WO1996040108A1 - Complexes metalliques polydentes et leurs procedes de preparation et d'utilisation - Google Patents

Complexes metalliques polydentes et leurs procedes de preparation et d'utilisation Download PDF

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WO1996040108A1
WO1996040108A1 PCT/US1996/010079 US9610079W WO9640108A1 WO 1996040108 A1 WO1996040108 A1 WO 1996040108A1 US 9610079 W US9610079 W US 9610079W WO 9640108 A1 WO9640108 A1 WO 9640108A1
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multidentate
metal complex
iii
complexes
cationic metal
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PCT/US1996/010079
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English (en)
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David R. Piwnica-Worms
Vijay Sharma
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Washington University
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Priority to JP9502188A priority Critical patent/JPH11507641A/ja
Priority to MX9709471A priority patent/MX9709471A/es
Priority to AU64771/96A priority patent/AU6477196A/en
Priority to EP96924270A priority patent/EP0831806A1/fr
Publication of WO1996040108A1 publication Critical patent/WO1996040108A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to multidentate metal complexes, and their use in therapeutic treatments.
  • Malaria is one of the most serious and widespread infectious diseases, resulting annually in more than 400 million cases and 2.5 million deaths worldwide, primarily in tropical countries (Muller and Baker, Medical
  • Antimalarial chemotherapy has historically targeted prophylactic, suppressive treatment and/or clinical cure.
  • Antimalarial agents can be roughly divided into several distinct groups based on mechanisms of action.
  • the first group characterized by older, well known agents such as chloroquine, primaquine, quinine and their derivatives, demonstrates rapid cytological action.
  • chloroquine is the most common, well-tolerated, and cost-effective drug for prophylaxis and therapy of malaria (Muller and Baker, Medical Parasitology, Gower Medical Publishing, London (1990)). These agents are thought to selectively accumulate within the parasite and interfere with digestive vacuole function.
  • chloroquine-sensitive strains of Plasmodium parasites concentrate chloroquine approximately 800-fold in their digestive vacuoles (Veignie and Moreau, Ann. Trop. Med. Parasitol 85:229 (1991)).
  • the digestive vacuole is a crucial organelle for the parasite, responsible for degradation of the host's hemoglobin, the main source of nutrients for the parasite.
  • the digestive vacuole has lysosomal characteristics such as an acid pH and a high content of acid hydrolases (Goldberg and Slater, Parasitol Today c?:280 (1992)).
  • Hemoglobin degradation inside the digestive vacuole generates large amounts of toxic heme which is detoxified by polymerization into pigment bodies by heme polymerase, an enzyme found exclusively in the digestive vacuole.
  • Chloroquine inhibits the heme polymerase in vitro at concentrations which are found in the digestive vacuole of chloroquine-sensitive parasites, explaii-ing the observation that accumulation of chloroquine in the digestive vacuole disturbs the formation of pigment bodies (Slater and Cerami, Nature
  • the second major group of agents represented by chloroguanide, pvrimetharnine, sulfonamides, and derivatives, has mechanisms of action that are slower in onset and target the synthesis of folinic acid (folate) from para- aminobenzoic acid (PABA).
  • These agents tend to interfere with the incorporation of PABA into folate, a synthetic process which does not occur in host mammalian cells, or to inhibit parasitic dihydrofolate reductase. Acquired or natural drug resistance to these agents can be demonstrated readily in the laboratory and, like the first group, has been reported from every major endemic malarial region.
  • U.S. Patent No. 5,270,037 discloses the treatment of malaria with compositions comprising an interferon.
  • WO93/00082 discloses the use of hydroamate derivatives which are useful for removal of iron (III) from mammalian cells and treatment of disorders caused by pathogenic organisms, such as Plasmodium falciparum that causes malaria. See also, EPA 214,933 EPA 214,101, Iheanacho et al, Trans. R. Soc. Trop. Med. Hyg.
  • the present invention arises from the inventors' discovery that certain intact metal complexes comprising multidentate ligands are effective as anti ⁇ malarial agents.
  • the invention relates to new compositions of matter that can be used for treating malaria and treatment of malaria with these compositions.
  • the complexes can be used in the treatment of other diseases as described herein, such as cancer and diseases attributed to multidrug resistance transporters.
  • the invention also relates to pharmaceutical compositions containing the complexes of the invention.
  • the invention also relates to a multidentate cationic metal complex having the following formula /:
  • M is Fe, In, Ga or Al; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M); A and B are oxygen, or in the case where Ar is 2-pyridyl or optionally substituted 2-pyrrolyl, a shared pair of electrons on the nitrogen; Ar is optionally substituted phenyl, optionally substituted naphthyl, optionally substituted 2-pyridyl or optionally substituted 2-pyrrolyl; and
  • X is (CHR 2 ) p [NR 3 (CHR 4 ) q ]., wherein p and q are independently 1, 2, 3, 4, 5 or 6; r is 0, 1 or 2; and R 2 , R 3 and R 4 are independently hydrogen, lower alkyl or phenyl, or two adjacent R 2 or R 4 groups represent a double bond or a fused benzene ring where p or q, respectively, is greater than 2; with the proviso that where r is 1 or 2, then there are 1 or two additional sites of coordination to the metal.
  • the complex optionally further comprises -A", where -A is a pharmaceutically acceptable anion.
  • the invention also relates to a multidentate cationic metal complex according to formula/ with proviso (A), wherein at least one of the substituents of Ar comprises boron.
  • the invention also provides a multidentate cationic metal complex according to formula /with proviso (B), wherein at least one of substituents of Ar comprises a linkage to a pharmaceutically active substituent.
  • the invention also provides a a multidentate cationic metal complex according to formula /, wherein X is optionally substituted phenyl.
  • X when having a substituted phenyl, has substituents as lower alkyl groups, with methyl groups preferred at the meta and para positions relative to the N bonds.
  • the invention further relates to a multidentate cationic metal complex selected from the group consisting of N,N'-bis[3-(2-hydroxy-3- memoxybe ⁇ r2ylirr ⁇ mo)propyl]emylenediar-rine M(III) + A", N,N'-bis[3-(2-hydroxy-
  • the invention also relates to a multidentate cationic metal complex selected from the group consisting of N,N'-bis[3-(2-hydroxy-3-fluoro- criz-zylirm ⁇ )propyl]ethylene-diamine M(III) + A", N,N'-bis[3-(2-hydroxy-3- fluorobeiizylarnino)propyl]-ethylene diamine M(III) + A", N,N'-bis[3-(2-hydroxy- 5-bromobenzyl-immo)propyl]e ylenediarnineM(III) + A",N,N'-bis[3-(2-hydroxy- 5-bromoben2ylirnmo)propyl]emylenediamine M(III) + A', N,N'-bis[3-(2-hydroxy- 5-bromobe ⁇ rzylamino)propyl]ethylenediamine M(III) + A", wherein M(III) is Fe(IH), In(lTI),
  • the invention also relates to a method for treating malaria comprising administering an effective amount of a multidentate cationic metal complex according to formula /.
  • the invention also relates to a method of treating cancer, comprising administering to an ariimal in need thereof an effective amount of a multidentate cationic metal complex according to formula/.
