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WO2000050117A2 - Composes, compositions et procedes pour la therapie photodynamique - Google Patents

Composes, compositions et procedes pour la therapie photodynamique Download PDF

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Publication number
WO2000050117A2
WO2000050117A2 PCT/US2000/004915 US0004915W WO0050117A2 WO 2000050117 A2 WO2000050117 A2 WO 2000050117A2 US 0004915 W US0004915 W US 0004915W WO 0050117 A2 WO0050117 A2 WO 0050117A2
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hydrogen
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WO2000050117A3 (fr
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Jeffrey M. Zaleski
Diwan Singh RAWAT
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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Priority to US09/913,924 priority Critical patent/US6828439B1/en
Priority to AU35032/00A priority patent/AU3503200A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/50—Organo-phosphines
    • C07F9/5027—Polyphosphines

Definitions

  • the present invention pertains generally to photodynamic therapy, as might be particularly useful in the treatment of cancers, viruses, and bacteria.
  • Photodynamic therapy has been used widely in the treatment of a variety of cancers, including breast metastases, gynecological tumors, cutaneous cancers, Karposi's sarcoma, and papillomatosis .
  • a chro ophoric dye molecule e.g., porphyrins, chlorins, pheophorbides, and phthalocyanines
  • the chromophore-containing dye molecule in its natural or ground state is a singlet (i.e., x ⁇ ) such that the two electrons in the highest occupied molecular orbital are paired.
  • the photoexcited ⁇ * state of the dye molecule decays non-radiatively to a triplet (i.e., 3 ⁇ *) state, which is lower in energy than the photoexcited singlet state of the dye molecule.
  • the dye molecule is then reacted with oxygen, which in its ground state is a triplet (i.e.,
  • Photofrin® which is commercially available from QLT Phototherapeutics of Vancouver, British Columbia, is an example of a porphyrin type of compound (i.e., hematoporphyrin dimer) that utilizes this bimolecular triplet state mechanism to achieve photodynamic therapy.
  • Photofrin® has been used to treat esophageal, lung, bladder, gastric, and cervical cancers.
  • some PDT studies have also been successful in treating viruses, such as papillomavirus, HIV, herpes simplex virus, measles and simian virus.
  • Another class of anticancer agents pertain to calicheamicin and esperamicin enediynes .
  • One feature of these systems is the unusual (Z) - 1 , 5-diyne-3- ene unit that undergoes Bergman cyclization to produce a 1, 4-benzenoid diradical .
  • This species provides the thermodynamic driving force for the DNA-cleaving reaction by promoting H-atom abstraction from the deoxyribose ring. Formation of the diradical intermediate can be triggered by the presence of reducing agents such as NADPH or dithiothrietol .
  • dynemicin-A is unique in that in addition to the reactive enediyne moiety, it also contains an anthraquinone chromophore that is responsible for the deep violet color of the molecule.
  • the proposed mechanism of action of dynemicin-A suggests that reduction of the anthraquinone subunit induces epoxide ring opening, followed by tautomerization and Bergman cyclization of the enediyne linkage to produce a reactive benzene diradical intermediate that affords DNA-strand scission through H-atom abstraction.
  • the present invention provides novel compounds, compositions, and methods for photodynamic therapy which function by a unimolecular mechanism.
  • the inventive compounds, compositions, and methods do not require the presence of oxygen as a co-reagent to function in photodynamic therapy. Since it has been found that the compounds, compositions, and methods of the present invention perturb DNA (as described below) , it is expected that the compounds, compositions, and methods of the present invention have significant utility in treating cancers as well as infections caused by microorganisms .
  • the compounds, compositions, and methods involve the formation of radical species for treating the cancers and/or infections.
  • the radicals are photochemically-induced (e.g., at visible wavelengths above 400 nm) .
  • a therapeutically effective amount of a compound capable of forming a radical upon exposure to light by a unimolecular mechanism e.g., in the absence of oxygen
  • is administered e.g., by injection
  • the compound is then irradiated at the site of action so as to induce radical formation.
  • the compounds can include metalloenediynes (i.e., transition metal complexes with metal chelating enediyne ligands) and/or transition metal complexes that bear at least one diazo functional group, such as, but not limited to, a terminal diazo group or as in a triazine (also generally referred to herein as "transition metal diazo compounds” or “transition metal diazo complexes” ) .
  • metalloenediynes i.e., transition metal complexes with metal chelating enediyne ligands
  • transition metal complexes that bear at least one diazo functional group, such as, but not limited to, a terminal diazo group or as in a triazine (also generally referred to herein as "transition metal diazo compounds” or “transition metal diazo complexes” ) .
  • the compounds, compositions, and methods of the present invention can be used in photodynamic therapy such that the presence of oxygen is not required in conjunction with light.
  • the compounds, compositions, and methods of the present invention can even function in hypoxic environments without compromising the selectivity of optical initiation.
  • desired compounds pursuant to the present invention exhibit high air and thermal stability, as well as high water solubility, which are particularly desirable attributes for photonucleases for photodynamic therapy applications.
  • the inventive compounds, compositions, and methods also permit triggering of the photochemical reactivity in a controlled manner for biological applications.
  • desired compounds can be synthetically obtained readily.
  • FIG. 1 schematically illustrates the synthesis of 1, 2, -bis (pyridine-3 -oxy) oct-4-ene-2, 6-diyne, i.e., compound "1", by nucleophilic S N 2 substitution.
  • FIG. 2 schematically illustrates metal complexation reactions for the preparation of (1 , 2-bis (pyridine-3- oxy) oct-4-ene-2 , 6-diyne) copper (I) , i.e., compound "2" and (1, 2-bis (pyridine 3-oxy) oct-4-ene-2 , 6- diyne) copper (II) , i.e., compound "3".
  • FIG. 3 illustrates a UN-Vis absorption spectrum of the enediyne ligand (compound 1), identified as “A”; (1, 2-bis (pyridine-3 -oxy) oct-4-ene-2 , 6-diyne) copper (I) , identified as “B” ; and (1 , 2-bis (pyridine 3-oxy) oct-4-ene- 2 , 6-diyne) copper (II) , identified as "C” .
  • FIG. 4A represents the crystal structure of the Pd (0) bis [1, 2-bis (diphenylphosphinoethynyl) benzene] enediyne compound .
  • FIG. 4B represents a thermal ellipsoidal plot of the compound shown in FIG. 4A illustrating the planarity of the enediyne ligand and out-of-plane disposition (0.89 A) of the Pd(O) center (phenyl rings removed for clarity) .
  • FIG. 5A shows the chemical structure of 3-Hydroxy- 1, 2, 3-benzotriazine-4 (3H) -one, also identified herein as compound "4" .
  • FIG. 5B shows the chemical structure of Fe(3- Hydroxy-1, 2, 3-benzotriazine-4 (3H) -one) 3 , also identified herein as compound "5" .
  • FIG. 6 illustrates an energy level diagram showing the relative energies of the optical LMCT transitions of compound 5 and the charge-separated Fe 2+ /L *+ state.
  • FIG. 7 depicts electronic absorption profiles of anaerobic photolyses of 0.1 nM acetonitrile solutions of compound 5 at: (a) 345 nm; and (b) 455 nm.
  • FIG. 8 illustrates the photoinduced DNA-cleavage of 30 ⁇ M/bp of pUC 118 plasmid DNA by 300 ⁇ M of compound 5 following 400 nm photolysis for 12 hours at 20 °C pursuant to 2 % agarose gel electrophoresis.
  • FIG. 9 illustrates the photoinduced DNA-cleavage of 50 ⁇ M pUC 118 plasmid DNA by compound 2 following 400 nm photolysis for 12 hours at 20 °C pursuant to 2 % agarose gel electrophoresis.
  • FIG. 10 illustrates DNA-cleavage of 50 ⁇ M pUC 118 plasmid DNA by bis (9-diazo-4 , 5-diazafluorene) copper (II) nitrate following 455 nm photolysis for 1 hour at 20 °C pursuant to 2 % agarose gel electrophoresis .
