regression lines for the predictions.
  • the anthrapyrazoles are identified by the numbers inside the symbols and losoxantrone, piroxantrone and mitoxantrone are identified as L, P, and M, respectively.
  • FIG. 12 Concentration dependence of ⁇ T m for the bisanthrapyrazoles binding to DNA.
  • FIG. 12A Concentration dependence of ⁇ T m for the bisanthrapyrazoles IA, IB, 1C, ID, and IE and parent monomer AP9 for comparison. The solid straight lines are linear least squares fits to the data.
  • FIG. 12B Concentration dependence of the slopes ⁇ SEM calculated from the data in (FIG. 12A). Except for IA (NS, not significant) the slopes of the plots in (FIG. 12A) for 2, 3, 4, and 5 are all significantly (***p ⁇ 0.001) different than the parent AP9. Compounds with slopes approximately twice that of the parent monomer AP9 indicate that they formed bisintercalation complexes with DNA.
  • FIG. 13 Effect of bisanthrapyrazoles IA, IB, 1C, ID, and IE on the topoisomerase Il ⁇ -mediated cleavage of supercoiled pBR322 DNA.
  • This fluorescent image of the ethidium bromide-stained gel shows that topoisomerase Il ⁇ (Topo Il ⁇ ) relaxed supercoiled pBR322 plasmid DNA (SC) to relaxed (RLX) DNA (lane 2, the band running slightly ahead of the SC band).
  • pBR322 DNA in the absence of topoisomerase Il ⁇ is shown in lane 1.
  • Topoisomerase Il ⁇ was present in the reaction mixture for all other lanes.
  • etoposide treatment 100 ⁇ M
  • N nicked circular
  • BisAP identifies the bisanthrapyrazoles IA, IB, 1C, ID, and IE. Based on densitometry, none of the bisanthrapyrazoles (50 ⁇ M) produced any significant amount of linear DNA above control levels (lane 2). Some of the fluorescent bisanthrapyrazoles present in the gel (lanes 3, 5, and 7) partially obscured the linear DNA band.
  • FIG. 14 Docking of the protonated bisanthrapyrazole IB into DNA.
  • the highest scoring structure of the strongest DNA-binding bisanthrapyrazole IB (CPK structure) is shown docked into DNA (stick structure).
  • the H-atoms of the DNA are not shown for clarity.
  • the DNA structure is 1DA9 from the Protein Data Bank and is a DNA- (doxorubicin ⁇ X-ray structure in which two doxorubicin molecules are bound to a 6-base pair piece of DNA. Both doxorubicin molecules were removed and IB was docked into its place with the genetic algorithm docking program GOLD.
  • the present invention is based on the finding that certain bisanthrapyrazoles likely behave as DNA intercalating agents and topoisomerase Il ⁇ inhibitors, and show potent inhibition of cancer cells.
  • these bisanthrapyrazoles comprise two anthrapyrazoles operatively linked together.
  • the linkage comprises one or more ester and/or amide groups.
  • Some of these bisanthrapyrazoles bind to DNA more strongly than doxorubicin, a known DNA intercalator and anticancer drug.
  • the present invention provides for novel anti-cancer agents, their syntheses and methods of using them in chemotherapeutic treatments.
  • Molecular modeling methods are also set forth herein that provide insight into how compounds bind to DNA and what the strength of that bind is.
  • Topoisomerase is an isomerase enzyme that alters the supercoiling of DNA.
  • the double -helical configuration of DNA strands makes them difficult to separate, and yet they must be separated by helicase proteins if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated.
  • so-called circular DNA in which double helical DNA is bent around and joined in a circle, the two strands are topologically linked, or knotted. Otherwise identical loops of DNA having different numbers of twists are topoisomers, and cannot be interconverted by any process that does not involve the breaking of DNA strands.
  • Topoisomerases catalyze and guide the unknotting of DNA. This unlinking activity is termed "decatenation.”
  • Topoisomerases are classified into two types separated by the number of strands cut in one round of action. Topoisomerase I cuts one strand, passes the other through it then reanneals the cut strand. Topoisomerase II cuts both strands, and passes an unbroken double strand through it then reanneals the cut strand.
  • Mammalian topoisomerase II has been further classified into types Il ⁇ and Il ⁇ . Some chemotherapy drugs work by interfering with topoisomerases in cancer cells. (Kornberg and Baker, DNA Replication, W. H. Freeman and Company, New York,
  • topoisomerase I is inhibited by irinotecan
  • Topoisomerase II is inhibited by etoposide
  • topoisomerase II poisons Drugs acting on topoisomerase II are divided into two main categories, topoisomerase II poisons and topoisomerase II catalytic inhibitors.
  • the topoisomerase II poisons are associated with their ability to stabilize the enzyme-
  • Topoisomerase poisons may bind to DNA, the topoisomerase, or either molecule at or near the region of the enzyme.
  • Many topoisomerase poisons such as the anthracyclines (discussed below) and actinomycin D, are relatively planar hydrophobic compounds that intercalate DNA.
  • Nonintercalating DNA binders can also poison topoisomerase (Chen, et al., 1993). However, DNA binding is neither a necessary nor sufficient condition for topoisomerase poisoning (Bast, Kufe, Pollock, Weichselbaum, Holland, Frei and Gansler, Eds., Cancer Medicine, 5 th Edition, BC Decker Inc., Hamilton, Ontario, 2000). Several antitumor agents in clinical use have potent activity as mammalian topoisomerase II poisons.
  • topoisomerase II catalytic inhibitors are an entirely different group of drugs. They act by interfering with the overall catalytic function, which can be accomplished in at least two ways. One is the inhibition of the initial binding of topoisomerase II to DNA, as in the case of chloroquine (Jensen et al., 1994) and aclarubicin (Sehested and Jensen, 1996; S ⁇ rensen et al., 1992).
  • topoisomerase II in its closed-clamp step after religation, as appears to be the case for the ICRF- 187 and its analogs (Tanabe et al., 1991; Berger et al., 1996; Roca et al., 1994; Roca et al., 1996). While the mechanisms of topoisomerase II poisons and inhibitors are different, one agent can act as both a topoisomerase II inhibitor and as a topoisomerase II poison.
  • Doxorubicin (Adriamycin®) and daunorubicin are anthracycline antibiotics that intercalate with DNA and are also topoisomerase Il ⁇ poisons.
  • Bisanthrapyrazoles of the present invention generally comprise two anthrapyrazoles joined directly or via a linker.
  • the bisanthrapyrazoles of the present invention may, in certain embodiments, be exemplified by the generic structure of formula (II):
  • R 1 -R 7 and R 12 -R 1S are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more OfY 1 -R 1 , R 1 -R 2 , R 2 -R 3 , R 3 -R 4 , R5-R6, R6-R7, R7- Z 1 , Y 2 -R 12 , R 12 -R 13 , R 13 -R 14 , R14-R15, R16-R17, R17-R18 or R 18 -Z 2 together forms a cyclic group, or any combination of one or more of these groups; R 8 and R49 are each independently oxygen or sulfur; D, E,
  • the present invention concerns methods for identifying further bisanthrapyrazoles, which are potential DNA intercalators, topoisomerase I or II inhibitors, and/or anti-cancer agents. It is contemplated that such identification will prove useful in the general identification of any compound that will serve the purpose of interacting with DNA in a manner similar to the exemplary bisanthrapyrazoles disclosed herein.
  • the molecular modeling protocol explained in Example 2 provides one means of identifying additional intercalating bisanthrapyrazoles.