  • the invention also relates to a method for the treatment of diseases attributed to the multidrug family of transporters, comprising administrating to an animal in need thereof an effective amount of a multidentate cationic metal complex according to formula /, wherein formula / with proviso (A) is preferred, wherein at least one of the substituents of Ar comprises boron.
  • the invention also relates to a method of potentiating photodynamic therapy in the treatment of cancer, comprising (a) admimstering to an animal in need thereof an effective amount of a multidentate cationic metal complex according to formula /, and (b) exposing cancer cells of the animal to wavelengths of light effective to kill said cancer cell.
  • the invention also relates to a method of treating cancer by boron-neutron therapy, comprising (a) administering to an animal in need thereof an effective amount of a multidentate cationic metal complex according to formula /; and (b) exposing cancer cells of said animal to a neutron beam effective to kill said cancer cells.
  • the invention also relates to a method of enhancing the oral abso ⁇ tion or cell accumulation of a pharmacologically active substance, comprising adi-iinistering to an animal in need thereof an effective amount of a multidentate cationic metal complex according to formula/, with proviso (B), wherein at least one of substituents of Ar comprises a linkage to a pharmaceutically active substituent.
  • the hatched lines represent coordination to the metal (III) cation (M).
  • Figure 2 Cytotoxicity of a MENPBI complex in KB-3-1 and KB-8-5 Cells.
  • Figure 3 Structure of the hexadentate (N 4 O 2 ) ethylenedi-amine- bis( ropyl(R-benzylimino)) ligand metal(III) monocationic complex shown in the trans phenolic configuration.
  • Figure 4A-B Effect of 4,6-dimethoxy-ENBPI Fe(III) complex on intraerythrocytic P. falciparum in culture.
  • Figure 4 A Concentration-effect curve of antimalarial activity: chloroquin sensitive (HB3) and resistant (FCR-3, Indo-1, Dd2) strains were grown in the absence or presence of various concentrations of inhibitor. Growth inhibition was measured by the 3 H- hypoxanthine incorporation assay. Data are shown as mean values of triplicate deterrriinations; error bars (when larger than symbol) represent ⁇ SEM.
  • Figure 4B Time course of antimalarial activity: parasites at the early (ring) stage (a) were incubated with 5 ⁇ M inhibitor or with the DMSO vehicle.
  • Figure 5 Inhibition of heme polymerization by 4,6-dimethoxy-ENBPI Fe(III) complex. Heme polymerization was assayed by pre-formed hemozoin nucleation (open circles) or histidine-rich protein Il-facilitated reaction (closed circles) in the presence of various concentrations of drug. Data are from representative experiments; values are the means of two independent determinations.
  • Figure 6 General structure of hexadentate R-ENBPI ligands.
  • Figure 7 Pathway for the synthesis of R-ENBPI metal complexes.
  • Figure 8A-B Proton decoupled ,3 C NMR spectra (75.4 MHz) of H 3 Mabi (1) (Figure 8A); and H 3 DMabi (2) ( Figure 8B) in CDC1 3 at room temperature. Chemical shifts are reported in ppm referenced to TMS.
  • Figure 9A-L Cell survival studies and LC 50 determination. Survival of parental KB-3-1 (O, (triangle), D) and multidrug resistant KB-8-5 (•, T, ⁇ ) cells in increasing concentrations of R-ENBPI metal(III) complexes (O, •), 25 ⁇ M colchicine ((triangle), T) or metal(III) ions (B, ⁇ ).
  • Figure 9A-D, 9E-H, 91- L represent A1(III), Fe(III), Ga(III), and In(III) complexes of: (i) 3-MeO-R- ENBPI ( Figure 9A-D), (ii) 4,6-diMeO-R-ENBPI ( Figure 9E- ); and (iii) the free metals ( Figure 9I-L), respectively.
  • Each point represents the mean of triplicate determinations; bars represent ⁇ SEM when larger than the symbol.
  • the invention is related to the discovery that intact metal complexes comprising multidentate ligands are useful for treating or preventing malaria.
  • the multidentate ligands that may be used in the present invention include the
  • the multidentate ligands are bi- or polydentate salicylaldiamines.
  • the disclosed multidentate ligand metal complexes can have the formula/:
  • M is Fe, In, Ga or Al; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • a and B are oxygen or nitrogen, or in the case where Ar is 2-pyridyl or 2-pyrrolyl, a shared pair of electrons on the nitrogen;
  • Ar is optionally substituted phenyl, naphthyl, 2-pyridyl or 2-pyrrolyl;
  • X is (CHR 2 ) p [NR 3 (CHR') q ] r , wherein R 2 R 3 and R 4 are independently hydrogen, lower alkyl or phenyl, or wherein two adjacent R 2 or R groups represent a double bond or a fused benzene ring where p or q, respectively, is greater than 2; p and q are independently 1, 2, 3, 4, 5 or 6; and r is 0, 1 or 2; with the proviso that where r is 1 or 2, then the multidentate ligand coordinates
  • the complex optionally further comprises -A", where -A is a pharmaceutically acceptable anion.
  • X can alternatively be optionally substituted phenyl, wherein the phenyl, when substituted, has lower alkyl substituents, with methyl groups preferred at the meta and para positions relative to the N bonds.
  • Optional substituents of Ar optionally further comprise a boron atom (as proviso (A)) or a linkage to a pharmaceutically active substance (as proviso (B)).
  • the multidentate ligands fall within the general category of hexadentate N 4 O 2 amine phenol ligands and the analogous Schiff base phenol ligands.
  • Such complexes may be referred to as Mfe hylene diamine N,N'- bis(p_ropyl)(2-hydroxy R-b_ei-zylamino)] complexes or "MENPBA-complexes” and Mfethylene diamine N,N'-bis(j3ropyl)(2-hydroxy R-benzylimino)] complexes or "MENPBI-complexes", where R represents alkyl or substituted alkyl or aryl or substituted aryl moieties and M represents any metal conferring the desired hexadentate coordination chemistry and biological activities.
  • the components of the alkylene backbone scaffold could readily contain unsaturated carbon chains and still confer the desired properties. The most preferred embodiments contain
  • the preferred complexes are overall (1+) monocations.
  • the anion counter ion of the metal complexes of the present invention can alternatively be any pharmaceutically acceptable anion including chloride, bromide, iodide, sulfate, phosphate, acetate, fumarate, succinate, citrate, tartrate, and the like.
  • the multidentate ligand complexes may be prepared as follows.
  • the method involves the condensation of a salicylaldehyde, a 2- pyridinecarboxaldehyde, or a 2-pyrrolidinecarboxaldehyde, with a diamine to give a Schiff base.
  • the Schiff base itself can be complexed with a metal salt and administered as an anti-malarial, anti-cancer agent or pharmaceutical enhancer, or reduced with sodium borohydride or cyanoborohydride to give the resulting amine, which may also be complexed with the metal salt and used as an antimalarial, anti-cancer agent or pharmaceutical enhancer.