  • FIG. 11 represents the crystal structure of the bis(9-diazo-4, 5-diazafluorene) copper (II) nitrate compound .
  • FIG. 12 illustrates an exemplary metalloenediyne- porphyrin synthesis, in accordance with the present invention.
  • FIG. 13 illustrates an exemplary synthetic approach for preparing novel Ru(II) metalloenediynes, in accordance with the present invention.
  • FIG. 14 illustrates the synthesis of the compound shown in FIGS. 4A and 4B (i.e., Pd(dppeb) 2 ).
  • the present invention is predicated, at least in part, on providing compounds, compositions, and methods for photodynamic therapy in which radicals (including diradicals and/or monoradical cations or anions) are formed in the presence of light .
  • radicals including diradicals and/or monoradical cations or anions
  • the term "radical” refers to at least one unpaired electron, and in the case of two unpaired electrons, they can be on the same carbon center or different carbon centers. Accordingly, the terms “radicals” and “diradicals” encompass carbenes in the context of the present invention.
  • the compounds form intermediates (that bear the radicals) in the presence of light by a unimolecular mechanism. • By virtue of the unimolecular mechanism, the presence of oxygen is not required in the photodynamic therapy applications of the inventive compounds, compositions, and methods.
  • the radicals perform hydrogen atom abstraction (also referred to as "H atom abstraction”) .
  • the radical-containing intermediates perform hydrogen atom abstraction (also referred to as "H atom abstraction") .
  • the radical-containing intermediates perform hydrogen atom abstraction (also referred to as "H atom abstraction") .
  • the compounds, compositions, and methods of the present invention can be utilized in photodynamic therapy applications involving, for example, the treatment of cancers and/or infections caused by microorganisms.
  • the types of cancer that can be treated using the inventive compounds, compositions, and methods include, but are not limited to, breast metastases, gynecological tumors, cutaneous cancers, Karposi's sarcoma, papillomatosis, and the like.
  • the compounds, compositions, and methods of the present invention can also be utilized to combat microorganisms, such as, for example, fungi, bacteria, viruses, protozoa, and the like.
  • the inventive compounds can also be used to study the life cycle of, for example, cancers, viruses, and microorganisms.
  • the skilled artisan can infer that the interacting molecule (to which the inventive compounds bind) is critical or important in regulating life cycle, cell permeability, or the like.
  • the inventive compounds may also be useful for determining the. mechanism of action of other compounds that work on the same cancer, virus, organism, or the like, for example, if one obtains the same result with or without addition of other pharmacoactive compounds, then a strong suggestion is that the two compounds regulate the same event.
  • the inventive compound can be used as a topical antiseptic on laboratory and other surfaces.
  • the inventive compounds can be used in separating non-transformed cells from a population of transformed cells.
  • the compounds, compositions, and methods of the present invention can be used locally to treat cancers and/or infections caused by microorganisms . For example, after determining a locus in a patient (e.g., a mammal) in which a microorganism or cancer is situated, a compound which is capable of forming a radical-containing intermediate by a unimolecular mechanism upon exposure to light is locally delivered to the locus (e.g., by injection) .
  • the compound can be combined with a suitable pharmaceutically acceptable carrier (as will be appreciated readily by one of ordinary skill in the art), especially injectable carriers, to form a pharmaceutical composition.
  • Visible radiation can then be initiated at the locus in the presence of the compound (as will be appreciated by one of ordinary skill in the art, for example, by a fiber optic probe connected to an external illuminating source or otherwise surgically exposing the locus to light at the desired wavelength) to generate the radical photochemically .
  • the wavelength of the light is preferably at least about 400 nm, more preferably at least about 600 nm, and even more preferably at least about 700 nm, such that tissue penetration is enhanced.
  • compounds that generate radicals at wavelengths of at least 400 nm preferably at least about 600 nm, more preferably at least 700 nm
  • suitable absorptivity e.g., exhibiting extinction coefficients of at least about 10 M ' 1 , preferably at least about 100 M ' 1 , more preferably at least about 1000 M "1 cm "1 ) are preferred.
  • compounds that are particularly useful in the context of the present invention for forming a radical-containing intermediate by a unimolecular mechanism include, but are not limited to, metalloenediyne complexes (comprising transition metals) as well as transition metal diazo complexes. Both the metalloenediynes and the transition metal diazos form radicals in the presence of light without requiring a co- reagent (such as oxygen)..
  • the transition metals utilized in the metalloenediyne and transition metal diazo complexes preferably are redox active transition metals, such as, for example, Cu(I), Cu(II), Ru, V, Ti, Zr, lanthanides, Fe(III), Pt(II), Pd(0), and Pd(II) .
  • the enediyne and diazo functional groups include the sites of radical formation, the other portions of the respective metalloenediyne and transition metal diazo molecules are selected to optimize the photactivation properties of the compounds useful in the context of the present invention.
  • the transition metal of both the metalloenediyne and diazo complexes are selected to enhance the photoselectivity and thermal properties (e.g., desirably, the radicals form in the presence of light at physiological temperatures) of the compounds. While not wishing to be bound by any particular theory, it is believed that the thermal chemistry and photactivation properties of the novel metalloenediyne and transition metal diazo complexes are based on metal complex geometry and/or metal ligand charge-transfer states.
  • the transition metal is reducing (i.e., putting electron density in the ⁇ orbital) or oxidizing the enediyne linkage (i.e., removing an electron from the triple bond) .
  • the transition metal reduces the bond order and converts -the triple bond to a double bond thereby converting the compound to a diradical-containing transition state made of three double bonds from which the cyclization can be completed (e.g., by way of H-atom abstraction as perhaps from a DNA backbone) .
  • redox active transition metals are particularly desirable.
  • the transition metal diazo complexes it is also believed that the transition metal governs the energy of the optical absorption band of the diazo functional group.
  • the transition metal is selected to activate the di-nitrogen release that results in the formation of the radical- containing intermediate, which has the ability to perform H-atom abstraction with, for example, DNA.
  • redox active transition metals are particularly desirable for activating di-nitrogen release.
  • metalloenediynes in accordance with an aspect of the present invention, a novel compound is provided having the following formula:
  • M is a metal selected from Ti, V, Mn,
  • n is an integer from 1-3.
  • L is a ligand of the formula:
  • a and A 1 are optionally present spacers which can be the same or different and each is independently (CR 12 R 13 ) m , wherein m is an integer from 0 to 6 (e.g., 0 to 3) . If present, A and/or A 1 optionally be substituted. For example, A and A 1 can be optionally substituted such that
  • R and R can be the same or different and each is hydrogen, halogen, amino, nitro, cyano, azido, a solubilizing group, or an organic group, such as, for example, an alkyl (e.g., C ⁇ . -C 6 ) or an aryl (e.g., phenyl).
  • a solubilizing group include, but are not limited to, a hydroxyl, an amino or acid addition salt thereof, an ammonium salt (e.g., quaternary ammonium salt), sulfonic acid or salt thereof, or carboxylic acid or salt thereof .
  • R 12 and/or R 13 is an organic group (e.g., alkyl or aryl)
  • the organic group can be unsubstituted or substituted with, for example, a halogen, nitro, cyano, azido, an organic group (e.g., alkyl or aryl) or solubilizing group, such as, for example, a hydroxyl, an amino or acid addition salt thereof, an ammonium salt (e.g., quaternary ammonium salt) , sulfonic acid or salt thereof, or carboxylic acid or salt thereof .
  • a halogen nitro, cyano, azido
  • an organic group e.g., alkyl or aryl
  • solubilizing group such as, for example, a hydroxyl, an amino or acid addition salt thereof, an ammonium salt (e.g., quaternary ammonium salt) , sulfonic acid or salt thereof, or carboxylic acid or salt thereof .
  • B and B 1 are the same or different and each is a nitrogen-, oxygen-, sulfur-, or phosphorus-containing substituent capable of complexing with M.