  • An alternative method for testing whether a bisanthrapyrazole is an intercalating agent is by using viscosity assays as described by Suh and Chaires (1995). Such assays are well known to those of skill in the art.
  • plots of the cubed root of the relative viscosity (( ⁇ / ⁇ o) m ) versus the binding ratio (bound drug/DNA bp) ought to have a slope of 1.0.
  • the slope is expected to be twice that observed for monointercalators, an expectation that has been verified for a variety of bisintercalating compounds (Wakelin, 1986).
  • the viscosity of a DNA solution is measured according to assays well known to those of skill in the art (Suh and Chaires, 1995) and compared to the viscosity of a known intercalator, such as daunorubicin. If the viscosity of the candidate substance is higher than that of the control monointercalator then the candidate substance is likely to be an effective bisintercalator.
  • the most useful pharmacological compounds for identification through application of, e.g., molecular modeling will be compounds that are further metabolized before they are therapeutically active (i.e., "prodrugs").
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
  • the fragment may be an anthrapyrazole.
  • anthrapyrazole Preparations of various anthrapyrazoles are known to those of skill in the art. See, e.g., Begleiter et al., 2006; Liang et al, 2006; Showalter et al, 1987; and Showalter et al, 1986 (each of these references are incorporated herein in their entirety).
  • an anthrapyrazole such as that of formula (I) above and incorporate a hydroxyl moiety at any OfR 1 -R 7 or R 1O , such as at R 5 .
  • Bisanthrapyrazoles may be synthesized wherein instead of ester linkages bridging the two anthrapyrazoles, amide linkages are used.
  • Such linkages could be generated by, for example, the scheme shown below:
  • MsCI mesyl chloride
  • DMF dimelthyl formamide
  • Example 17 A non-limiting example of the preparation of an amide-linked bisanthrapyrazole of the present invention is presented in Example 17.
  • An "arm” is schemtically shown using the core skeleton structure of formula (A): arm
  • Conjugation of first and second bisanthrapyrazoles is not restricted to linkages between their "arms.” Conjugation may take place at any position available for conjugation on either anthrapyrazole, such as a terminal end of the "arm” (which includes the terminal group of any branched group stemming from the backbone of the "arm"), any ring atom of rings that make up the anthrapyrazoles, or any substituent on any of the rings that make up the anthrapyrazoles.
  • a linkage may form between substitutions on the aromatic rings of the first and second anthrapyrazoles (see Begleiter et ah, 2006; Liang et ah, 2006).
  • the first and second anthrapyrazoles are the same. In certain embodiments, the first and second anthrapyrazoles are different. In the latter case, the different bisanthrapyrazoles that would form in the reaction would require separation. Separation techniques are well-known in the art and include, for example, column chromatography, HPLC, crystallization and dialysis. In certain embodiments, the present invention provides for substantially pure bisanthrapyrazoles. As used herein, the term "substantially pure" indicates that the bisanthrapyrazoles of interest constitutes the predominant species in a mixture (that is, greater than 50%, or, in other words, at least 50% pure).
  • a substantially pure bisanthrapyrazoles may be about or at least about 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 99.9% pure.
  • a substantially pure bisanthrapyrazoles excludes any naturally occurring mixtures that contain the compound.
  • a substantially pure bisanthrapyrazoles may also, or in the alternative, refer to the percent enantiomeric excess (% ee) of the compound.
  • a substantially pure bisanthrapyrazoles may exhibit a % ee of about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or higher, or any range derivable therein, such as about 10% to about 62%.
  • bisanthrapyrazoles are prepared as salts, such as dihydrochloride salts.
  • the bisanthrapyrazoles are fairly soluble, such as when they exist in their dihydrochloride salt form: in such a case, the dihydrochloride salt may be more soluble than the bisanthrapyrazole compound in its non-salt form.
  • the invention also contemplates formation of compounds that comprise three or more anthrapyrazoles.
  • a third anthrapyrazole which may be the same or different than the anthrapyrazoles which make up the bisanthrapyrazole, may be linked to the bisanthrapyrazole.
  • “derivative” refers to a chemically modified compound that still retains the desired effects of the compound prior to the chemical modification
  • “bisanthrapyrazole derivatives” therefore, refers to a chemically modified compound that still retains the desired effects of the parent bisanthrapyrazole prior to its chemical modification. Such effects may be enhanced (e.g. , slightly more effective, twice as effective, etc.) or diminished (e.g., slightly less effective, 2-fold less effective, etc.) relative to the unmodified bisanthrapyrazole, but may still be considered a bisanthrapyrazole derivative. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule.
  • Non-limiting examples of the types of modifications that can be made to the compounds and structures disclosed herein include the addition or removal of lower unsubstituted alkyls such as methyl, ethyl, propyl, or substituted lower alkyls such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, imide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfenyl, sulfonyl, sulfoxido, sulfonamide, phosphate, phosphono, phosphoryl groups, and halide substituents.
  • lower unsubstituted alkyls such as methyl, ethyl, propyl, or substituted lower alkyls such as hydroxymethyl or aminomethyl groups
  • carboxyl groups and carbonyl groups hydroxyls; nitro, amino, amide, imide,
  • Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group.
  • heteroatoms such as N, S, or O can be substituted into the structure instead of a carbon atom.
  • Another aspect of the present invention contemplates a method of preparing a bisanthrapyrazole comprising preparing a first anthrapyrazole, preparing a second anthrapyrazole, and conjugating the first anthrapyrazole to the second anthrapyrazole either directly or through a linker.
  • the linker may join the two anthrapyrazoles at any two or more atoms of either anthrapyrazole.
  • the linker may be any type of linker described herein, and known to those of skill in the art.
  • the joined atoms of the first and second anthrapyrazoles may be any two atoms as described herein (e.g., the Z atoms of the compounds of core skeleton formula (A), or the Z 1 atoms of the anthrapyrazoles of the compound of formula (I)).
  • one of the two anthrapyrazoles comprises a nucleophile and the other comprises a leaving group, and the two anthrapyrazoles are linked together via reaction of the nucleophile and the atom to which the leaving group is attached.
  • Also contemplated by the present invention is a method of preparing a compound of formula (II) comprising: preparing a first anthrapyrazole of formula (I):
  • R 1 -R 7 are each independently hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more Of Y 1 -R 1 , R 1 - R 2 , R 2 -R 3 , R3-R4, R5-R6, R6-R7 or R 7 -Z 1 together forms a cyclic group, or any combination of one or more of these groups; Rs is either oxygen or sulfur; R 9 is either not present or is a linker; and R 1O is either hydrogen, alkyl, a nucleophile or a leaving group; D, E, F, G, H, I, J, K and L are each independently carbon, -CH or nitrogen; Y 1 is selected from the group consisting
  • R 12 -R 18 are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, oxo, hydroxy, silyl, phosphoro, acyl, aryl, acetyl, carbonyl, cyano, amido, amino, ester, NO, NO 2 , azido, sulfo, or a protecting group, or any one or more of Y 2 -R 12 , R 12 -R 13 , R13-R14, R14-R15, R16-R17, R17-R18 or R 18 -Z 2 together forms a cyclic group, or any combination of one or more of these groups;
  • R 49 is either oxygen or sulfur;
  • R 21 is either not present or is a linker; and
  • R 22 is either H, alkyl, a nucleophile or a leaving group;
  • M, P, Q, R, T, U V, W and X are each independently selected from the group consisting of carbon
  • the conjugation may take place via any method known to those of skill in the art, and may take place between two or more atoms on either anthrapyrazole.