  • Non-limiting examples of diamines which can be used in the present invention include, but are not limited to ethylenediamine, bis(3- aminopropyl)ethylenediamine, bis(3 -aminopropyl)- 1 ,3 -propanediamine, 1 ,3 - bis(aminomethyl)cyclohexane, bis(2-aminoethyl)-l,3-propanediamine, bis(2- aminopropyl)- 1,4-butanediamine, bis(2-aminopropyl)ethylenediamine, bis(2- anrmoethyl)ethylenediamine, 1,5,9-triazanonane, l,4,9,12-tetraazododec-6-ene, 1,2-propylenediamine, 1,5-pentanediamine, 1,6-hexanediamine, 2-methyl-2- an inoethylamine, 3-methyl-3-aminopropylamine, 4-methyl-4-aminobutylamine, 5-methyl
  • salicylaldehyde derivatives which may be used have the formula//:
  • R 3 , R 4 , R 5 and R are independently hydrogen, halo, alkyl, alkoxy or nitro.
  • 5-bromosalicylaldehyde 5- chlorosalicylaldehyde, 3-fluorosalicylaldehyde, 3 -methoxysalicylaldehyde, 3- ethoxysalicylaldehyde, 4,6-dimethoxysalicylaldehyde, 5-bromo-3- methoxysalicylaldehyde and 5-nitrosalicyladehyde, which are commercially available, as well as salicylaldehyde-5-sulfonate described by Evans and
  • R 3 , R 4 , R 5 and R 5 are independently hydrogen, halo, alkyl, alkoxy or nitro.
  • R 3 , R 4 , R 5 and R 6 are independently hydrogen, halo, alkyl, alkoxy or nitro.
  • bis(3-aminopropyl)ethylenediamine and the corresponding substituted salicylaldehyde, pyridocarboxaldehyde, or pyrrolidine- carboxaldehyde (1:2 ratio) are dissolved in methanol, stirred for 30 minutes, and treated with dropwise addition of sodium methoxide dissolved in methanol. After 30 minutes, the solution is treated with dropwise addition of M(III) salt (e.g., hydrated ferric nitrate) dissolved in methanol, stirred for 1 hour, and then filtered. Volatiles are removed under reduced pressure, residue is dissolved in hot water, and potassium hexafluorophosphate dissolved in water is added.
  • M(III) salt e.g., hydrated ferric nitrate
  • MENPBI-M(III) e.g., MENPBI-Fe(III)
  • Analysis and characterization of substituted free ligands and M + -complexes may be performed by routine analytical 'H-NMR, 13 C-NMR, and/or GC-mass spectrometry.
  • EMPBI or ENPBA complexes according to formulae V-XII can have overall neutral or positive charge, and when charged and optionally include A", as a pharmaceutically acceptable anion.
  • Preferred monocationic substituted ENPBI-complexes according to the present invention have the following formula V:
  • M is Fe +3 , In +3 , Ga +3 , orAl +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 and R 2 are independently hydrogen or alkyl
  • R 3 is methoxy or R 3 is ethoxy, or R 4 and R 6 are methoxy.
  • lower alkoxy is intended C 1-6 alkoxy groups.
  • halo is intended fluoro, chloro, bromo or iodo.
  • alkyl is intended a straight or branched chain hydrocarbon group, preferably containing one to six carbon atoms.
  • Preferred R 3 groups include OCH 3 , OC 2 H 5 , OC 3 H 7 , and halogens.
  • R 4 and R 6 groups are OCH 3 and OC 2 H 5 .
  • Preferred substituted metallofalkylene diamine N,N'-bis(alkyl)(2-hydroxy R-benzylimino)] complexes have formula VI:
  • M is Fe +3 , In +3 , Ga +3 , orAl +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 is hydrogen or alkyl
  • Especially preferred substituted MENPBI-complexes have the following formula VII:
  • M is Fe or Al; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 3 is OCH 3 or OC 2 H 5 ;
  • R 4 and R 6 are OCH 3 ;
  • R 1 and R 5 are H; and n is 2; n' is 3;
  • M is Fe +3 , In +3 , Ga +3 orAl +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 is hydrogen or alkyl
  • R 3 , R 4 , R 5 and R 6 are independently a lower alkoxy, halo or nitro group
  • MisFe +3 ,In +3 ,Ga +3 ,orAl +3 the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 is hydrogen or alkyl
  • R 3 , R 4 , R 5 and R 6 are independently a lower alkoxy, halo or nitro group
  • M is Fe +3 , In +3 , Ga +3 , orAl +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 is hydrogen or alkyl
  • R 3 , R 4 , R 5 and R 6 are independently a lower alkoxy, halo or nitro group
  • R 7 and R 8 are independently hydrogen or alkyl
  • M is Fe +3 , In +3 , Ga +3 , or Al +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M); R 1 is hydrogen or alkyl;
  • R 3 , R 4 , R 5 and R 6 are independently a lower alkoxy, halo or nitro group;
  • R 7 , R 8 , R ⁇ and R 12 are independently hydrogen or lower alkyl;
  • R 9 and R'° are independently hydrogen, a lower alkyl, a lower alkoxy, halo or nitro group; and
  • n 1, 2, 3, 4, 5 or 6; and wherein R" and R 12 are preferably methyl.
  • Other preferred ENPBI complexes have the following formula XII:
  • M is Fe +3 , In +3 , Ga +3 , orAl +3 ; the dashed lines represent independently a single or a double bond; the hatched lines represent coordination to the metal cation (M);
  • R 1 is hydrogen or alkyl
  • R 3 , R 4 , R 5 and R 6 are independently a lower alkoxy, halo or nitro group
  • Especially preferred substituted MENPBA-complexes have structures analogous to formulas /, V, VI, VIII, IX, X, XI, and XII, but the Schiff base is reduced to an amine.
  • the metal complexes of the present invention may be co-administered with one or more known antimalarial substances.
  • the known antimalarial substances can be divided into the following 6 main groups on the basis of their chemical compositions: 1) the 9-aminoacridines (e.g., mepacrine),
  • the biguanides with an inhibiting effect on dihydrofolic acid reductase e.g., chloroproguanil, cycloguanil, proguanil
  • diaminopyrimidines e.g., pyrimethamine
  • sulphones such as dapsone, sulphonamides, sulphanilamides and antibiotics such as tetracycline may also be used as antimalarial agents.
  • the known antimalarial agents can be divided into the following categories:
  • the first group includes, for example, proguanil, pyrimethamine and primaquine and derivatives thereof, and also sulphanilamides, sulphonamides and tetracyclines.
  • the second group includes, for example, 8-aminoquinolines, such as primaquine and its analogues and derivatives, floxacrine, cycloguanil, dapsone and quinazolines.
  • Substances active against the blood schizonts include, in particular, 4-aminoacridines such as mepacrine, and the 4-aminoquinolines such as chloroquine or chloroquinesulphate, quinine, amodiaquine and mepacrine, mefloquine, and related compounds such as halofantrene, pyrimethamine, proguanil, primaquine and the sulphanilamides and sulphonamides, particularly in conjunction with pyrimethamine.
  • 4-aminoacridines such as mepacrine
  • the 4-aminoquinolines such as chloroquine or chloroquinesulphate, quinine, amodiaquine and mepacrine, mefloquine, and related compounds such as halofantrene, pyrimethamine, proguanil, primaquine and the sulphanilamides and sulphonamides, particularly in conjunction with pyrimethamine.
  • sesquiterpene lactones based on the compound artemisinine, and the semisynthetic derivatives thereof, such as artesunate, artemether, piperaquine, hydroxypiperaquine, pyronaridine, halofantrene and, generally, the biguanides and quinine salts.