  • the dotted line between B and B 1 represents an optional covalent bond.
  • the N- , 0-, S-, or P- containing groups capable of complexing with M can be cyclic or non- cyclic and can be substituted or unsubstituted.
  • R 1 and R 2 can be the same or different and each independently can be hydrogen, a linear or branched alkyl (e.g., Cx-Cs) , an aralkyl, an aryl, a halogen, a nitro, or a cyano, or R 1 and R 2 together with the carbons to which they are bonded can comprise an aryl, a heterocycle, a cycloalkeny . l, or a macrocycle, wherein R 1 and R 2 can be unsubstituted or substituted.
  • a linear or branched alkyl e.g., Cx-Cs
  • an aralkyl e.g., an aralkyl
  • an aryl e.g., a halogen, a nitro, or a cyano
  • R 1 and R 2 together with the carbons to which they are bonded can comprise an aryl, a heterocycle, a cycloalkeny . l,
  • cycloalkenyls can be in the form of a cyclohexene ring, cycloheptene ring, cycloheptadiene ring, cyclohexadiene ring, cyclooctene ring, cyclooctodiene ring, cyclooctotriene ring, or the like.
  • M can be complexed with at least one additional ligand other than a ligand of the formula:
  • a dimer an oligomer, or a polymer of the metalloenediyne compound.
  • At least one of B and B 1 is a nitrogen-containing group capable of complexing with M, such as, for example, a nitrogen-containing group characterized by one of the following formulas :
  • M is copper (i.e., Cu(I) or Cu(II)), m is 1, n is 2, and/or R 1 and R 2 are hydrogen.
  • B or B 1 is a phosphorus- containing group capable of complexing with M.
  • the phosphorus-containing group can be a diarylphosphine such as a diphenylphosphine .
  • Some exemplary compounds are of the formulas :
  • Additional exemplary enediyne ligands include, but are not limited to, ligands characterized by the following formulas:
  • R 1 and R 2 are independently selected from the group consisting of hydrogen, an alkyl (e.g., C ⁇ -C 6 ) , an aryl, and an aralkyl, or, alternatively, when R 1 and R 2 (together with the carbons to which they are bonded) comprise an aryl or heterocycle, at least one of R 1 or R 2 can be optionally substituted with a substituent such as, for example, a halogen, a nitro, a cyano.
  • a substituent such as, for example, a halogen, a nitro, a cyano.
  • B and B 1 are not both diphenylphosphine groups, although this embodiment may be used in the methods and compositions of the present invention.
  • M can be optionally complexed with at least one additional auxiliary ligand other than an enediyne.
  • the additional ligand can be, for example, a substituent of the formula:
  • Q 2 is an aryl, a heterocycle, a macrocycle, or a C 2 -C 6 (e.g., C 2 -C 3 ) alkyl spacer, wherein the aryl, heterocycle, or macrocycle is monocyclic or polycyclic and Q 2 is unsubstituted or substituted.
  • at least one hydrogen of the alkyl spacer is optionally substituted, for example, with a C ⁇ . -C 6 alkyl.
  • Q 2 is an aryl.
  • the heterocycle can be bicyclic or polycyclic.
  • a polycyclic heterocycle suitable for Q 2 is characterized by the formula:
  • the additional ligand can be a substituent characterized by the formula:
  • the inventive compound can include at least one additional ligand of the formula:
  • more than one additional ligand can be incorporated into a compound according to the invention.
  • the optional at least one additional ligand can also include a ligand of the formula:
  • R 11 is a hydrogen or a straight chain or branched alkyl ; d is zero or 1 ;
  • B 2 and B 3 are the same or different and are independently selected from nitrogen and sulfur;
  • Z is a contiguous linker which, together with X, B 2 , and the carbons to which they are bonded, forms a 5- or a 6- membered heterocyclic ring;
  • L is a contiguous linker which, together with Y, B 3 , and the carbons to which they are bonded, forms a 5- or a 6- membered heterocyclic ring; the dotted lines represent double bonds optionally present in said 5- or 6- membered heterocyclic ring;
  • X is N, NR 5 , or CR 5 , wherein R 5 is hydrogen, halogen, or straight chain or branched alkyl;
  • Y is N, NR 4 , or CR 4 , wherein R 4 is hydrogen, halogen, or straight chain or branched alkyl;
  • Q 1 is an organic moiety which includes a diazo group capable of photochemically forming a radical species by the loss of N 2 .
  • the at least one additional ligand can include at least one ligand of the formula: wherein a-d are the same or different and each is hydrogen, halogen, alkyl, OR 10 SR 10 nitro, and cyano, wherein R 10 is hydrogen or straight chain or branched alkyl .
  • M when n is 1 or 2, M can be optionally complexed with at least one additional ligand that comprises a macrocycle, such as, for example, a porphyrin, a porphyrazine, a chlorin, a phthalocyanine, a texaphrin, a cyclam, or a crown ether.
  • a macrocycle such as, for example, a porphyrin, a porphyrazine, a chlorin, a phthalocyanine, a texaphrin, a cyclam, or a crown ether.
  • An especially preferred macrocycle is a porphyrin or a porphyrazine.
  • the macrocycle can be a porphyrazine of the formula :
  • thiols located within the brackets defining the porphyrazine are uncomplexed (e.g., with an additional metal) or are optionally complexed with at least one additional metal complex of the formula:
  • M 1 and M 2 can be the same or different and each can be a metal selected from the group consisting of Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu, Zr, Nb, Mo, Te, Ru, Rh, Pd, Ag, Sn, Ta, W, Re, Os, Ir, Pt , and Au.
  • Q 3 is an enediyne of the formula :
  • a and A 1 are the same or different and each is independently (CR 12 R 13 ) m , wherein m is an integer from 0 to 6 and wherein R 12 and R 13 are the same or different and each is hydrogen, halogen, nitro, cyano, azido, an optionally substituted organic group, or a solubilizing group ; n is an integer from 1-3;
  • B and B 1 are the same or different and each is a substituent comprising a nitrogen-, oxygen-, sulfur-, or phosphorus-containing group capable of complexing with M, wherein a covalent bond can be optionally present between B and B 1 ;
  • R 1 and R 2 are the same or different and each is independently a hydrogen, a linear or branched alkyl (e.g., C ⁇ -C 6 ) , an aralkyl, an aryl, a halogen, a nitro, or a cyano, or R 1 and R 2 together with the carbons to which they are bonded comprise an aryl, a heterocycle, or a macrocycle, wherein R 1 and R 2 is unsubstituted or substituted.
  • a linear or branched alkyl e.g., C ⁇ -C 6
  • R 1 and R 2 are the same or different and each is independently a hydrogen, a linear or branched alkyl (e.g., C ⁇ -C 6 ) , an aralkyl, an aryl, a halogen, a nitro, or a cyano, or R 1 and R 2 together with the carbons to which they are bonded comprise an aryl, a heterocycle, or a
  • the metalloenediyne can be in the form of a dimer, an oligomer, or a polymer.
  • the compound can be, for example, a dimer of the formula : wherein Q is an enediyne of the formula:
  • the metalloenediyne is one of the following compounds:
  • transition metal diazo complexes in accordance with another aspect of the present invention, a novel compound is provided having the following formula :
  • M is a metal that is capable of complexing (as indicated) , and is preferably selected from the group consisting of Ti, V, Mn, Fe, Co, Ni , Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu, Zr, Nb, Mo, Te, Ru, Rh, Pd, Ag, Sn, Ta, W, Re, Os, Ir, Pt , and Au. Meanwhile, n is an integer from 1 to 3.
  • the respective ring systems comprising B 2 and B 3 can be the same or different and can be separated by a bond or a one-carbon spacer (e.g., CH 2 R 1:L ) . As a result, it is preferred that d is zero or 1.
  • B 2 and B 3 can be the same or different and are independently selected from nitrogen and sulfur.
  • R 11 can be hydrogen or a straight chain or branched alkyl (e.g., C ⁇ -C 6 ) .