  • the conjugation step comprises conjugating said first anthrapyrazole to said second anthrapyrazole through a linker that joins the Z 1 and Z 2 positions of each anthrapyrazole.
  • the linker comprises an alkyl group, a substituted alkyl group, a heterocyclyl group, an oxo group, a sulfo group, a thioether group, an aryl group, an amide group, a sulfonamide group, a carbonyl group, a thiocarbonyl group, a secondary amine group, a tertiary amine group, an ester group, a thioester group, a sulfonyl group, or any combination of one or more of these groups.
  • the linker may comprise an alkyl group, at least one ester, and/or at least one amide.
  • R 1 O and R 22 are selected from the group consisting of a nucleophile and a leaving group, wherein R 1 O ⁇ R 22 .
  • the bisanthrapyrazoles of the present invention are likely bisintercalators. Antitumor activity and topoisomerase Il ⁇ decatenation activity of the bisanthrapyrazoles were each explored. These studies indicated that these compounds exert their cytotoxic effects by acting as topoisomerase II decatenation activity inhibitors (Begleiter et al, 2006; Liang et al, 2006). This is keeping with the inhibition activities of loxoxantrone and piroxantrone, but in contrast to the dual topoisomerase Il ⁇ inhibitor/poison behavior displayed by other monoanthrapyrazoles (Begleiter et al, 2006; Liang et al, 2006).
  • the present invention is directed to a method for determining the ability of a bisanthrapyrazole to inhibit the growth of cancer cells.
  • the method includes generally the steps of:
  • Cancer cells known to be responsive to DNA intercalators are well-known in the art.
  • Inhibition of growth of cancer cells may be measured by, for example, the cell culture and cytotoxicity assay as set forth in Example 3 below, comprising the MTS assay. See also Liang et al., 2006. Inhibition of growth of cancer cells can also be measured by the MTT assay. Growth assays as measured by the MTT assay are well known in the art. Assays may be conducted as described by Mosmann et al, 1983; Rubinstein et al., 1990; and Green et al., 1984 (each incorporated herein by reference). Therefore, if a candidate substance exhibited inhibition in this type of study, it would likely be a suitable compound for use in the present invention.
  • a significant inhibition in growth is represented by decreases of about or at least about 30% (e.g., at least about or about 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to uninhibited, with more significant decreases also being possible.
  • Quantitative in vitro testing of the bisanthrapyrazoles discussed herein is not a requirement of the invention as it is generally envisioned that the agents will often be selected on the basis of their known properties or by structural and/or functional comparison to those bisanthrapyrazoles or anthrapyrazoles already demonstrated to be effective. Therefore, the effective amounts will often be those amounts proposed to be safe for administration to animals in another context.
  • the present invention also contemplates molecular modeling methods used to estimate the binding strength of a compound to DNA.
  • This method also may be used, for example, to model how a compound may bind to DNA.
  • Any compound may be studied and in particular embodiments, intercalators or putative intercalators may be modeled.
  • a series of anthrapyrazole and bisanthrapyrazole compounds that are analogs of piroxantrone and losoxantrone were synthesized and their cell growth inhibitory effects, DNA binding, topoisomerase Il ⁇ -mediated (EC 5.99.1.3) cleavage of DNA and inhibition of DNA topoisomerase Il ⁇ decatenation catalytic activities were determined.
  • Structure -based three- dimensional quantitative structure-activity analyses (3D-QSAR) were carried out on the aligned structures of the anthrapyrazoles and bisanthrapyrazoles docked into DNA using comparative molecular field analysis (CoMFA) and comparative molecular similarity index (CoMSIA) analyses in order to determine the structural features responsible for their activity. Both CoMFA and CoMSIA analyses yielded statistically significant models upon partial least squares analyses.
  • the 3D-QSAR analyses showed that hydrogen bond donor interactions and electrostatic interactions with the protonated amino side chains of the anthrapyrazoles led to high cell growth inhibitory activity.
  • the present invention also discusses a study of a series of anthrapyrazole compounds showed that they potently inhibited the growth of K562 cells.
  • the parent compounds of this group losoxantrone and piroxantrone, which have both been tried in clinical trials (Showalter et al, 1986; Judson, 1992; Gogas and Mansi, 1996; Diab et al, 1999; Talbot et al, 1991; Ingle et al, 1994) likely exert their cell growth inhibitory effects by acting as DNA topoisomerase II poisons (Leteurtre et al, 1994; Capranico et al, 1994).
  • topoisomerase II was not the primary mechanism by which these compounds act. This result does not rule out that the anthrapyrazoles were acting as topoisomerase II poisons. Intercalating compounds typically inhibit the catalytic activity of topoisomerase II, presumably by interfering with the formation of the DNA-topoisomerase II complex (Fortune and Osheroff, 2000).
  • anthrapyrazoles were catalytic inhibitors of topoisomerase II, they likely exerted their cell growth inhibitory activity, at least in part, through their ability to act as topoisomerase II poisons.
  • QSAR correlation and 3D-QSAR analyses showed the importance of anthrapyrazole-DNA van der Waals interactions, while the 3D-QSAR CoMFA and CoMSIA analysis showed that hydrogen bond donor interactions and electrostatic interactions with the protonated amino side chains of the anthrapyrazoles led to high cell growth inhibitory activity.
  • Certain bisanthrapyrazoles were designed using molecular modeling and docking into DNA in order to determine the optimal linker length for optimal binding to DNA and selection for their subsequent synthesis.
  • An X-ray structure (1DA9) (Leonard et ah, 1993) of two molecules of doxorubicin separated by 4 base pairs bound to duplex DNA was used for the docking (FIG. 14).
  • Five bisanthrapyrazoles based on AP9 with 1-5 methylene linkers (IA, IB, 1C, ID and IE) were synthesized and chemically and biologically characterized.
  • DNA melting temperatures ⁇ T m (Table 5) were determined as a measure of strength of DNA binding and were significantly increased over that of the parent AP9, a result which suggests that the bisanthrapyrazoles IB, 1C, ID and IE formed bisintercalation complexes with DNA.
  • concentration dependence of ⁇ T m for compounds IB, 1C, ID and IE (FIGS. 12A and 12B) indicated that these compounds formed mainly bisintercalation complexes with DNA.
  • Our docking results predicted IA to have too short a linker to form a bisintercalation complex. This was confirmed by the results in FIG. 12B in which compound IA and monomeric AP9 had the same slope and thus both formed mono intercalation complexes.
  • the bisanthrapyrazoles have a relatively large number of rotatable bonds (e.g., up to 20 for IE) and thus are highly conformational ⁇ flexible molecules that can adopt large numbers of low energy configurations. This high degree of conformational flexibility makes it computationally difficult to obtain the best possible GOLDScore. This fact likely explains the lack of a smooth change in GOLDScore as the number of linker groups is varied (see, e.g., FIG. 3). Again considering the size of the bisanthrapyrazoles and the large number of rotatable bonds, the docking produced chemically reasonable bisintercalation complexes as shown in FIG. 14.
  • the bisanthrapyrazoles were docked into a single X-ray structure (PDB ID: 1DA9) which does not have the set of base pairs which necessarily leads to optimum binding to DNA.
  • the base pair specificity of the bisanthrapyrazoles for calf thymus DNA is unknown.
  • Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically effective amount of a bisanthrapyrazole for chemotherapeutic purposes.
  • the bisanthrapyrazoles of this invention may be administered to kill tumor cells by any method that allows contact of the active ingredient with the agent's site of action in the tumor. They can be administered by any conventional methods available for use in conjunction with pharmaceuticals, either as individual therapeutically active ingredients or in a combination of therapeutically active ingredients. They may be administered alone, but are generally administered with a pharmaceutically acceptable carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • the bisanthrapyrazoles may be extensively purified and/or dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Such methods are well-known in the art.