  • the schizonticides mentioned above are effective against the schizonts of, for example, P. vivax, P. malariae or P. ovale, but not against the mature gametocytes.
  • the 8-aminoquinolines, such as primaquine and quinocide, are also effective against the gametocytes.
  • Proguanil, primaquine and pyrimethamine are also sporonticidal agents.
  • chloroproguanil e.g., as a salt of embonic acid
  • pamaquine plasmocide
  • totaquine spirogermanium
  • febrifugine brusatol
  • bruceine-A bruceine-B
  • bruceine-C yadanziolide-A
  • tebuquine enpirolin
  • eurycomanone 3-(4-imidazolyl)-2- (pivaloylamido)-propionylhydrazide, cinchonidine
  • cucurbitacine tripynadine, 5-ethylthioribose, arteether (ethylether analogues of artemether), artenilic acid, pyrexol, atalaphillinine, diformyldapsone, bruceantine, nitroquine, octanoylprim
  • 2-propionyl-pyridinethiosemicarbazones tebuquine, 2,6-bis(l- piperidmylmethyl)-4-((7-(trifluoromethyl)-4-quinolinyl)amino)-phenol, primary phosphoric acid esters of 4'-chloro-5-(l,l-dimethylethyl)-3-(((l,l- dime ylethyl)amino)methyl)-( 1 , 1 '-biphenyl-2-ol, N4-(2,6-dimethoxy-4-methyl- 5-(3-trifluoromethyl)-phenoxy-8-quinolinyl)- 1 ,4-pentanediamine, N,N-diethyl-
  • the present invention also relates to the use of these complexes for their cytotoxic activity on other cell types.
  • cell types include cancer cells such as CNS tumors, breast cancer, lung, head and neck, cancer lymphoma, leukemias, ovarian carcinoma, sarcomas, renal cell carcinoma, and prostate cancer.
  • the invention is also related to a method of treating cancer comprising contacting said cells with an effective amount of one of the multidentate cationic metal complexes of the present invention. Such contacting will typically be carried out by administration in vivo to an animal.
  • the present invention also relates to the use of these metal complexes as potentiators of photodynamic therapy.
  • Light-absorbing chemicals such as the metal complexes of the present invention are selectively retained by cancer cells.
  • the cancer cells are killed when exposed to certain wavelengths of light [(400 to 800 nm)].
  • the metal complexes are administered to the animal and the complex-treated cells are exposed to laser beams of the appropriate wavelengths through an endoscope.
  • Such an approach works best with cancer cells that are localized (and not in circulation, e.g., leukemia cells) and where the cancer cells are easily accessible. See, Harrison's Principles of Internal Medicine, 11th. Edition, Braunwald et al. (eds.), McGraw-Hill Book Co, New York, N. Y., pp.441 (1987).
  • Examples of such cancer cells include CNS tumors, breast cancer, lung, head and neck, lymphoma, and melanoma.
  • the present invention also relates to the use of these metal complexes which comprise boron atoms in boron neutron therapy.
  • metal complexes can be prepared by reaction of a compound having formula ///, wherein one of R 3 , R 4 or R 5 is a nitro group, with a reducing agent such as H 2 Pd or Sn/HCl to give the corresponding aniline.
  • This amine may be coupled with the active ester of trimethylamine-borane carboxylic acid (an ⁇ -boron analog of betaine, available from Sigma), in the presence of a dehydration agent, to give the boron-substituted complex.
  • a catechol can be converted to salicylaldehyde using protection of one hydroxy group through methoxymethylether (MOM) and the other hydroxy group as acetate (acetic anhydride) exploiting the ortho directing ability of MOM to make salicylaldehyde, and then deprotecting the acetate group (NaOH, MeOH). Subsequently, react with trialkyl borane and deprotect MOM in the final step.
  • MOM methoxymethylether
  • acetic anhydride acetic anhydride
  • Examples of the active esters which may be used in the practice of the invention include the hemi-succinate esters of N-hydroxy succinimide, sulfo-N- hydroxysuccinimide, hydroxybenzotriazole, and rj-nitrophenol.
  • Examples of dehydration agents include dicyclohexylcarbodiimide (DCC), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI).
  • DCC dicyclohexylcarbodiimide
  • EDC l-(3- dimethylaminopropyl)-3-ethylcarbodiimide
  • EDCI l-(3- dimethylaminopropyl)-3-ethylcarbodiimide methiodide
  • the boron-containing complexes can be administered to a patient suffering, for example, from one of the tumors mentioned above.
  • the complexes will be taken up by the cells.
  • the boron atoms When exposed to neutron therapy, the boron atoms will absorb the neutrons and release the energy thereof, thereby killing the cancer cells.
  • the metal complexes of the present invention may also be used as pharmacologically active substance delivery tools to enhance oral absorption and cell accumulation of linked prodrugs.
  • the pharmacologically active substances are linked through the benzene ring to a peptide, oligonucleotide, peptidomimetic, peptide nucleic acid analog or derivative thereof.
  • the lipophilic cationic properties of the metal complexes of the present invention provide a thermodynamic driving force that pull the linked pharmacologically active substance across membrane bilayers of cells, in response to the negative inner membrane potential.
  • the invention also relates to the use of the complexes of the present invention in the preparation of pharmacologically active substance-complex conjugates which can be used to target cells.
  • the conjugates may be prepared by reaction of the aniline mentioned above with a bifunctional linking group which is capable of linking the aniline amine to a pharmacologically active substance.
  • the present invention is also directed to complex/pharmacologically active substance conjugates which comprise a multidentate ligand-metal complex of the invention linked to a pharmacologically active substance, wherein the linkage does not interfere substantially with the ability of the pharmacologically active substance to carry out its expected pharmacological action.
  • linker group is intended one or more bifunctional molecules which can be used to covalently couple the complex to the pharmacologically active substance and which do not interfere with the pharmacologic action of the pharmacologically active substance in vivo.
  • linker groups which can be used to link the complex to the pharmacologically active substance may comprise
  • salicylaldehyde derivatives can be synthesized by exploiting the presence of halogen at 5 position through coupling reaction involving protected derivative (Formula II) and 3-bromoquinoline (Aldrich) using
  • the pharmacologically active substance is linked to the anilino derivative of the complex by the reaction with succinic anhydride to give the hemi-succinamide.
  • the resulting hemi-succinamide may then be converted to an active ester with a dehydration agent, followed by reaction of the active ester with a nucleophilic functional group on the pharmacologically active substance.
  • Examples of the active esters which may be used in the practice of the invention include the hemi-succinate esters of N-hydroxysuccinimide, sulfo-N-hydroxy- succinimide, hydroxybenzotriazole, andrj-nitrophenol.
  • Examples of dehydration agents include dicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI).
  • a Grignard reagent may be prepared by reaction with magnesium, followed by condensation with a carbonyl-containing pharmacologically active substance.
  • a pharmacologically active substances containing a carbonyl group are progesterone, rolipram, rolicyprine, and progabide.
  • Pharmacologically active substances which can be conjugated to the complexes of the present invention include, but are not limited to, enzymes, such as transferases, hydrolases, isomerases, proteases, ligases, kinases, and oxidoreductases such as esterases, phosphatases, glycosidases and peptidases; enzyme inhibitors such as leupeptin, chymostatin and pepstatin and growth factors such as tumor angiogenesis factor.