  • L and Z are contiguous linkers, which, together with Y, B 3 , and the carbons to which they are attached (in the case of L) or together with X, B 2 , and the carbons to which they are bonded (in the case Z) form a 5 or 6- membered heterocyclic ring.
  • X is N, NR 5 , or CR 5 , wherein R 5 is hydrogen, halogen, or C ⁇ -C 6 straight chain or branched alkyl.
  • Y is N, NR 4 , or CR 4 , wherein R 4 is hydrogen, halogen, or C ⁇ -C 6 straight chain or branched alkyl.
  • the dotted lines represent double bonds optionally present in the 5- or 6- membered heterocyclic ring.
  • L is CH
  • Y and B 3 are each nitrogen
  • the dotted lines represent double bonds in the ring comprising L, Y and B 3
  • the resulting ring is a five membered heterocylic ring, namely, imidazole .
  • Y is CH
  • B 3 is nitrogen
  • the dotted lines represent double bonds in the ring comprising L, Y and B 3
  • the resulting ring is a six-membered heterocyclic ring, namely, pyridine.
  • Q 1 represents an organic moiety which includes a diazo group (such as, but not limited to, a terminal diazo or a diazo that is endocyclically situated, as perhaps in a triazine) which is capable of photochemically forming a radical by the loss of N 2 .
  • a diazo group such as, but not limited to, a terminal diazo or a diazo that is endocyclically situated, as perhaps in a triazine
  • M can be complexed with at least one additional auxiliary ligand other than a diazo-containing ligand, e.g., other than a ligand of the formula :
  • the metal is platinum, n is 1, and/or at least one of B 2 and B 3 is nitrogen.
  • Q 1 can be, for example, a substituent represented by the formula:
  • R 6 -R 9 are the same or different and each can be, for example, hydrogen, halogen, cyano, nitro, and straight chain or branched alkyl (e.g., C ⁇ -C 6 ) .
  • the dotted lines in the ring defining Q 1 represent double bonds that are optionally present in the ring.
  • Q 1 can be a substituent represented by the formula:
  • M can be optionally complexed with at least one additional ligand, such as an additional ligand of the formula: wherein B 4 and B 5 are the same or different and each is nitrogen, oxygen, sulfur, or phosphorus; and Q 2 is an aryl, a heterocycle, a macrocycle, or a C 2 -C 3 alkyl spacer, wherein at least one hydrogen of the alkyl spacer is optionally substituted with an alkyl (e.g., C ⁇ -C 6 ) •
  • Q 2 is an aryl.
  • the additional ligand can be of the formula :
  • a-d are the same or different and each is selected from hydrogen or straight chain or branched alkyl (e.g. , C ⁇ -C 6 ) .
  • the at least one additional ligand can also include, for example, at least one ligand of the formula
  • a-d are the same or different and each is hydrogen, halogen, alkyl, OR 10 SR 10 nitro, and cyano, wherein R 10 is hydrogen or straight chain or branched alkyl .
  • the additional ligand can be a macrocycle, such as, for example, a porphyrin, a porphyrazine, a chlorin, a phthalocyanine, a texaphrin, a cyclam, or a crown ether.
  • a preferred macrocycle is a porphyrin or a porphyrazine, such as, but not limited to, a porphyrazine of the formula:
  • thiols located within the brackets defining the porphyrazine are uncomplexed or are optionally complexed with at least one additional metal complex of the formula :
  • M is a metal selected from the group consisting of Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu, Zr, Nb, Mo, Te, Ru, Rh, Pd, Ag, Sn, Ta, W, Re, Os, Ir, Pt, and Au; d is zero or 1;
  • R 11 is a hydrogen or a straight chain or branched alkyl (e.g. , C ⁇ -C 6 ) ;
  • B 2 and B 3 are the same or different and are independently selected from nitrogen and sulfur;
  • Z is a contiguous linker which, together with X, B 2 , and the carbons to which they are bonded, forms a 5- or a 6- membered heterocyclic ring
  • L is a contiguous linker which, together with Y, B 3 , and the carbons to which they are bonded, forms a 5- or a 6- membered heterocyclic ring
  • the dotted, lines represent double bonds optionally present in the 5- or 6- membered heterocyclic ring;
  • X is N, NR 5 , or CR 5 , wherein R 5 is hydrogen, halogen, or straight chain or branched alkyl (e.g., C ⁇ -C 6 ) ;
  • Y is N, NR 4 , or CR 4 , wherein R 4 is hydrogen, halogen, or straight chain or branched alkyl (e.g., C ⁇ -C 6 ) ; and
  • Q 1 is an organic moiety which includes a diazo group capable of photochemically forming a radical species by the loss of N 2 .
  • a novel transition metal diazo compound is provided characterized by the following formula:
  • M is a metal selected from the group consisting of Ti , V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu, Zr, Nb, Mo, Te, Ru, Rh, Pd, Ag, Sn, Ta, W, Re, Os, Ir, Pt, and Au.
  • n is an integer from 1 to 3.
  • a-d can be the same or different and each can be hydrogen, halogen, C ⁇ -C ⁇ alkyl,
  • M can be complexed, if desired, with at least one additional auxiliary ligand other than a ligand of the formula:
  • M is iron and n is 3.
  • examples of optional additional ligands to which M can be complexed include, but are not limited to, an additional ligand represented by the formula :
  • B 4 and B 5 are the same or different and each is nitrogen, oxygen, sulfur, or phosphorus; and Q 2 is an aryl, a heterocycle, a macrocycle, or an alkyl spacer (e.g., C 2 -C 3 ) , wherein at least one hydrogen of the alkyl spacer is optionally substituted with an alkyl (e.g., Ci- C 6 ) .
  • Q 2 is an aryl.
  • the additional ligand can be of the formula :
  • a-d are the same or different and each is selected from hydrogen or a straight chain or branched alkyl (e.g. , C ⁇ -C 6 ) .
  • the additional ligand can be a macrocycle, such as, for example, a porphyrin, a porphyrazine, a chlorin, a phthalocyanine, a texaphrin, a cyclam, or a crown ether.
  • a preferred macrocycle is a porphyrin or a porphyrazine, such as a porphyrazine of. the formula:
  • thiols located within the brackets defining the porphyrazine are uncomplexed or are optionally complexed with at least one additional metal complex of the formula :
  • M 1 and M 2 are the same or different and each is a metal selected from the group consisting of Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu, Zr, Nb, Mo, Te, Ru, Rh, Pd, Ag, Sn, Ta, W, Re, Os, Ir, Pt, and Au; and
  • M is a metal selected from the group consisting of Ti, V, Mn, Fe, Co, Ni, Cu, Zn, Ga, Tb, Eu, Gd, Dy, Lu , Zr , Nb , Mo , Te , Ru , Rh , Pd, Ag , Sn , Ta , W, Re , Os , Ir , Pt , and Au ; and a-d are the same or different and each is hydrogen, halogen, alkyl (e.g., C ⁇ -C 6 ) , OR 10 , SR 10 , nitro, and cyano, wherein R 10 is hydrogen or straight chain or branched alkyl (e.g. , C ⁇ -C 6 ) •
  • the present invention further provides a method of treating cancer comprising administering to a patient a therapeutically effective amount (e.g., an anticancer effective amount, such as an antitumor effective amount) of at least one compound or composition of the present invention, optionally in combination with an anticancer effective amount of at least one additional anticancer compound other than a compound of the present invention.
  • a therapeutically effective amount e.g., an anticancer effective amount, such as an antitumor effective amount
  • the compound or composition can be administered, for example, orally, intramuscularly, subcutaneously, or intravenously.
  • the composition can be present as a solution suitable, for example, for intravenous injection or infusion.
  • the composition also can be present in unit dosage form, such as, for example, a tablet or capsule.
  • the therapeutically effective amount is the dose necessary to achieve an "effective level" of the active compound in the individual patient.