  • the active compounds will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below.
  • Aqueous compositions of the present invention will typically have an effective amount of anthrapyrazole to kill or slow the growth of cancer cells. Further the potential recognition of genes can be accomplished by the synthesis of bisanthrapyrazoles with specific structures that allow for the recognition of specific parts of DNA. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • any bisanthrapyrazole can be provided in prodrug form, meaning that an environment to which a bisanthrapyrazole is exposed alters the prodrug into an active, or more active, form. It is contemplated that the term "precursor" covers compounds that are considered “prodrugs.” In certain embodiments, a bisanthrapyrazole may act as a prodrug by in situ release of the individual anthrapyrazoles.
  • compositions of the present invention comprise an effective amount of one or more candidate substances ⁇ e.g., a bisanthrapyrazole) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the candidate substance may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination
  • the composition is administered to a subject using a drug delivery device.
  • a drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of a bisanthrapyrazole.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the dose can be repeated as needed as determined by those of ordinary skill in the art.
  • a single dose is contemplated.
  • two or more doses are contemplated.
  • the time interval between doses can be any time interval as determined by those of ordinary skill in the art.
  • the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.
  • compositions may comprise, for example, at least about 0.1% of a bisanthrapyrazole.
  • the bisanthrapyrazole may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the composition may comprise various antioxidants to retard oxidation of one or more component.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens ⁇ e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
  • the candidate substance may be formulated into a composition in a free base, neutral, or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol ⁇ e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids ⁇ e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • 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 by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained.
  • the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation.
  • various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.
  • the candidate substance is prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof.
  • the oral composition may be prepared as a syrup or elixir.
  • a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the fore
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina, or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.
  • the bisanthrapyrazole may be combined with traditional drugs. It is contemplated that this type of combination therapy may be used in vitro or in vivo.
  • an anti-cancer agent may be used in combination with a bisanthrapyrazole.
  • bisanthrapyrazoles of the present invention may be provided in a combined amount with an effective amount of an anti-cancer agent to reduce or block DNA replication in cancerous cells ⁇ e.g., tissues, tumors). This process may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit. This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.
  • the compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks.
  • the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance.
  • one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 2 hours
  • a bisanthrapyrazole is "A” and a second agent, such as an anti-cancer agent, is "B":
  • Typical yield was about 2.6 g, 46%.
  • AP-7 HCl can be converted to the free base by taking up the crude mixture in CHCI3 and extracting with excess NaOH solution.
  • AP-9 synthesis The AP-7 free base (1.70 g, 5 mmol) was dissolved in formic acid (3.8 mL) and 37% aqueous formaldehyde (1.1 mL) was added. The mixture was heated to 110 0 C for 4 h - gas evolution begins at 95°C. After cooling to room temperature the mixture was poured onto cold water - CHCI3 and the pH brought to ⁇ 13 with 2N NaOH. The chloroform phase was separated, and the aqueous phase washed with 2 x 20 niL chloroform. The chloroform phases were dried and concentrated and the red residue recrystallized from methanol affording about 1.23 g orange needles, mp 119.5-121°C.
  • the genetic algorithm-based molecular modeling program CCDC GOLD was used in docking bisanthrapyrazoles 1A-1E and AP-9 into the Protein Data Bank Id9a.pdb DNA(doxorubicin)2 x-ray structure. Doxorubicin itself docked back into the DNA with a rms of 1.9 A. As shown in FIG. 1, ID docked into the DNA minor groove and bound at the two doxorubicin binding sites. See also Example 14.
  • Topoisomerase Il ⁇ kDNA decatenation assay Topoisomerase Il ⁇ decatenates kDNA in an ATP-dependent reaction to yield individual minicircles of DNA.
  • kDNA was obtained from TopoGEN, Inc. (Port Orange, Florida). After reaction samples were centrifuged at 8000 g for 15 min, 20 ⁇ l of the supernatant were added to PicoGreen® dye in a 96-well plate. The fluorescence, which was proportional to the amount of kDNA, was measured in a fluorescence plate reader.
  • Topoisomerase Il ⁇ cleavage assay pBR322 plasmid DNA (MBI Fermentas, Burlington, Ontario, Canada) was incubated with a compound and purified human topoisomerase Il ⁇ and the reaction was stopped with SDS to trap the cleavable complexes. The linear DNA produced was separated by gel electrophoresis. Cell culture and cytotoxicity assay. K562 and KTVP.5 cell lines and topoisomerase Il ⁇ were a each gift from Jack Yalowich, University of Pittsburgh, Pittsburgh, Pennsylvania. K562 cells and etoposide-resistant KTVP.5 cells were grown in D-MEM/FCS. Cell growth was determined by MTS assay at 72 hr. DNA ⁇ T m determination.
  • the strength of compound binding to sonicated calf thymus DNA was measured by determining the effect of 2 ⁇ M drug on the DNA melting temperature.
  • ⁇ T m was estimated from the maximum in the first derivative of the 280 nm absorbance in a temperature-programmed cell compartment.
  • K562 cells are a cell line of human erythroleukemia cells.
  • KTVP.5 cells are a K562 cell line with acquired resistance to etoposide because they contain one-fifth the amount of topoisomerase Il ⁇ .
  • the bisanthrapyrazoles inhibited the growth of human leukemia K562 and low content topoisomerase Il ⁇ mutant K/VP.5 cells in the low micromolar concentration range (FIG. 4).
  • the IC50 values only varied from 1.1 to 3.3 ⁇ M compared to 5.7 ⁇ M for AP-9.
  • the IC 50 values only varied 3-fold.
  • the K562 IC 50 values were also poorly correlated with ⁇ T m (FIG. 5). Together these results suggest that the ester-linked bisanthrapyrazoles may have been hydro lyzed by intercellular esterases to give AP-9.
  • the resulting anthrapyrazole(s) may induce the cytotoxic effects discussed herein.
  • any of the anthrapyrazoles discussed in Liang et al. , 2006 that form upon cleavage of a bisanthrapyrazole would behave as a topoisomerase Il ⁇ poison. In situ production of a topoisomerase Il ⁇ poison would likely produce a more potent drug.
  • the present invention contemplates bisanthrapyrazoles as prodrugs.
  • Treatment with the anthrapyrazoles or the bisanthrapyrazoles of the present invention may be similar to the treatment regimes of other drugs, such as DNA intercalators ⁇ e.g., the anthracyclines and their derivatives).
  • DNA intercalators e.g., the anthracyclines and their derivatives.
  • standard treatment with doxorubicin is described in Remington 's Pharmaceutical Sciences as follows.
  • bisanthrapyrazoles may be studied or anthrapyrazoles. Reference to bisanthrapyrazoles is made in Examples 4-7 for simplicity.
  • Doxorubicin is administered intravenously to adults at 60 to 75 mg/m 2 at 21- day intervals or 25 to 30 mg/m on each of 2 or 3 successive days repeated at 3- or 4- week intervals or 20 mg/m once a week.
  • the lowest dose should be used in elderly patients, when there is prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs.
  • the dose should be reduced by 50% if the serum bilirubin lies between 1.2 and 3 mg/dL and by 75% if above 3 mg/dL.
  • the lifetime total dose should not exceed 550 mg/m 2 in patients with normal heart function and 400 mg/m 2 in patients with normal heart function and 400 mg/m on each of 3 consecutive days, repeated every 4 weeks. Prescribing limits are as with adults. It has been reported that a 96-hr continuous infusion is as effective as and much less toxic than the same dose given by bolus injections.