  • enzymes such as transferases, hydrolases, isomerases, proteases, ligases, kinases, and oxidoreductases such as esterases, phosphatases, glycosidases and peptidases
  • enzyme inhibitors such as leupeptin, chymostatin and pepstatin
  • growth factors such as tumor angiogenesis factor.
  • Suitable pharmacologically active substances are fat-soluble steroids such as progesterone, estrogens and androgens, as well as the fat soluble vitamins
  • the pharmacologically active substance may be an anti-inflammatory agent (e.g., indomethacin, flurbiprofen, ketoprofen, ibuprofen, phenylbutazone), antibiotics (e.g., beta-lactams, aminoglycosides, macrolides, tetracyclines, pryridonecarboxylic acids, phosphomycin), anti-tumor agents (e.g., adriamycin, cisplatin, bleomycin, mitomycin, fluorouricil, vinblastine, vincristine), amino acids (e.g., ascorbic acid, N-acetyltryptophan), antifungal agents, prostaglandins, vitamins, steroids, and antiviral agents (AZT, DDI, acyclovir, idoxuridine, amantadine, and vidarabine).
  • antibiotics e.g., beta-lactams, aminoglycosides, macro
  • compositions according to the invention may be administered by means of the pharmaceutical or galenic formulations known and used by those skilled in the art for the particular method of administration, but preferably those used for parenteral administration, especially for intravenous, intramuscular, subcutaneous, intracutaneous, intraarticular, intrathecal, intraperitoneal infusion or injection, including continuous infusions or intermittent infusions with the pumps available to those skilled in the art, or the administration by means of micro-encapsulated preparations, e.g., based on liposomes, e.g., according to EP-A-213,523.
  • a ready-to-use solution for the purposes of the invention may for example be prepared by dissolving the metal complex in water or in phosphate-buffered physiological saline solution (pH 7 to 7.5), optionally supplemented with Tween and/or gelatine or an burnin, before the solution being transferred under sterile conditions into suitable containers (e.g., syringes, ampoules, bags).
  • suitable containers e.g., syringes, ampoules, bags.
  • the quantity of metal complex to be administered for the purposes of the invention will be determined in accordance with the dosages known in the art, the severity of the disease, the response rate and the further course of the disease and side effects. Generally speaking, the dosage must be adjusted according to individual criteria.
  • the metal complex is administered to an animal at an effective dosage level of from about 50 mg to 500 mg per day.
  • the method of administration and dosage will depend on the therapy plans known for the above-mentioned antimalarial agents, including also liposome-based microencapsulated antimalarial substances, e.g., according to EP-A-213,523 or EP-A- 152,379 and also, for example, according to EP-A-354,442 or EP-B-56,781, to name just some of the numerous published patent literature.
  • liposome-based microencapsulated antimalarial substances e.g., according to EP-A-213,523 or EP-A- 152,379 and also, for example, according to EP-A-354,442 or EP-B-56,781, to name just some of the numerous published patent literature.
  • the metal complexes according to the invention and conventional antimalarial substance can be administered either by simultaneous administration or by consecutive or sequential administration by suitable route, the individual active substances being provided and administered either separately, e.g., in the form of a kit or directly together.
  • the active substance components which are present separately or either indirectly or directly together may be provided both as dry substances and as solutions, while microencapsulated forms are also possible in which the active substance components may be used directly together, indirectly as a liposome mixture or as separate systems for administration. It is advantageous for the two active substance components, the antimalarial drug and metal complex, to be administered simultaneously.
  • any animal may be treated with the pharmaceutical compositions of the present invention.
  • such animal is a human, however, the invention is not intended to be so limited.
  • the following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All patents and publications cited herein are incorporated by reference herein in their entirety.
  • the potency of these novel metallopharmaceuticals to inhibit the intraerythrocytic growth of the malarial parasite in vitro was determined with the hypoxanthine incorporation method. Because growth of parasites in culture parallels incorporation of radiolabeled hypoxanthine into cellular DNA, antimalarial agents characteristically decrease hypoxanthine radioactivity found within the cell pellets. For the following data, 100% inhibition represents complete eradication of the parasite.
  • Drug-sensitive P. falciparum clone HB3 and chloroquine-resistant P. falciparum clones FCR-3 and Indo were grown at 37°C under 3% oxygen/3% carbon dioxide in RPMI medium using 5% human red blood cells (Trager and Jensen, Science 193:673 (1976)) supplemented with 10% human plasma (Hui et al, Trans. R. Soc. Trop. Med. Hyg. 625:625 (1984)). Synchronization was attained by treatment with sorbitol (Lambros and Vandenberg, J. Parasitol.
  • Late ring-stage cultures at 10% parasitemia were grown in triplicate in the presence of various concentrations of MENPBI-complexes or MENPBA- complexes (diluted in RPMI medium) for 16 h. At the end of this period, l ⁇ Ci
  • ring-stage cultures were incubated with or without addition of lO ⁇ M MENPBI-complexes for 6-21 h. At the designated times, aliquots of cultured parasites were removed and blood smears prepared using Giemsa stain.
  • Late ring-stage cultures at 2% parasitemia were grown for 16 h in the presence of various combinations of the MENPBI-complexes (Francis et al,
  • [ 3 H]hypoxanthine incorporation was measured as previously described (Desjardins et al. , Agents Chemother. 75:710 (1979)).
  • the DMSO vehicle for the drug was diluted in RPMI 1640 to the same extent and added to a similar culture. This had no effect on parasite hypoxanthine incorporation.
  • Figure 1 shows concentration-effect curves for the R 3 -methoxy, R - ethoxy, or R 4 ,R 6 -dimethoxy substituted MENPBI-Fe(III) complexes. All three monocationic Fe(III) complexes were potent inhibitors of the drug-sensitive HB3 strain of P. falciparum. Lineweaver-Burk plots of the inhibition curves revealed
  • IC JO values half-maximal inhibition concentrations of 2.9 ⁇ M, 0.8 ⁇ M, and 1.1 ⁇ M, respectively.
  • the In(III) analog of the 4,6-dimethoxy MENPBI-complex showed only minimal antimalarial activity at all doses (data not shown).
  • a chemical structure-activity survey of the antimalarial action of these novel metallopharmaceuticals when present at concentrations of 1 ⁇ M and 5 ⁇ M was performed with drug-sensitive HB3 and two chloroquine-resistant P. falciparum clones, FCR-3 and Indo (Table 1).
  • CQR Chloroquine-Resistant
  • Figure 2 shows examples of concentration-effect curves for the R 4 ,R 6 - dimethoxy substituted MENPBI-Fe(III) complex in a cytotoxicity assay with human KB renal cell carcinoma cells.
  • the MENPBI-Fe(III) complex was a potent cytotoxic agent in the parental KB-3-1 cells demonstrating an IC 50 of approximately 8 ⁇ M.
  • the derived multidrug-resistant KB-8-5 cells showed no evidence of cytotoxic effects at concentrations up to 100 ⁇ M, indicating that expression of the human MDRl P-glycoprotein transporter modulated the cytotoxic action of the MENPBI-complex.