  • the therapeutically effective amount can be defined, for example, as that amount required to be administered to an individual patient to achieve an anticancer effective level of a compound of the present invention to kill or inhibit the growth of the cancer; the effective level might be chosen, for example, as that level to kill or inhibit the growth of tumor cells in a screening assay. Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending upon interindividual differences in pharmacokinetics, drug distribution, and metabolism.
  • the "effective level” can be defined, for example, as the level desired in the patient that corresponds to a concentration of a compound of the present invention which kills or inhibits the growth of human cancers in an assay which can predict for clinical anticancer activity of chemical compounds.
  • the "effective level” for compounds of the present invention can vary when these compounds are used in combination with other anticancer compounds or combinations thereof.
  • the "effective level" can be defined, for example, as that concentration of the compound of the present invention needed to inhibit markers of the cancer in the patient's blood, or which slows or stops the growth of the patient's cancer, or which causes the patient's cancer to regress or disappear, or which renders the patient asymptomatic to the particular cancer, or which renders an improvement in the patient's subjective sense of condition. Since a fixed . "anticancer effective amount" is used as the preferred endpoint for dosing, the actual dose and schedule for drug administration for each patient can vary depending upon interindividual differences in pharmacokinetics, drug disposition, and metabolism. Moreover, the dose can vary when the compound is used in combination with other drugs .
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired effective level in the individual patient.
  • One skilled in the art also can readily determine and use an appropriate indicator of the effective level of the compounds of the present invention by a direct (e.g., analytical chemistry) and/or indirect (e.g., with clinical chemistry indicators) analysis of appropriate patient samples (e.g., blood and/or tissues), or by direct or indirect observations of the shrinkage or inhibition of growth of the individual patient's tumor.
  • a direct e.g., analytical chemistry
  • indirect e.g., with clinical chemistry indicators
  • the present method of treating cancer using the compounds of the present invention can be made more effective by administering other anticancer compounds along with the compound of the present invention.
  • other anticancer compounds include, but are not limited to, all of the known anticancer compounds approved for marketing in the United States and those that will become approved in the f ture. See, for example, Table 1 and Table 2 of Boyd "The Future of Drug Development", Current Therapy in Oncology, Section I. Introduction to Cancer Therapy (J.E. Niederhuber, ed.), Chapter 2, by B.C. Decker, Inc., Philadelphia, 1993, pp. 11-22.
  • these other anticancer compounds include doxorubicin, bleomycin, vincristine, vinblastine, VP-16, VW-26, cisplatin, procarbazine, and taxol for solid tumors in general; alkylating agents, such as BCNU, CCNU, methyl-CCNU and DTIC, for brain or kidney cancers; and antimetabolites such as 5-FU and methotrexate for colon cancer.
  • the present invention further provides a method of treating infections by viruses and microorganisms in a host, e.g., a mammal.
  • a host e.g., a mammal.
  • the specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host .
  • the dose administered should be a "therapeutically effective amount” or an amount necessary to achieve an "effective level” in the individual patient. Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule may vary, depending upon interindividual differences in pharmacokinetics, drug distribution, and metabolism.
  • the "effective level” may be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more compounds of the invention which inhibits a microorganism or virus such as HIV in an assay known to predict for clinical antiviral activity of chemical compounds.
  • the "effective level” for compounds which are the subject of the present invention also may vary when the compositions of the present invention are used in combination with AZT or other known antiviral compounds or combinations thereof.
  • One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective concentration" in the individual patient .
  • an appropriate indicator of the "effective concentration" of the compounds of the present invention by a direct (e.g., analytical chemical analysis) or indirect (e.g., with surrogate indicators such as p24 or RT) analysis of appropriate patient samples (e.g., blood and/or tissues) .
  • a direct e.g., analytical chemical analysis
  • indirect e.g., with surrogate indicators such as p24 or RT
  • infected e.g., virally
  • a "mega- dosing" regimen wherein a large dose is administered, time is allowed for the compound to act, and then a suitable reagent is administered to the individual to inactivate the compound.
  • the pharmaceutical composition may contain other pharmaceuticals, in conjunction with the compounds of the invention to therapeutically treat acquired immunodeficiency syndrome (AIDS) .
  • additional pharmaceuticals include antiviral compounds, immunomodulators, immunostimulants, and antibiotics.
  • antiviral compounds include 3'- azido-2 ' , 3 ' -dideoxythymidine (AZT) , 2 ' 3 ' -dideoxyinosine (ddl) , 2'3' -dideoxycytidine (ddC) , 2 ' 3 ' -didehydro-2 ', 3 ' - dideoxythymidine (D4T) , 9- (1 , 3-dihydroxy-2- propoxymethyl) guanine (gancyclovir) , fluorinated dideoxynucleotides such as 3 ' -fluoro-2 ' , 3- dideoxythymidine, nonnucleoside compounds such as 6,11- dihydro-ll-cyclo
  • Exemplary immunomodulators and immunostimulants include various interleukins, CD4 , cytokines, antibody preparations, blood transfusions, and cell transfusions.
  • Exemplary antibiotics include antifungal agents, antibacterial agents, and anti-Pneumocystis carnii agents.
  • RT reverse transcriptase
  • ddC ddC
  • AZT ddl
  • ddA reverse transcriptase
  • anti-TAT agents ddC, AZT, ddl, ddA
  • a virustatic range of ddC is generally between 0.05 ⁇ M to 1.0 ⁇ M.
  • a range of about 0.005-0.25 mg/kg body weight is virustatic in most patients.
  • the preliminary dose ranges for oral administration are somewhat broader, for example 0.001 to 0.25 mg/kg given in one or more doses at intervals of 2 , 4, 6, 8, 12, etc. hours. Currently 0.01 mg/kg body weight ddC given every 8 hours is preferred.
  • the other antiviral compound for example, may be given at the same time as the compound of the invention or the dosing may be staggered as desired.
  • the two drugs also may be combined in a composition. Doses of each may be less when used in combination than when either is used alone.
  • alkyl means a straight-chain or branched alkyl substituent containing from, for example, about 1 to about 20 carbon atoms, preferably from about 1 to about 10 carbon atoms, more preferably from about 1 to about 8 carbon atoms, still more preferably from about 1 to about 6 carbon atoms.
  • substituents include methyl, ethyl, propyl, isopropyl, n- butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.
  • the alkyl group is preferably an alkyl group that promotes or enhances therapeutically desirable properties with respect to the compounds of the present invention, for example, anticancer or anti-microbial activity, metabolic stability, bioavailability, tissue distribution, improved pharmacokinetic properties, and the like, as will be appreciated by one of ordinary skill in the art.
  • aryl refers to an unsubstituted or substituted aromatic carbocyclic substituent, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl and naphthyl substituents, and the like.
  • aralkyl as utilized herein means alkyl as defined herein, wherein at least one hydrogen atom is replaced with an aryl substituent as defined herein.
  • Aralkyls include, for example, benzyl, phenethyl, or substituents of the formula:
  • At least one hydrogen on a given alkyl, aryl, heterocycle, or aralkyl substituent can be optionally substituted with an organic, inorganic, or functional group that is capable of forming a covalent bond with a carbon atom.
  • Suitable functional groups include, for example, an alkyl, halogen, a cyano, a nitro, an amino, an amide, a hydroxy (or an ester or ether thereof), and the like.
  • heterocycle or “heterocyclic” encompasses both heterocycloalkyls and heteroaryls .
  • heterocycloalkyl means a cycloalkyl substituent as defined herein (including polycyclics) , wherein at least one carbon which defines the carbocyclic skeleton is substituted with a heteroatom such as, for example, O, N, or S, optionally comprising one or more double bond within the ring, provided the ring is not heteroaryl as defined herein.
  • the heterocycloalkyl preferably has 3 to about 10 atoms (members) in the carbocyclic skeleton of each ring, preferably about 4 to about 7 atoms, more preferably 5 to 6 atoms.
  • heterocycloalkyl substituents include epoxy, aziridyl, oxetanyl, tetrahydrofuranyl, dihydrofuranyl, piperadyl, piperidinyl, pyperazyl, piperazinyl, pyranyl, morpholinyl, and the like.