  • EXAMPLE 5 In Vivo Prevention of Tumor Development Using Bisanthrapyrazoles
  • a mouse model of human cancer with the histologic features and metastatic potential resembling tumors seen in humans is used.
  • the animals are treated with bisanthrapyrazoles of the present invention to determine the suppression of tumor development.
  • These studies are based on the discovery that bisanthrapyrazoles of the current invention have anti-cancer activity in cancer cells.
  • Bisanthrapyrazoles are tested in vivo for antitumor activity against murine leukemia L1210, P388 and P388 resistant to doxorubicin. In conjunction with these studies, the acute and sub-acute toxicity is studied in mice (LDlO, LD50, LD90). In a more advanced phase of testing, the antitumor activity of bisanthrapyrazoles against human xenografts is assessed and cardiotoxicity studies performed is done in a rat or rabbit model.
  • Two groups of mice of a suitable cancer model are treated with doses of bisanthrapyrazoles. Several combinations and concentrations of bisanthrapyrazoles are tested. Control mice are treated with buffer only. The effect of bisanthrapyrazoles on the development of tumors is compared with the control group by examination of tumor size, and histopathologic examination (tissue is cut and stained with hematoxylin and eosin) of the relevant tissue. With the chemopreventive potential of bisanthrapyrazoles 1A-1E and other bisanthrapyrazoles of the present invention, it is predicted that, unlike the control group of mice that develop tumors, the testing group of mice is resistant to tumor development.
  • This example describes a protocol to facilitate the treatment of cancer using bisanthrapyrazoles.
  • a cancer patient presenting cancer is treated using the following protocol. Patients may, but need not, have received previous chemo-, radio-, or gene therapeutic treatments. Optimally the patient exhibits adequate bone marrow function (defined as peripheral absolute granulocyte count of > 2,000/mm 3 and platelet count of 100, 000/mm 3 , adequate liver function (bilirubin 1.5 mg/dl) and adequate renal function (creatinine 1.5 mg/dl).
  • a composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants, and/or vehicles as desired.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intra-arterial injection, or infusion techniques. The bisanthrapyrazoles may be delivered to the patient before, after, or concurrently with any other anticancer agent(s), if desired.
  • a typical treatment course may comprise about six doses delivered over a 7 to
  • the regimen may be continued six doses every three weeks or on a less frequent (monthly, bimonthly, quarterly, etc.) basis.
  • compositions described in the present invention To kill cancer cells using the methods and compositions described in the present invention, one will generally contact a target cell with a bisanthrapyrazole of the present invention. These compositions are provided in an amount effective to kill or inhibit the proliferation of the cell.
  • agent(s) of the present invention it is contemplated that one would contact the cell with agent(s) of the present invention about every 6 hours to about every one week. In some situations however, it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, 7, or more) to several weeks (1, 2, 3, 4, 5, 6, 7, or more) lapse between respective administrations.
  • Regional delivery of a bisanthrapyrazole is an efficient method for delivering a therapeutically effective dose to counteract the clinical disease.
  • chemotherapy may be directed to a particular affected region.
  • systemic delivery of active agents may be appropriate.
  • the therapeutic composition of the present invention may be administered to the patient directly at the site of the tumor.
  • the volume of the composition should usually be sufficient to ensure that the tumor is contacted by the bisanthrapyrazole.
  • administration simply entails injection of the therapeutic composition into the tumor.
  • a catheter is inserted into the site of the tumor and the cavity may be continuously perfused for a desired period of time.
  • Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by the disappearance of all measurable disease for at least a month.
  • a partial response may be defined by a 50% or greater reduction of the sum of the products of perpendicular diameters of all evaluable tumor nodules or at least 1 month with no tumor sites showing enlargement.
  • a mixed response may be defined by a reduction of the product of perpendicular diameters of all measurable lesions by 50% or greater with progression in one or more sites.
  • treatment regimes may be altered in accordance with the knowledge gained from clinical trials, such as those described in Example 7.
  • Those of skill in the art are able to take the information disclosed in this specification and optimize treatment regimes based on the results from the trials.
  • This example is concerned with the development of human treatment protocols using the bisanthrapyrazoles. These compounds are of use in the clinical treatment of various cancers in which transformed or cancerous cells play a role.
  • the various elements of conducting a clinical trial, including patient treatment and monitoring, are known to those of skill in the art in light of the present disclosure.
  • the following information is being presented as a general guideline for studying bisanthrapyrazoles of the present invention in clinical trials.
  • Patients with cancer such as human metastatic breast and/or epithelial ovarian carcinoma, colon cancer, leukemia, or sarcoma are chosen for clinical study. Measurable disease is not required; however the patient must have easily accessible pleural effusion and/or ascites.
  • the patient may carry tumors that express a MDR (multi-drug resistant) phenotype.
  • patients may undergo placement of a Tenckhoff catheter, or other suitable device, in the pleural or peritoneal cavity and undergo serial sampling of pleural/peritoneal effusion.
  • a Tenckhoff catheter or other suitable device
  • Baseline cellularity, cytology, LDH, and appropriate markers in the fluid CEA, CA15-3, CA 125, pl85
  • ElA, pl85 may also be assessed and recorded.
  • the patient should exhibit a normal coagulation profile.
  • bisanthrapyrazoles may be administered.
  • the administration may be in the pleural/peritoneal cavity, directly into the tumor, or in a systemic manner.
  • the starting dose may be 0.5 mg/kg body weight.
  • Three patients may be treated at each dose level in the absence of grade > 3 toxicity.
  • Dose escalation may be done by 100% increments (0.5 mg, 1 mg, 2 mg, 4 mg) until drug related grade 2 toxicity is detected. Thereafter, dose escalation may proceed by 25% increments.
  • the administered dose may be fractionated equally into two infusions, separated by six hours, if the combined endotoxin levels determined for the lot of bisanthrapyrazole exceeds 5 EU/kg for any given patient.
  • the bisanthrapyrazoles may be administered over a short infusion time or at a steady rate of infusion over a 7 to 21 day period.
  • the bisanthrapyrazole infusion may be administered alone or in combination with, for example, another anti-cancer drug.
  • the infusion given at any dose level is dependent upon the toxicity achieved after each. Hence, if Grade II toxicity was reached after any single infusion, or at a particular period of time for a steady rate infusion, further doses should be withheld or the steady rate infusion stopped unless toxicity improved.
  • Increasing doses of bisanthrapyrazoles in combination with an anti-cancer drug is administered to groups of patients until approximately 60% of patients show unacceptable Grade III or IV toxicity in any category. Doses that are 2/3 of this value could be defined as the safe dose.
  • SMA- 12- 100 liver and renal function tests
  • coagulation profile and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms.
  • appropriate biological markers in serum should be monitored (e.g., CEA, CA 15-3, pi 85 for breast cancer, and CA 125, pi 85 for ovarian cancer).
  • the patients should be examined for appropriate tumor markers every 4 weeks, if initially abnormal, with twice weekly CBC, differential and platelet count for the 4 weeks; then, if no myelosuppression has been observed, then weekly. If any patient has prolonged myelosuppression, bone marrow examination is advised to rule out the possibility of tumor invasion of the marrow as the cause of pancytopenia.
  • a coagulation profile shall be obtained every 4 weeks.
  • An SMA-12-100 shall be performed weekly.
  • Pleural/peritoneal effusion may be sampled 72 hours after the first dose, weekly thereafter for the first two courses, then every 4 weeks until progression or off study.
  • Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by the disappearance of all measurable disease for at least a month. Whereas a partial response may be defined by a 50% or greater reduction of the sum of the products of perpendicular diameters of all evaluable tumor nodules or at least 1 month with no tumor sites showing enlargement. Similarly, a mixed response may be defined by a reduction of the product of perpendicular diameters of all measurable lesions by 50% or greater with progression in one or more sites.
  • Pleural/Peritoneal Fluids X X 5 X
  • chest X-rays may be performed at 72 hours after first dose, then prior to each treatment administration.
  • Fluids may be assessed 72 hours after the first dose, weekly for the first two courses and then every 4 weeks thereafter.
  • EXAMPLE 8 Materials and Methods for Examples 9-16 pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington,
  • kinetoplast plasmid DNA from TopoGEN (Columbus, OH).
  • HindIII was from Invitrogen (Burlington, Canada). Unless indicated, other chemicals were from Sigma (Oakville, Canada).
  • the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96® AQueous One Solution Cell Proliferation Assay kit was obtained from Promega (San Luis Obispo, CA). Losoxantrone and piroxantrone were obtained from the National Cancer Institute (Bethesda, MD). The linear least squares analysis was done with SigmaStat, (Systat, Point Richmond, CA).
  • AP-3, AP-7, AP-IO and AP- 12 have been described (Showalter et ah, 1987; Showalter et ah, 1986a).
  • the chloroanthrapyrazole derivatives AP-3 and AP-7 were prepared from the commercially available 1,4- and 1,5-dichloroanthroquinone by reaction with 2-[(hydrazinoethyl)amino]ethanol in refluxing acetonitrile (Showalter et ah, 1986b).
  • AP-12 was prepared by reaction of 1 ,4-dichloroanthroquinone with 2-hydrazinoethanol at 110 0 C.
  • the N-methylated derivatives AP-I and AP-9 were prepared by reaction of the corresponding unmethylated compounds (AP-3 and AP-7) with formaldehyde in formic acid at 110 0 C.
  • the N,N'- disubstituted anthrapyrazoles AP-IO, AP-I l, AP-13 and AP-14 were prepared from the corresponding chloroanthrapyrazoles by reaction with excess N,N-dimethylethylenediamine at reflux.
  • Chlorinated derivatives AP-2, AP -4, AP-6 and AP-8 were prepared from the corresponding hydroxy compounds by reaction with excess thionyl chloride at room temperature.
  • K/VP.5 cells a 26-fold etoposide -resistant K562-derived sub-line with decreased levels of topoisomerase Il ⁇ mRNA and protein
  • DMEM Dulbecco's Modified Eagle Medium, Invitrogen, Burlington, Canada
  • FCS fetal calf serum
  • the effect of converting the secondary amine group on the pyrazole side chain into a tertiary methyamine was mixed.
  • the growth inhibitory effect was increased, but for the AP-3/AP-1, AP-4/AP-2, AP-8/AP-6, AP-7/AP-9 and AP- 10/ AP- 11 pairs for this same substitution, growth inhibitory effects were either unchanged or slightly reduced. It can also be seen from the AP-IO/ AP-13, AP-11/AP- 14 pairs that moving the amino side chain from the 5- to the 7-ring position decreased or did not affect growth inhibitory effects, respectively.
  • topoisomerase II poisons One method by which cancer cells increase their resistance to topoisomerase II poisons is by lowering their level or activity of topoisomerase II (Ritke et al., 1994a; Fortune and Osheroff, 2000). With less topoisomerase II in the cell, cells produce fewer DNA strand breaks and topoisomerase II poisons are less lethal to cells. These cell lines provide a convenient way to test whether a drug that inhibits topoisomerase II acts as a topoisomerase II poison (Hasinoff et al., 2005).
  • KTVP.5 cell line with acquired resistance to etoposide contained one-fifth the topoisomerase Il ⁇ content of the parental K562 cells (Ritke and Yalowich, 1993; Ritke et al, 1994a; Ritke et al, 1994b; Fattman et al, 1996).
  • the resistance factor was calculated from the ratio of the IC 50 value for the K/VP.5 cell line divided by that for the K562 cell line. b Data from reference (Liang et al, 2006).
  • kDNA consists of highly catenated networks of circular DNA. Topoisomerase Il ⁇ decatenates kDNA in an ATP-dependent reaction to yield individual minicircles of DNA.
  • the 20 ⁇ l reaction mixture contained 0.5 mM ATP, 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl 2 , 30 ⁇ g/ml bovine serum albumin, 50 ng kDNA, test compound (0.5 ⁇ l in dimethyl sulfoxide) and 20 ng of topoisomerase Il ⁇ protein (the amount that gave approximately 80% decatenation).
  • test compound 0.5 ⁇ l in dimethyl sulfoxide
  • 20 ng of topoisomerase Il ⁇ protein the amount that gave approximately 80% decatenation.
  • the assay incubation was carried out at 37°C for 20 min and was terminated by the addition of 12 ⁇ l of 250 niM Na 2 EDTA. Samples were centrifuged at 8000 g at 25°C for 15 min and 20 ⁇ l of the supernatant was added to 180 ⁇ l of 600-fold diluted PicoGreen dye (Molecular Probes, Eugene, OR) in a 96-well plate. The fluorescence, which was proportional to the amount of kDNA, was measured in a Fluostar Galaxy (BMG, Durham, NC) fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
  • BMG Fluostar Galaxy
  • Topoisomerase Il-cleaved DNA complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate
  • the 20 ⁇ l cleavage assay reaction mixture contained 100 ⁇ M of the drug, 150 ng of topoisomerase Il ⁇ protein, 80 ng pBR322 plasmid DNA (MBI Fermentas, Burlington, Canada), 0.5 rnM ATP in assay buffer (10 rnM Tris-HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl 2 , 2.5% (v/v) glycerol, pH 8.0, and drug (0.5 ⁇ l in dimethyl sulfoxide).
  • the order of addition was assay buffer, DNA, drug, and then topoisomerase Il ⁇ .
  • the reaction mixture was incubated at 37°C for 10 min and quenched with 1% (v/v) SDS/25 mM Na 2 EDTA.
  • the reaction mixture was treated with 0.25 mg/ml proteinase K (Sigma) at 55°C for 30 min to digest the protein.
  • the linear pBR322 DNA cleaved by topoisomerase Il ⁇ was separated by electrophoresis (2 h at 8 V/cm) on a TAE (Tris base (4 mM)/glacial acetic acid (0.11% (v/v))/Na 2 EDTA (2 mM) buffer)/ethidium bromide (0.5 ⁇ g/ml)/agarose gel (1.2%, wt/v)). Ethidium bromide was used in the gel and running buffer in order that the inhibition of relaxation activity could be measured along with formation of cleaved linear DNA.
  • TAE Tris base (4 mM)/glacial acetic acid (0.11% (v/v))/Na 2 EDTA (2 mM) buffer
  • ethidium bromide 0.5 ⁇ g/ml
  • agarose gel (1.2%, wt/v)
  • the DNA in the gel was imaged by its fluorescence on a Alpha Innotech (San Leandro, CA) Fluorochem 8900 imaging system equipped with a 365 nm UV illuminator and a CCD camera.
  • doxorubicin and the other anthracyclines are thought to be cytotoxic by virtue of their ability to stabilize a covalent topoisomerase II-DNA intermediate (the cleavable complex) and act as what are called topoisomerase II poisons.