  • Human MDRl P-glycoprotein is a homologue of the P. falciparum pfrndrl gene product, Pghl, both being integral membrane proteins of the ATP-binding cassette (ABC) superfamily of membrane transporters (Wilson, C. et al, Science 244: 1184-1186
  • P-glycoprotein is an outwardly-directed drug transporter (Gottesman, M.M. and Pastan, I., Annual Review of Biochemistry 52:385-427
  • Pghl may contribute to chloroquine resistance in various clinical isolates of P. falciparum (Foote, S.J. etal, Nature 545:255-258 (1990)), although pfmdrl has been separated from chloroquine resistance in a genetic cross (Wellems, T.E., etal, Nature 545:253-255 (1990)).
  • pfmdrl has been separated from chloroquine resistance in a genetic cross.
  • Wellems, T.E., etal, Nature 545:253-255 (1990) Given the high degree of homology between the MDRl P-glycoprotein and Pghl, we hypothesized that selected analogues of the N 4 O 2 class of metal(III) compounds might be amenable to development as antimalarial chemotherapeutics.
  • the lipophilicity and molecular shape of the resulting complexes can be altered by variation of the substituents on the aromatic rings and hydrocarbon backbone independently.
  • These compounds comprise a new class of readily synthesized antimalarials that, as chloroquine, block hemozoin formation, but additionally are active on chloroquine-resistant strains.
  • Mabi and Dmabi were characterized by 'H and 13 C NMR spectroscopy, infrared (IR) spectroscopy, mass (FAB) spectrometry (LR and HRMS) at the Washington University Resource for Biomedical and Bioorganic Mass Spectrometry Facility (Sharma, V. et al, J. Chem. Med., submitted (1996)).
  • Metal complexes were obtained through the reactions of Mabi and Dmabi with the appropriate metal salts in ethanol as described previously (Sharma, V. et al, J. Chem. Med, submitted (1996)). Resulting metal(III) complexes were characterized by elemental analysis confirming purity and by 'H NMR (except for Fe(III) complexes), IR, and FAB-LRMS establishing structural identity.
  • Plasmodium falciparum strains (HB3, FCR-3, Indo-1, Dd2) were grown in intraerythrocytic culture by the method of Trager and Jensen (Trager, W. and Jensen, J.B., Science 193:673-675 (1976)). Cultures were maintained at 5% parasitemia, 2% hematocrit using human serum and erythrocytes, in a 3% oxygen/3% carbon dioxide atmosphere. Synchronization of developmental stage was achieved by sorbitol treatment (Lambros, C. and Vandenberg, J., J. Parasitol 55:418-420 (1979)).
  • IC 50 half-maximal inhibitory concentrations
  • HRP II histidine-rich protein II
  • the digestive vacuole proteases plasmepsins I and II and falcipain were purified and assayed using ,4 C-globin substrate as previously described (Gluzman, I. et al, J. Clin. Invest. 95:1602-1608 (1994)). Inhibitors were added as 1:1000 dilutions of a 10 mM DMSO stock.
  • Parasite death as measured by the 3 H-hypoxanthine incorporation assay correlated well with blood smear counts.
  • the drug was effective at the mid- trophozoite stage; more mature parasites were resistant and less mature parasites grew normally until they developed into trophozoites, at which point they were killed by the treatment ( Figure 4B).
  • Parasites cultured in the presence of drug showed greatly diminished hemozoin formation.
  • control parasites matured normally, developing a large digestive vacuole filled with hemozoin pigment, and then undergoing normal schizogony.
  • Chloroquine is modestly lipophilic and possesses titratable protons that confer a net cationic charge in acid environments.
  • diffusive transmembrane transport and drug trapping may account for concentrative accumulation of chloroquine within the digestive vacuole (reviewed in Slater, A., Pharmacol. Ther. 57:203- 235 (1993)).
  • the net cationic charge provided by protonation of the agent or binding to high affinity sites within the vacuole may prevent back-diffusion of the compound (Chou, A. etal, Biochem. 79:1543-1549
  • Acta 7747:262-266 (1993) may allow permeation across membranes and concentrative accumulation within cell interiors in response to the negative transmembrane potentials generated by living cells (Ritchie, RJ., Prog. Biophys. Molec. Biol 43:1-32 (1984)). Additional non- physicochemical processes could also contribute, particularly parasitic expression of the pfmdrl gene product, Pghl, a member of the ABC superfamily of membrane transporters (Wilson, C. etal, Science 244:1184-1186 (1989); Foote, S.J. et al, Nature 545:255-258 (1990); van Es, H. et al, Molec. and Cell Biol.
  • the present invention identifies a new class of antimalarial compounds with potent activity in Plasmodium culture, and significantly, against several chloroquine-resistant strains.
  • Antimalarial potency in culture correlated with their ability to inhibit heme polymerization, the most potent lead being the 4,6- dimethoxy- ENBPI Fe(III) complex.
  • Potency also correlated with the fold-drug resistance conferred by expression of MDRl P-glycoprotein on cells exposed to these complexes, one measure of the ability of these agents to be transported by the human gene product (Sharma, V. et al, J. Chem. Med., submitted (1996)). It is expected that accumulation of drug by the parasites is dependent on the
  • Pgh-1 Plasmodium P-glycoprotein homologue, Pgh-1, which is located on the digestive vacuole membrane (Cowman, A.F. et al, J. Cell Biol. 775:1033-1042 (1991)).
  • P-glycoprotein especially the R-ENBPI In(III) complexes, lacked significant parasitocidal activity in Plasmodium culture and did not inhibit hemozoin formation in vitro. It is unlikely that this can be attributed to differential demetallation reactions of the agents, since this study demonstrated poor correlation between metal(III) salts and intact metal(ffi) complexes for inhibition of hemozoin formation under acidic conditions and, furthermore, the agents have been documented by 'H-NMR and UV/VIS spectroscopy to be hydrolytically stable at neutral pH (37°C) for 72 hrs (51 -A). Molecular configuration may be relevant.
  • the IC 5 o values for culture inhibition are in some cases slightly lower than those for in vitro heme polymerization.
  • the conditions for heme polymerization in the digestive vacuole could be sufficiently different from those in the test tube to change the IC S0 value.
  • the compounds could be accumulated against a concentration gradient in the digestive vacuole.
  • the R-ENBPI metal(III) complexes had equivalent potency in the pre- formed hemozoin-initiated polymerization assay as in the HRP JJ-mediated assay.
  • the tetramine backbone can be varied and substituted in a variety of positions on the aromatic ring in this class of compounds.
  • the agents are relatively easy and inexpensive to synthesize, which is a crucial feature for success of a useful antimalarial drug.
  • varying the ring substituents provided new compounds that are effective in the high nanomolar range. It is encouraging to have a group of agents with scaffolds incorporating a biologically compatible metal like Fe(III) that appear to intercept the same molecular target as chloroquine, but are not subject to the same resistance mechanisms.
  • Table 2 shows the effect of various R-ENBPI metal(III) complexes and chloroquine on intraerythrocytic P. falciparum growth in culture.
  • the substituent nomenclature is referenced in Figures 3 and 6.
  • Agents were tested at the indicated concentrations against chloroquine-sensitive and -resistant strains by the 3 H-hypoxanthine incorporation method; values are presented as percent growth inhibition relative to control cultures without drug. Data are presented as the mean of triplicate determinations from representative experiments.
  • NAA no antimalarial activity
  • R 5 H.