  • heteroaryl means a substituent defined by an aromatic heterocyclic ring, as is commonly understood in the art, including monocyclic and polyclic heteroaryls.
  • Monocyclic heteroaryls include, for example, imidazole, thiazole, pyrazole, pyrrole, furane, pyrazoline, thiophene, oxazole, isoxazol, pyridine, pyridone, pyrimidine, pyrazine, and triazine substituents.
  • Polycyclic heteroaryls include, for example, quinoline, isoquinoline, indole, purine, benzimidazole, benzopyrrole, and benzothiazole substituents, which heteroaryl substituents are optionally substituted with one or more substituents selected from the group consisting of a halogen, an alkyl, alkoxy, amino, cyano, nitro, and the like. It will be appreciated that the heterocycloalkyl and heteroaryl substituents can be coupled to the compounds of the present invention via a heteroatom, such as nitrogen (e.g., 1-imidazolyl) .
  • a heteroatom such as nitrogen (e.g., 1-imidazolyl) .
  • polycyclic heterocyclic rings contain an aromatic ring and a non- aromatic ring.
  • polycyclic substituents include, for example, .benzotetrahydrofuranyl, benzopyrrolidinyl, benzotetrahydrothiophenyl, and the like.
  • macrocycle refers to an organic molecule (possibly complexed with one or more metals) having a large ring structure that contains at least about 15 carbons.
  • suitable macrocycles include porphyrins, porphyrazines, chlorins, phthalocyanines, texaphrins, cyclams, and crown ethers.
  • macrocycle- containing metalloenediyne or transition metal diazo complexes can often photochemically form radicals at wavelengths that exhibit enhanced tissue penetration of light, e.g., in the 700-850 nm range.
  • This example demonstrates a procedure for making a transition metal diazo compound identified as Cu(9-diazo- 4 , 5-diazafluorene) 2 (N0 3 ) 2 .
  • All synthetic preparations were performed under ambient oxygen conditions. All reagents were used as purchased without further purification.
  • 4,5-Diaza-9- fluorenone was synthesized by the oxidation of 1,10- phenathroline with KMn0 4 according to literature procedures, as described, for example, by L. J. Henderson, J.; Fronczek, F. R. ; and Cherry, W. R. , in J " . Ada . Chem . Soc . 1984, 106, 5876-5879.
  • 4,5-diaza-9- fluorenone hydrazone was prepared by refluxing a mixture of the fluorenone and hydrazine hydrate in methanolic solution with a catalytic amount of acetic acid to prevent azine formation, as described, for example, by Mlochowski, J. ; and Szulc, Z. in Polish J. Chem. 1983, 57, 33-39. It is to be noted that, in terms of safety, diazo compounds are highly reactive and potentially explosive upon photo- or thermal decomposition due to the copious production of N 2 gas. As such, adequate protective precautions should be taken when working with them.
  • Cu (diazodafene) 2 (N0 3 ) 9-diazo-4 , 5-diaza luorene (76 mg, 0.39 mmol) was dissolved in 15 mL of methanol, keeping the flask in the dark.
  • Cu (N0 3 ) 2 .2.5H 2 0 (46 mg, 0.20 mmol) was dissolved in 5 mL of methanol. Both solutions were filtered through glass wool, and the Cu(N0 3 ) 2 solution was added to the ligand solution dropwise. The solution turned from orange to deep green and the solution was s-tirred for about 1 minute. The reaction mixture was allowed to stand in refrigerator or freezer overnight and then filtered to removed the microcrystalline green solid.
  • the organic synthesis for the ligands can be carried out by adding tosylhydrazide to the 4 , 5-diazafluoren-9- one to form the tosylhydrazone, followed by removal of the tosylalcohol leaving group upon addition of base.
  • EXAMPLE II This example demonstrates an exemplary procedure for making the metalloenediyne complexes known as (1,2- bis (pyridine-3 -oxy) oct-4-ene-2, 6-diyne) copper (I) and (1, 2-bis (pyridine 3-oxy) oct-4-ene-2 , 6-diyne) copper (II) .
  • the general synthesis for the Cu(I) metal complex and Cu(II) metal complex, respectively, is illustrated in FIGS. 1 and 2.
  • the Cu(I) complex 2 was prepared by reacting two equivalents of compound 1 with one equivalent of Cu (CH 3 CN) PF 6 in a solution of acetonitrile under nitrogen.
  • the Cu(II) complex was prepared by a similar approach using one equivalent of Cu (N0 3 ) 2 • 2.5H 2 0 and two equivalents of the enediyne ligand to yield 3 as a light green powder. Characterization of 3: isolated yield: 65%. Melting point (356 °C (decomposes)).
  • Infrared (IR, KBr pellet) 3400, 3063,
  • the Bergman cyclization of 1-3 was studied by differential scanning calorimetry (DSC) at a heating rate of 10 °C per minute on a General V4.1C DuPont 2100 DSC differential scanning calorimeter.
  • the free ligand 1 exhibits an exothermic peak at 258 °C corresponding to Bergman cyclization of the enediyne moiety. This value compares favorably to those reported for both the 1,2- bis (diphenylphosphinoethynyl) benzene ligand (243 °C) disclosed by B.P. Warner, S.P. Millar, R.D. Broene, S.L.
  • complexes 2 (166 °C) and 3 (135 °C) each possess a single-phase transition at dramatically reduced temperatures.
  • the differences in the thermal reactivities of 2 and 3 can be attributed to contributions to the thermal barrier height from the geometry of the metal center.
  • the tetrahedral structure of the Cu(I) species would be expected to require more thermal energy to reach the transition state than the square planar Cu(II) complex due to the enhanced separation of the alkyne termini ("a" in FIG. 2) based on the ⁇ 109° N-Cu-N bond angles in the structure of analogous tetrakispyridine copper (I) complexes. See, e.g., K. Nilsson and A .
  • the thermal cyclization of 2 was also studied in solution under similar conditions by monitoring the disappearance of the olefinic protons (H a ) at 6.10 ppm and the appearance of the phenyl protons (H g , Hi) at 7.32 ppm.
  • the Cu(I) complex showed complete conversion to the Bergman cyclized product after heating at 90 °C for 8 hours.
  • the Cu(II) complex displays two LMCT transitions at 363 (1180 M ' 1 ) and 390 nm (820 M “1 cm “1 ) which are consistent with those observed for other CuN 4 constructs. Higher energy transitions appear in all three spectra in FIG. 3 and likely arise from ligand based ⁇ * transitions.
  • EXAMPLE III This Example demonstrates the preparation, and the X-ray crystal structure and thermal reactivity of, a mononuclear Pd(0) metalloenediyne compound with two chelating 1, 2-bis (diphenylphosphinoethynyl) benzene ligands (dppeb) , also referred to in this Example as "the compound” or "the complex.”
  • This unique structure as seen in FIGS. 4A and 4B and the synthesis of which is shown in FIG. 14, features a tetrahedral Pd center with four phosphorus atoms from two chelating ligands comprising the coordination sphere.
  • the structure is an example of a metal-chelated enediyne compound and represents a pre-transition state structural model for in si tu metal-assisted Bergman cyclization reactions. Additionally, the thermal reactivity of the compound in solution exhibits a marked increase in the temperature required for cyclization with respect to the Pd (dppeb) Cl 2 species. This is reflective of the increased distance of the alkyne termini promoted by the tetrahedral geometry of the d 10 Pd(0) oxidation state relative to the square planar geometry expected for the d 8 Pd(II) system.
  • the dppeb ligand was synthesized according to literature procedures, and the Pd(0) compound was prepared from a modified literature preparation. In particular, equivalents of the ligand were added to Pd(PPh 3 ) 2 Cl 2 in benzene. The solution was heated and stirred at 60 °C for about 16 hours in benzene to allow complexation and was then reduced with hydrazine. Crystals of the complex suitable for X-ray diffraction were obtained within 72 hours by slow evaporation from CH 2 C1 2 . The structure of the complex exhibits the typical tetrahedral coordination environment expected for a d 10 metal center with four phosphine ligands. The P—Pd—P bond angles nearly match the idealized 109.5° geometry.. Of more prominent interest are the unique structural features of the metal-chelated enediyne linkage.