  • Topoisomerase II alters DNA topology by catalyzing the passing of an intact DNA double helix through a transient double-stranded break made in a second helix and is critical for relieving torsional stress that occurs during replication and transcription and for daughter strand separation during mitosis (Fortune and Osheroff, 2000; Li and Liu, 2001).
  • DNA cleavage assay experiments (Burden et al, 2001) as the inventors previously described (Hasinoff et ah, 2006) were carried out using 100 ⁇ M etoposide as a control to see whether 100 ⁇ M of the test compounds stabilized the cleavable complex. As shown in FIG.
  • ⁇ T m DNA thermal melt temperature
  • sonicated calf thymus DNA 5 ⁇ g/ml
  • 10 mM Tris-HCl buffer (pH 7.5) in a Cary 1 (Varian, Mississauga, Canada) double beam spectrophotometer by measuring the absorbance increase at 260 nm upon the application of a temperature ramp of l°C/min.
  • the maximum of the first derivative of the absorbance-temperature curve was used to obtain the ⁇ T m .
  • Doxorubicin (2 ⁇ M) which is a strong DNA intercalator, was used as a positive control (Priebe et al., 2001). Under limiting conditions the value of ⁇ T m is directly proportional to the logarithm of the equilibrium constant for ligand binding to DNA (McGhee, 1976) and thus the value of ⁇ T m was used directly in the free energy correlation analyses.
  • Doxorubicin (2 ⁇ M) which is a well-known DNA intercalating drug, was used as a control and was observed to increase the ⁇ T m of sonicated DNA by 13.2 0 C from 71.0 0 C. Under limiting conditions the value of ⁇ T m is 90 directly proportional to the logarithm of the equilibrium constant for ligand binding to DNA (McGhee, 1976) and thus the value of ⁇ T m was used directly in the free energy correlation analyses.
  • the ⁇ T m was increased, but for the AP-3/AP-1, AP-4/AP-2, AP-8/AP-6, AP-7/AP-9 and AP-lO/AP-11 pairs for this substitution, the ⁇ T m values were essentially unchanged. It can also be seen from the AP-10/AP-13, AP-11 /AP- 14 pairs that moving the amino side chain from the 5- to the 7-ring position decreased ⁇ T m which suggests that having the two amino side chains on the same side of the anthrapyrazole as in losoxantrone, piroxantrone, and mitoxantrone favours binding to DNA.
  • the resistance factor was calculated from the ratio of the /C 50 value for the KTVP.5 cell line divided by that for the K562 cell (b) Bisanthrapyrazoles
  • the concentration dependence of ⁇ T m was measured from 0.1 to 2 ⁇ M anthrapyrazole or bisanthrapyrazole as described above in 10 mM Tris-HCl buffer (pH 8.0) at a DNA concentration of 20 ⁇ M (base pair basis).
  • the slopes of the plots for monomer AP9 were compared with that of the bisanthrapyrazoles IA- IE using a t-test comparison of the slopes. (Jones, D., Pharmaceutical Statistics; Pharmaceutical Press: London, 2002, p. 585).
  • FIG. 12A The slopes ⁇ SEM are plotted in FIG. 12B.
  • a comparison of the slopes by t-test showed that all bisanthrapyrazoles but compound 1 (p > 0.5, not significant) had slopes that were significantly different (p ⁇ 0.001) than monomer AP9. From the data in FIG.
  • the following protocol can be used to model DNA with both anthrapyrazoles and bisanthrapyrazoles to evalute the binding ability and positioning of these compounds. (Liang et ah, 2006, incorporated herein in its entirety).
  • the following methods can also be expanded to model any putative intercalator. While the protocols recited below pertain to anthrapyrazoles, they may be suitably modified to examine bisanthrapyrazoles and any other intercalator. Structures of tested anthrapyrazoles are shown:
  • AP9 and the bisanthrapyrazoles were docked into the doxorubicin binding site of a 6 bp x-ray crystal structure of 2 molecules of doxorubicin bound to double stranded DNA, d(TGGCCA)/doxorubicin, (world wide web at .rcsb.org/pdb/ ; PDB ID: 1DA9) (Leonard et al., 1993) using the genetic algorithm docking program GOLD version 2.2 or 3.1 (CCDC Software, Cambridge, UK) using the default GOLD parameters and atom types and with 100 starting runs (Verdonk et al., 2003). GOLDScore was used as the fitness function with flipping options of amide bonds, planar and pyramidal nitrogens and internal hydrogen bonds being allowed. No early termination was allowed.
  • the 1DA9 x-ray structure shows that the first and second base pairs buckle out to accommodate bound doxorubicin (Leonard et al., 1993; Berman et al., 2000).
  • the 1DA9 x-ray structure of the doxorubicin-DNA complex was used for the docking experiments, rather than constructing DNA in SYBYL because it was reasoned that this DNA structure would be a more realistic model for binding of anthrapyrazoles and bisanthrapyrazoles because of their structural similarity to doxorubicin.
  • the protonated anthrapyrazoles were first geometry optimized with the Tripos force field using a conjugate gradient with a convergence criterion of 0.01 kcal/mol and Gasteiger-Huckel charges and a distance-dependent dielectric constant.
  • the SYBYL CONFORT module was then used to find the lowest energy conformation.
  • the DNA structure was prepared by removing one of the bound doxorubicin molecules and removing all water molecules to avoid potential interference with the docking. Hydrogens were added to the DNA and the SYBYL Biopolymer module and sometimes used to add Kollman-All charges to the DNA.
  • the binding site was defined as being within 5 A of the reference ligand or defined using an atom in the center of the DNA molecule and was large enough that it encompassed both of the doxorubicin-binding sites.
  • the anthrapyrazoles, losoxantrone, piroxantrone and mitoxantrone were all docked into the DNA structure to obtain the top 10 scoring GOLDScore structures for each molecule. Each of these structures were then rescored using a local optimization
  • the GOLD fitness function (GOLDScore) is the sum of its four components: DNA-ligand hydrogen bond energy (external H-bond); DNA-ligand van der Waals
  • vdw energy (external vdw); ligand internal vdw energy (internal vdw); and ligand torsional strain energy (internal torsion) and were output for QSAR correlation analysis.
  • the ligand intramolecular hydrogen bond energy (internal H-bond) term was zero for each docked ligand as there were no internal H-bonds found in the docked structures.
  • doxorubicin was docked back into the DNA structure with a heavy atom root-mean-squared distance of 1.3 A compared to the x-ray structure (Leonard et ah, 1993). Values of 2.0 A or less in the extensive GOLD test set are considered to be good (Verdonk et ah, 2003).
  • FIG. 9A The structure of AP-10, the most potent anthrapyrazole, docked into DNA is shown in FIG. 9A.
  • AP-10 shows both stacking interactions and H-bond interactions through both amino side chains with the DNA base pairs between which it intercalates. Additionally, the side chain hydroxyl group forms an H-bond with the oxygen of a DNA sugar residue.
  • FIG. 9B The conformation of all the anthrapyrazoles and mitoxantrone docked into DNA have been superimposed in FIG. 9B. Most of the anthrapyrazoles docked into the DNA with a similar configuration, both with respect to the pyrazole rings and the amino side chains. However, some of the docked anthrapyrazoles were slightly rotated about the central DNA axis.
  • FIG. 1OB shows that an increase in the internal energy of the anthrapyrazoles decreases cell growth inhibitory properties (and also in weaker binding to DNA as shown in FIG. 10D).
  • the internal energy term reflects the internal torsional and van der Waals increases in energy due to conformational changes that the anthrapyrazoles must make to be able to dock into DNA. Thus, it is reasonable that an increase in the internal energy is negatively correlated with both ⁇ T m and growth inhibitory effects.