  • Table 3 shows the IC 50 values for inhibition of parasite growth and hemozoin formation that are tabulated from this study and compared to MDR1- mediated drug resistance values recalculated from data in Sharma, V. et al, J. Chem. Med., submitted (1996). Derived from studies in human KB cells, the
  • MDRl data indicate the fold-resistance (IC 50 in MDR cells IC 50 in drug-sensitive cells; Ford, J.M. and Hait, W.N., Pharmacol. Rev. 42:155-199 (1990)) that expression of MDRl P-glycoprotein conferred on the cytotoxic potency of the indicate metal(III) complexes.
  • Table 4 shows data for the inhibition of heme polymerization by metal salts. Heme polymerization was assayed by the pre-formed hemozoin nucleation reaction (see Methods) in the absence or presence of 10 ⁇ M concentrations of the indicated metal salts. Data are mean values of triplicate determinations from representative experiments.
  • these hexadentate Schiff-base ligands with the general structure shown in Figure 6, or according to formula / offer tremendous flexibility, since their binding affinities can be varied by inserting appropriate donor atoms to match the requirements of various incoming metals.
  • the lipophilicity and molecular shape of the resulting complexes can be altered by variation of the substituents on the aromatic rings and hydrocarbon backbone independently.
  • Heptadentate compounds 3 and 4 precursors ( Figure 7) of the desired hexadentate ligands, were synthesized by condensation of the appropriate amine and three equivalents of substituted salicylaldehyde in ethanol ( Figure 7).
  • condensation of the amine with three equivalents of substituted salicylaldehyde in dry methylene chloride in the presence of activated molecular sieves provided spectroscopically identical compounds.
  • NMR spectrum showed 20 resonance signals (Figure 8), including the characteristic carbon resonance at ⁇ 165.2 for the imine carbon and ⁇ 89.3 for the benzylic carbon, consistent with the proposed structure for precursor 3.
  • the ⁇ NMR of precursor 4 in CDC1 3 also showed the characteristic imine proton singlet at ⁇ 8.25, aromatic protons at ⁇ 5.99, 5.91, 5.84, and 5.56 in a ratio of 1:1:2:2, for the middle and outer aromatic rings, respectively, a proton assigned to the benzylic position at ⁇ 4.32, different methoxy groups at ⁇ 3.77, 3.76, 3.71, and 3.70 in a ratio of 2:2:1:1, respectively, and protons for the hydrocarbon backbone in the form of a series of multiplets at ⁇ 3.52, 3.35, 2.75-2.30, and 1.80.
  • Proton- decoupled 13 C NMR spectrum of precursor 4 in CDC1 3 showed 20 resonance signals instead of 22 ( Figure 8), presumably because of overlapping methoxy signals.
  • the heptadentate precursors When treated with trivalent metals in aqueous conditions, the heptadentate precursors, whether isolated as 3 and 4 or formed as Schiff base byproducts during condensation reactions of appropriate salicylaldehydes and amines, split off the middle ring, thereby decreasing steric demands on the resultant ligands and providing an N 4 O 2 hexadentate donor core (Sarma, B.D. and Bailar, J.C., J.
  • A1(III) and In(III) complexes were isolated by treatment of precursors 3 and 4 with their appropriate hydrated salts in the presence of base. Formation of metal complexes with the ligands ( Figure 6) derived from precursors 3 and 4 and main group metal cations should be governed by the electronic and steric demands of the incoming metals relative to the flexibility of the donor core. Due to the nearly identical six-coordinate ionic radii of Fe +3 and Ga +3 (0.65 A and 0.62A, respectively (Shannon, R.D., Acta Cryst. A32:751-767 (1976))), ligands with high affinity for Fe +3 tend to have high affinity for Ga +3 , often resulting in similar coordination chemistry.
  • Al +3 (0.5lA)and In +3 (0.81 A) were selected as two extreme limits to analyze the impact of the central metal core on the organic scaffold and to evaluate their overall effect on biological activity.
  • complexation of heptadentate compounds 3 and 4 with their corresponding trivalent metal ions lead to cleavage of the five- membered imidazolidine ring, thereby exposing the inner secondary amine nitrogens for coordination to the metals. This imparted some degree of pre- organization to the N 4 O 2 donor core to meet the demands of the incoming metal ion as the ligands wrapped around the coordination sphere.
  • Infrared (IR) spectra as KBr pellets of A1(III), Fe(III), Ga(H ⁇ ), and In(III) metal complexes were essentially identical, with only marginal differences in a few selected bands. Broad bands between 3250-3100 cm 1 and 1550-1560 cm" 1 were assigned to stretching and bending modes, respectively, of the coordinated amine nitrogens. All complexes, except 3b, showed a broad band between 3600-
  • the R-ENBPI derivatives of precursors 3 and 4 (Figure 7), containing an N 4 O 2 donor core with the general structure shown in Figure 6, wrapped around the central metal ion to provide four six-membered rings and one five-membered ring.
  • two six-membered and three five-membered rings are created by compounds containing bis ethylene groups (Sarma, B.D. and Bailar, J.C., J. Am. Chem. Soc. 77:5476-5480 (1955)) instead of bis propylene groups.
  • the propylene backbone links adjacent donor atoms so that they must be placed cis to each other, therefore generating angles between adjacent donor atoms close to 90°.
  • Ga(III) complexes but suggest the cis configuration for In(III) complexes (Sharma, V. etal, J. Nucl. Med, submitted (1996)).
  • P-glycoprotein encoded by the human multidrug resistance (MDRl) gene, is an integral plasma membrane transporter which renders tumors resistant to chemotherapy by transporting chemotherapeutic agents out of cells (Gros, P. et al, Nature 525:728-731 (1986); Shen, D.W. etal, Science 252:643-645 (1986);
  • P-glycoprotein is thought to be one potent mechanism of cancer chemotherapeutic failure in patients.
  • Many drug substrates and modulators (inhibitors) of P-glycoprotein are lipophilic cationic compounds
  • Somatic Cell Mol. Genet. 11:117-126 (1985)) express no immunodetectable and modest levels of MDRl P-glycoprotein, respectively, as determined by immunoblots of plasma membrane preparations with the anti-P-glycoprotein monoclonal antibodies C219 (Dolci, E.D. etal, InternationalJournal of Cancer 54:302-308 (1993)) and C494.
  • Cytotoxic potency was determined by computer fitting of survival curves and determination of an LC 50 .
  • (3-MeO-R- ENBPI)Fe(III) (3b, see Experimental) demonstrated an LC 50 of 9 ⁇ M, the most potent cytotoxic activity for the 3-MeO-R-ENBPI series ( Figure 9A-D).
  • Metal(III) complexes of 3-MeO-R-ENBPI showed a rank order cytotoxic potency of Fe(III) > A1(IH) > In(III) > Ga(III) (Table 5).
  • MDRl P- glycoprotein by the KB-8-5 tumor cells conferred dramatic protection from the cytotoxic action of (3-MeO-R-ENBPI)Fe(III) (3b), with concentrations of 3b as high as 100 ⁇ M demonstrating no significant cytotoxicity. Additionally, MDRl P-glycoprotein conferred modest protection (2-3 fold) from the cytotoxic action of 3a and 3c, but conferred no significant protection from 3d. Similarly, in KB- 3-1 cells, metal(III) complexes of 4,6-diMeO-R-ENBPI showed a rank order cytotoxic potency of Fe(III) > Al(III) > Ga(III) » In(III) ( Figure 9E-H, and
  • Fe(III) ions did not show any evidence of cytotoxicity at any concentration up to 100 ⁇ M or any differential effect between drug-sensitive and MDR cells.