  • the thermal reactivity of the complex was as follows. Upon heating at 102 °C with 5 equivalents of cyclohexadiene as H-donor for 11 hours, the solution, originally orange, converted to a deep purple indicating that some reaction took place. The reaction was followed by 31 P NMR analysis. The chemical shift of the complex was -5.71 ppm while that of the free ligand was -32.1 ppm. After 11 hours, there was evidence of the complex, free ligand, and 2 other resonances at -10.6 ppm and 25.4 ppm. Heating for 19 hours showed a decrease in the complex and growth in all other resonances. A shift downfield was expected for the cyclized product and this could help explain the resonance at 25.4 ppm.
  • the bite-angle of the resulting cyclized product, 1, 2-bis (diphenylphosphino) naphthalene would be smaller than that which would be required for tetrahedral geometry. This dissociation/reassociation may be slow enough to be detected on the NMR timescale.
  • the crystal structure of the complex provides supporting evidence for this hypothesis as three unique inter-ligand phenyl ring interactions are observed.
  • the closest contact derives from a coplanar slipped ⁇ -stacking interaction between two aromatic rings at a ⁇ 3.3 A inter-ring separation.
  • In a perpendicular orientation to one of these partners is an additional phenyl substituent from the second phosphine of the adjacent ligand at ⁇ 3.6 A.
  • a third eclipsed ⁇ interaction was observed which yielded atomic separations ranging from 4.3-6.3 A.
  • this Example reports the first X-ray crystal structure of a chelated metalloenediyne compound.
  • Metal chelation imposes direct steric influences upon the enediyne ligand as evidenced by the pronounced distortion from linearity of the alkyne carbons.
  • the local tetrahedral geometry about the Pd(0) center versus square planar Pd(II) forces the alkyne termini to a separation distance of ⁇ 3.47 A, which yields an enediyne species that is stable at room temperature.
  • the Pd(II) analog can be thermally activated at temperatures (e.g., 5 °C) at which the compound described in the present example does not undergo thermal activation.
  • EXAMPLE IV This Example demonstrates the preparation of an Fe(III) triazine complex known as Fe (3-Hydroxy-l, 2 , 3 , - benzotriazine-4 (3H) -one) 3 , shown in FIG. 5B, hereinafter compound "5" .
  • Fe(III) is an ideal choice as a prototype because it is a highly active redox metal that is known to be a powerful excited state oxidant, often forming Fe(II) with ligand fragmentation.
  • the electronic absorption spectrum of 4 in acetonitrile exhibits pronounced ⁇ - ⁇ * transitions in the 300 nm region and a shoulder at 325 nm corresponding to the forbidden diazo n- ⁇ * excitation.
  • the LMCT excited state of 5 is best described as a charge-separated Fe 2+ / ligand radical.
  • the cyclic voltammogram (CV) of 4 demonstrates an irreversible oxidation wave at a peak potential of +1.7 V vs. Ag/AgCl (E ox (4)), consistent with rapid denitrogenation.
  • the CV of 5 demonstrates a reversible Fe(III)/(II) redox couple with a half potential of -0.3 V vs. Ag/AgCl (B red (5)) .
  • the minimum energy required to produce the charge-separated excited state is approximately -2.48 eV, or 500 nm.
  • FIG. 8 illustrates the agarose gel electrophoresis of photolysis products of 300 ⁇ M 5 in the presence of pUC 118 plasmid DNA (30 ⁇ M/bp). Solutions were irradiated for 12 hours in 1:9 DMS0:Tris buffer (20 mM, pH 7.55) at 400 nm. Photolyses were performed at 400 NM (rather than 455 NM) due to a soluatochronic blue shift in the optical absorption spectrum.
  • Photolyses produced a mixture of linear and nicked forms (lane 3), while a similar thermal incubation effected no DNA cleavage (lane 4) .
  • Photolysis of the plasmid alone yielded a small amount of form II (lane 5) , as did photolysis of 900 ⁇ M free ligand (lane 6) .
  • These results demonstrate that although the photoreaction of 5 did not produce exclusively linear DNA, it is the only species in FIG. 8 to produce any form III DNA and to completely consume the supercoiled form I .
  • this Example describes the facile preparation of a novel transition metal triazine compound that demonstrates unique photoreactivity .
  • the overall strategy of metal complex activation via low energy optical excitation of LMCT transitions is indeed operative, as reactivity is observed beyond the cutoff wavelength of the free ligand.
  • Photolyses in acetonitrile induce ligand decomposition through loss of N 2 , resulting in the generation of ligand-based radical intermediates.
  • Irradiation of the Fe(III) complex in the presence of plasmid DNA affords both single- and double- strand cleavage, with significantly greater efficiency than photolysis of free ligand in threefold higher concentration.
  • This Example demonstrates the photochemical DNA- cleaving ability of the novel Cu(I) metalloenediyne complex identified herein as (1, 2-bis (pyridine-3-oxy) oct- 4-ene-2 , 6-diyne) copper (I) , also identified as [Cu (bped) 2] + , in an anaerobic aqueous solution (10 mM Tris, pH 7.6) .
  • TLC thin layer chromatography
  • Form I was supercoiled plasmid.
  • Form II was a nicked plasmid (i.e., one of the two DNA strands was cut) .
  • Form III was a linear plasmid (i.e., both strands were cut) .
  • the three plasmid forms were then subjected to eight lanes (wells) of varying conditions. Lane 1 related to the DNA forms alone. Lane 2 involved an EcoRl (restriction enzyme) digest of the DNA, which was used as a marker. Lane 3 subjected the DNA forms to the [Cu(bped)2] + complex in the presence of light for 12 hours at a wavelength of 400 nm.
  • Lane 4 subjected the DNA forms to light alone, without the [Cu(bped)2] + complex.
  • Lane 5 the DNA forms were subjected to the [Cu(bped)2] + complex and 12 hour incubation in air.
  • Lane 6 subjected the DNA forms to the [Cu(bped)2] + complex and 12 hour incubation in N 2 .
  • the DNA forms were subjected to the copper II complex, i.e., [Cu (bped) 2] 2+ , and 12 hour incubation in N 2 .
  • the results show that photolysis of the [Cu(bped)2] + complex (and the [Cu (bped) 2] 2+ complex, gel not shown) completely consumed the supercoiled plasmid, thereby rendering a mixture of mainly linear DNA, with a measurable amount of nicked plasmid (as seen in lane 3 of FIG. 9) . Meanwhile, the thermal controls show predominantly unreacted supercoiled DNA, with a small amount of nicked plasmid (as seen in lanes 4-6 of Fig. 9) .
  • EXAMPLE VI This Example demonstrates the photochemical DNA- cleaving ability of the novel Cu(II) diazo complex that possesses two 9-diazo-4 , 5-diazafluorene ligands, identified herein as bis (9-diazo-4 , 5- diazafluorene) copper (II) nitrate.
  • FIG. 10 illustrates the DNA cleavage of 50 ⁇ M pUCll ⁇ by this compound following 455 nm photolysis . for 1 hour at 20 . °C (agarose gel, 2%) .
  • Form I was supercoiled plasmid.
  • Form II was nicked plasmid.
  • Form III was linear plasmid.
  • the three plasmid forms were subjected to 11 lanes of varying conditions. Lane 1 pertained to the DNA forms alone . Lane 2 involved an EcoRl digest of the DNA, which was used as a marker. In lane 3, the DNA was subjected for 1 hour to light at a wavelength of 455 nm. In lane 4, the DNA was subjected to 25 ⁇ m Cu(II) (diazafluorenone) 2 2+ for 1 hour incubation.
  • the DNA was subjected to 25 ⁇ m Cu(II) (diazafluorenone) 2+ in the presence of light at a wavelength of 455 nm for 1 hour.