  • the following protocol can be used to model DNA with both anthrapyrazoles and bisanthrapyrazoles to evalute the binding ability and positioning of these compounds. (Liang et ah, 2006, incorporated herein in its entirety).
  • the following methods can also be expanded to model any putative intercalator. While the protocols recited below pertain to anthrapyrazoles, they may be suitably modified to examine bisanthrapyrazoles and any other intercalator.
  • CoMFA and CoMSIA analysis requires that the 3D structures of the molecules be aligned to a core conformational template that is their presumed active form. The molecular conformations which were used for alignment were the conformers that had the best GOLDScore value after docking by GOLD.
  • the energy cutoff was 30 kcal/mol.
  • the alignment and lattice box used for the CoMFA calculation were also used to calculate similarity index fields for the CoMSIA analysis. Steric, electrostatic, hydrophobic, hydrogen bond donor and acceptor fields were evaluated in CoMSIA analysis. Similarity indices were computed using a probe atom with +1 charge, radius 1 A, hydrophobicity +1, hydrogen bond donating +1, hydrogen bond acceptor +1, attenuation factor ⁇ 0.3 for the Gaussian-type distance.
  • a partial least-squares (PLS) statistical approach which is an extension of multiple regression analysis in which the original variables are replaced by a set of their linear combinations, was used to obtain the 3D-QSAR results.
  • All models were investigated using the leave-one-out (LOO) method, which is a cross-validated partial least-squares method.
  • LOO leave-one-out
  • the CoMFA and CoMSIA descriptors were used as independent variables and pIC ⁇ or ⁇ T m were used as dependent variables to derive 3D-QSAR models.
  • the q (cross-validated correlation coefficient r ) and the optimum number of components (N) were obtained by the LOO method.
  • the final model (non- cross-validated conventional analysis) was developed and yielded the non-cross- validated correlation coefficient r 2 with the optimum number of components. Because it has been shown that losoxantrone was most closely related to the anthracenedione mitoxantrone and other topoisomerase II poisons in the NCI COMPARE analysis (Leteurtre et al, 1994), and because mitoxantrone has a core structure and side chains the same or similar to the other anthrapyrazoles, it was included in the analysis of the other 15 anthrapyrazoles studied.
  • topoisomerase Il ⁇ /C50 The lack of correlation with topoisomerase Il ⁇ /C50 does not mean that these compounds did not act on topoisomerase II as topoisomerase II poisons, but only that they did not act solely through their inhibition of the catalytic activity of topoisomerase Il ⁇ .
  • bisAP9_n 11 1 , 2, 3, 4, 5
  • Catalytic Hydrogenation of AP9-N3 - AP-9-N3 (0.438 g, 1.15 mmol) was dissolved in methanol (100 niL) and hydrochloric acid (2.530 mmol). Palladium on charcoal (0.082 g, 0.767 mmol) was added and the mixture was placed under a positive hydrogen atmosphere for 2 hours. The mixture was then filtered through a Celite pad. The methanol filtrate was condensed, extracted into chloroform with base, dried over sodium sulfate, condensed and dried on a vacuum pump, affording 0.485 g (1.367 mmol) of a yellow solid in 119% yield.
  • Electrospray ionization mass spectra were acquired on an Applied Biosystems API 2000 Triple Quadrupole mass spectrometer (Thornhill, Toronto, Canada) equipped with a syringe pump. Samples ( ⁇ 1 mM in acetonitrile) were injected into the ion source at a flow rate of 5 ⁇ L/min.
  • AP9 (7-chloro-2-[2-[(2- hydroxyethyl)methylamino] ethyl] anthra[l,9-c ⁇ i]pyrazol-6(2H)-one) was prepared from 1,5-dichloroanthraquinone as previously described (Liang et al, 2006) (incorporated herein by reference in its entirety).

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Abstract

L'invention porte sur de nouveaux bisanthrapyrazoles qui agissent vraisemblablement comme bisintercalateurs et inhibiteurs de la topoisomérase Ilα. Ces composés manifestent une inhibition puissante de la croissance des cellules cancéreuses, et certains bisanthrapyrazoles se lient à l'ADN plus étroitement que la doxorubicine, un médicament anticancer d'intercalation connu. L'invention porte également sur des techniques de modélisation moléculaire qui permettent de prédire la force de liaison à l'ADN de divers intercalateurs.
PCT/IB2008/002784 2007-04-13 2008-03-12 Bisanthrapyrazoles comme agents anticancer WO2009004496A2 (fr)

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US9266886B2 (en) 2014-02-03 2016-02-23 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US9481674B1 (en) 2016-06-10 2016-11-01 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US9663515B2 (en) 2014-11-05 2017-05-30 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US9796710B2 (en) 2014-10-14 2017-10-24 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US9845308B2 (en) 2014-11-05 2017-12-19 Vitae Pharmaceuticals, Inc. Isoindoline inhibitors of ROR-gamma
US10301261B2 (en) 2015-08-05 2019-05-28 Vitae Pharmaceuticals, Llc Substituted indoles as modulators of ROR-gamma
US10829481B2 (en) 2016-01-29 2020-11-10 Vitae Pharmaceuticals, Llc Benzimidazole derivatives as modulators of ROR-gamma
US10913739B2 (en) 2017-07-24 2021-02-09 Vitae Pharmaceuticals, LLC (121374) Inhibitors of RORγ
US11008340B2 (en) 2015-11-20 2021-05-18 Vitae Pharmaceuticals, Llc Modulators of ROR-gamma
US11186573B2 (en) 2017-07-24 2021-11-30 Vitae Pharmaceuticals, Llc Inhibitors of ROR gamma

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CN102603785A (zh) * 2012-02-27 2012-07-25 上海大学 萘并或蒽并吡唑衍生物及其合成方法
US10047085B2 (en) 2014-02-03 2018-08-14 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US9266886B2 (en) 2014-02-03 2016-02-23 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US11535614B2 (en) 2014-02-03 2022-12-27 Vitae Pharmaceuticals, Llc Dihydropyrrolopyridine inhibitors of ROR-gamma
US9624217B2 (en) 2014-02-03 2017-04-18 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US10807980B2 (en) 2014-02-03 2020-10-20 Vitae Pharmaceuticals, Llc Dihydropyrrolopyridine inhibitors of ROR-gamma
US10399976B2 (en) 2014-02-03 2019-09-03 Vitae Pharmaceuticals, Llc Dihydropyrrolopyridine inhibitors of ROR-gamma
US9796710B2 (en) 2014-10-14 2017-10-24 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
US10087184B2 (en) 2014-10-14 2018-10-02 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of RORγ
US9845308B2 (en) 2014-11-05 2017-12-19 Vitae Pharmaceuticals, Inc. Isoindoline inhibitors of ROR-gamma
US11001583B2 (en) 2014-11-05 2021-05-11 Vitae Pharmaceuticals, Llc Dihydropyrrolopyridine inhibitors of ROR-gamma
US9663515B2 (en) 2014-11-05 2017-05-30 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
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US10829448B2 (en) 2015-08-05 2020-11-10 Vitae Pharmaceuticals, Llc Substituted benzoimidazoles as modulators of ROR-γ
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US10829481B2 (en) 2016-01-29 2020-11-10 Vitae Pharmaceuticals, Llc Benzimidazole derivatives as modulators of ROR-gamma
US9481674B1 (en) 2016-06-10 2016-11-01 Vitae Pharmaceuticals, Inc. Dihydropyrrolopyridine inhibitors of ROR-gamma
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