  • Lane 6 subjected the DNA to 25 ⁇ m of 9-diazafluorene in the presence of light at a wavelength of 455 nm for 1 hour.
  • the DNA was subjected to 25 ⁇ m 9-diazo-4 , 5-diazafluorene in the presence of light at a wavelength of 455 nm for 1 hour.
  • the DNA was subjected to 25 ⁇ m bis(9-diazo- 4 , 5-diazafluorene) copper (II) nitrate rapid quench.
  • the DNA was subjected to 25 ⁇ m bis (9-diazo-4 , 5- diazafluorene) copper (II) nitrate for 1 hour incubation.
  • the DNA was subjected to 25 ⁇ m bis(9-diazo- 4 , 5-diazafluorene) copper (II) nitrate for 1 hour incubation at 37 °C.
  • the DNA was subjected to 25 ⁇ m bis (9-diazo-4 , 5-diazafluorene) copper (II) nitrate for 1 hour at a wavelength of 455 nm.
  • Example VII This example illustrates the preparation of electronic modulating metalloenediynes with extended ⁇ structures, i.e., netalloenediynes having large chromophoric porphyrazine ligands.
  • metalloenediynes are advantageous because the macrocycle has established x ⁇ * and 3 ⁇ * states ( ⁇ 2.2 and 1.5 eV) with high extinction coefficients (typically 70,000 M "1 cm “1 at 405 nm, and 20,000 M “1 cm “1 at 580-750 nm) that can be used to probe the photochemical " reactivity of the adjacent enediyne ligand.
  • extinction coefficients typically 70,000 M "1 cm “1 at 405 nm, and 20,000 M “1 cm “1 at 580-750 nm
  • the large, near-IR absorbing chromophore can be forced to non- radiatively dissipate the photochemical energy into heat, which may provide a photo-thermal temperature jump strategy for activating the enediyne ligand to undergo Bergman cyclization.
  • Magnesium acts as a template to facilitate cyclization of the porphyrazine (tetraazaporphyrin) complex, which may be demetallated under mild conditions. Demetallation and nickel complexation are benchtop reactions common to porphyrinoid chemistry.
  • the thiolate anion 30 is susceptible to air oxidation but can be manipulated effectively using standard Schlenk techniques .
  • Nickel diphosphine units have been shown to chelate directly to the in situ octathiolate anion 30.
  • the resulting complex (es) 31 is/are both air- and water-stable. Our efforts to date have now prepared 29 on the gram scale and are poised to proceed with the final capping reaction and formation of the metalloenediyne porphyrazine complexes.
  • Ni(dppe) 2 structures are structures known to date, it is apparent that many more potential diimine dithiolate and diphosphine dithiolate analogs can be readily prepared with Pt, Cu, Ni, and Fe and other metals pursuant to the invention using our current enediyne ligands. These compounds have a number of major advantages .
  • these compounds exhibit strong electronic communication between the central metal ion and the metal bound at the periphery.
  • DSC and NMR characterization of the thermal reactivities of systems in this class will reflect the importance of electronic contributions from adjacent ligands to the thermal reactivity of the enediyne.
  • these compounds provide a mechanism to use long-lived triplet states, or higher energy singlet states to drive the enediyne cyclization reaction photochemically with systematic variations in excitation energies.
  • the ability to tune the optical excitation energy and lifetime/energy of the excited state is critical to understanding how potential redox induced enediyne cyclization proceeds.
  • the triplet state of the tetraazaporphyrin lies lower in energy than the charge separated state for direct photoredox enediyne cyclization involving Ru(II) and Pt(II) centers bound at the periphery.
  • These metals typically have metal centered redox couples above 1.0 V and when combined with the energy required to reduce or oxidize the enediyne, approximately 2.4 eV will be required for this reaction. In other words, this redox state lies uphill from the 3 ⁇ * state (-1.8 eV) of the macrocycle. However, this is not true for the excited ⁇ " ⁇ * states of the tetraazaporphyrin since higher energy fluorescence can be observed from these systems.
  • Photoinduced preparation of the 3 ⁇ * state (-1.8 eV) of macrocycle will reduce the Cu(II) to Cu(I) ( ⁇ -0.3 V vs. SCE ) followed by oxidation of the enediyne bound to the copper center (+1.4 V vs. SCE) . Coupling the potentials for those steps, it will cost - 1.7 V to carry out this reaction, which is nearly 734 nm. Therefore, this process is thermodynamically favored from the 3 ⁇ * state of the system and will allow us to investigate the ability to tune the redox properties of these complexes to learn whether redox chemistry is indeed at the heart of many enediyne cyclization reactions.
  • the large chromophore of the porphyrazine also generates opportunities for driving Bergman cyclization using optical absorption-temperature jump concepts for systems with metals at the periphery that have low thermal barriers to cyclization.
  • Pt (II) /Ni (II) conjugates may now be reactive upon 3 ⁇ * excitation, not because of redox induced cyclization, but rather via thermal heating of the porphryrazine upon optical excitation in the red spectral region. As discussed above, in these experiments, the 3 ⁇ * state will not possess sufficient energy to energy to activate the enediyne via a direct redox reaction.
  • This Example describes the preparation of Ru(II) metalloenediyne complexes (six-coordinate) using nitrogen and phosphorous chelating enediyne ligands described above. From the perspective of thermal chemistry, the 6- coordinate structures will likely show diminished barriers to thermal cyclization. This in itself is a valuable study as Ru has not been examined to date as a metal cofactor to assist enediyne cyclization. However, much more valuable information can be gained about the excited state mechanism for Bergman cyclization as Ru(II) diimine compounds have an established history of MLCT photochemistry, and Ru(II) binds nitrogen and phosphorous ligands well. Moreover, by careful selection of compounds, we are able to photochemically drive redox equivalents to or away from the ligand supporting the enediyne functionality.
  • the color of the complex indicates that the Ru(II) bpy MLCT transition is blue shifted due to the stabilization of the Ru d-orbitals by the ⁇ - donating phosphine ligand.
  • Emission from the complex is weak relative to Ru(bpy) 3 2+ -
  • the weak emission profile is also blue shifted relative to that of Ru(bpy) 3 + .
  • DSC measurements can be made and can provide immediate feedback regarding the thermal phase transition temperatures for these novel complexes. Since this complex, and those with the diimine enediyne ligands, will not be paramagnetic, 1 H, 13 C and 31 P NMR will also be a very useful tool for studying the thermal reactivities of these complexes.
  • the oxybipyridyl enediyne ligand has an irreversible reduction at -1.4 V that will make this ligand the site of MLCT photoreduction.
  • these photooxidation and photoreduction schemes allow us to determine whether the photochemical cyclization of the enediyne bound to the metal is a redox driven process, or simply governed by electronic modulation within the excited state. This is accomplished first using NMR to monitor the progress of the photoreaction in the presence of 1,4- cyclohexadiene, with subsequent separation and characterization of the photochemical products as we have demonstrated in our copper metalloenediyne photocyclization studies. We then employ transient absorption and luminescence lifetime measurements to evaluate the kinetics for the photoreaction process.

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Abstract

L'invention concerne de nouveaux composés et de nouvelles compositions, et des procédés particulièrement utiles en thérapie photodynamique. En particulier, les composés et les compositions selon l'invention concernent la formation d'espèces radicales cytotoxiques en présence de lumière. De manière significative, les composés, les compositions et les procédés selon l'invention ne nécessitent pas la présence d'oxygène en thérapie photodynamique et, de ce fait, se fondent sur un mécanisme unimoléculaire pour produire les radicaux. Les composés, les compositions et les procédés selon l'invention peuvent être utilisés, par exemple, pour traiter les cancers ainsi que des infections provoquées par des microorganismes comme des protozoaires, des champignons, des bactéries et des virus.
PCT/US2000/004915 1999-02-26 2000-02-25 Composes, compositions et procedes pour la therapie photodynamique Ceased WO2000050117A2 (fr)

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