  • Ga(III) and In(III) salts demonstrated modest toxic activity at 100 ⁇ M with a trend towards increased potency in the drug-resistant cell line, opposite to effects observed with the metal complexes.
  • precursors 3 and 4 when added to the aqueous buffer (thereby likely providing the free hexadentate ligand) demonstrated no significant cytotoxic activity except at the highest test concentration of 100 ⁇ M where modest activity was observed equally in both cell lines (data not shown).
  • the preferential potency of the Fe(III)-complexes may be attributable to specific conformational dimensions of the complex, but cannot exclude the possibility of acid hydrolysis of these agents within subcellular organelles such as lysosomes. While these metallopharmaceuticals were designed to exploit electronegative compartments such as mitochondria as a cytotoxic target, further experiments are required to explore the pharmacological sites, mechanism(s) of action, stereochemistry of drug activity and impact of co- administration of MDR modulators on their anticancer potency.
  • Bis(N,N'-aminopropyl)ethylenediamine, 4,6-dimethoxy salicylaldehyde and O-vanillin (3-methoxy salicylaldehyde) were obtained from Aldrich Chemical Co.
  • a 1(111)-, Fe(III)-, Ga(III)-, and In(III)-acetylacetonates and their hydrated salts were purchased from Mathey- Johnson/Alfa Chemical Co. and Aldrich.
  • the ⁇ and 13 C NMR spectra were recorded on a GEMINI 300 HHz spectrometer; chemical shifts are reported in ⁇ (ppm) with reference to TMS.
  • IR spectra were recorded on a Perkin-Elmer 1710 Fourier transform spectrophotometer.
  • Mass spectra were obtained from the Washington University Resource for Biomedical and Bioorganic Mass Spectrometry with 3-nitrobenzyl alcohol or thioglycerol as a matrix. Elemental analyses (C, H, N) were performed by Galbraith Laboratories, Knoxville, TN.
  • Molar conductance (K, ⁇ " 1 mol *1 cm 2 ) was determined with a portable conductivity meter, (Orion Research, model 120) at 25°C in acetonitrile with 0.37 mM solutions of each complex. Aqueous stabilities of the complexes were determined by suspending 5-10 mg of sample in 2 ml of H 2 O (pH 7.5) in a sealed vial. Samples were stirred for 72 h (37°C), evaporated and residue analyzed by U VIS (Beckman 620) or ⁇ NMR spectroscopy in DMSO-d 6 .
  • N,N'-bis(aminopropyl)ethylenediamine (286 mg, 1.64 mmol) dissolved in ethanol (5 ml) was added to a stirred solution of o-vanillin (748 mg, 4.92 mmol) dissolved in ethanol (10 ml).
  • the reaction mixture was heated to reflux for 3 h. After cooling to room temperature, volatiles were removed through rotatory evaporation and the residue dried under reduced pressure for two days to yield a bright yellow solid 1 (900 mg, 1.56 mmol, 95% yield).
  • ligand was dissolved in methanol (3 ml), treated dropwise with potassium hydroxide and heated to reflux for 20 min. While hot, the appropriate hydrated salt dissolved in methanol (5 ml) containing water (2ml) was added, refluxed for 1 h, cooled and filtered. The filtrate was slowly evaporated at room temperature to yield a microcrystalline solid, which was separated, washed with water, followed by cold methanol, then washed with ether and dried under reduced pressure overnight.
  • Another method involved mixing equimolar quantities of the appropriate ligand and metal acetylacetonates in ethanol. Contents were refluxed for 1 h, then the reaction mixture was treated while hot with an equimolar amount of Kl dissolved in water, and then refluxed for an additional 5-1 Omin. The resulting mixture was cooled to room temperature and a microcrystalline compound precipitated out. This was separated, washed with cold ethanol, then washed with ether and dried under reduced pressure.
  • Monolayers of parental KB-3-1 and multidrug-resistant KB-8-5 cell lines were routinely grown in DMEM (GIBCO, Grand Island, NY) supplemented with
  • Multidrug-resistant cells were cultured in drug-free media for 96 h prior to cytotoxicity assays. Cytotoxicity potencies of R-ENBPI metal-complexes, metal salts or colchicine were determined in 96-well microtiter plates as described (Piwnica-Worms, D. etal, Cancer Res. 53:1-8 (1993)). Cells
  • SRB sulforhodamine B
  • S cell protein
  • S max cell protein in control buffer
  • S min residual cell protein at highest drug toxicity
  • ⁇ is the slope
  • C cytotoxic agent concentration
  • LC 50 represents the half-maximal cytotoxic concentration.

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Abstract

Les complexes métalliques polydentés décrits répondent à la formule (I), dans laquelle M désigne Fe, In, Ga ou Al; les lignes interrompues désignent indépendamment une liaison simple ou double; les lignes hachurées désignent une coordination avec le cation métallique (M); A et B désignent oxygène, ou lorsque Ar désigne pyridyle-2 ou pyrrolyle-2, une paire partagée d'électrons sur l'azote; Ar désigne phényle, naphtyle, pyridyle-2 ou pyrrolyle-2, le cas échéant substitués, à condition qu'au moins un des substituants comprenne un atome de bore; X désigne alternativement (i) (CHR2)p[NR3(CHR4)q]r, où p et q valent indépendamment 1, 2, 3, 4, 5 ou 6; r vaut 0, 1 ou 2; et R?2, R3 et R4¿ désignent indépendamment les uns des autres hydrogène, alkyle inférieur ou phényle, ou deux groupes R2 ou R4 adjacents désignent une liaison double ou un anneau benzène fusionné dans lequel p ou q, respectivement, sont supérieurs à 2; à condition que, lorsque r vaut 1 ou 2, un ou deux sites additionnels de coordination avec le métal soient présents; ou (ii) phényle, éventuellement substitué par des alkyles inférieurs. Une des deux conditions suivantes peut s'appliquer: (A) au moins un des substituants d'Ar comprend du bore, ou (B) au moins un des substituants d'Ar comprend une liaison avec un substituant pharmaceutiquement actif.
PCT/US1996/010079 1995-06-07 1996-06-07 Complexes metalliques polydentes et leurs procedes de preparation et d'utilisation WO1996040108A1 (fr)

Priority Applications (4)

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JP9502188A JPH11507641A (ja) 1995-06-07 1996-06-07 多座金属錯体並びにこれらを製造する方法及びこれらの使用
MX9709471A MX9709471A (es) 1995-06-07 1996-06-07 Complejos metalicos multidentados y metodos para preparar y usar los mismos.
AU64771/96A AU6477196A (en) 1995-06-07 1996-06-07 Multidentate metal complexes and methods of making and using thereof
EP96924270A EP0831806A1 (fr) 1995-06-07 1996-06-07 Complexes metalliques polydentes et leurs procedes de preparation et d'utilisation

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US48799195A 1995-06-07 1995-06-07
US08/487,991 1995-06-07

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CA (1) CA2224115A1 (fr)
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WO (1) WO1996040108A1 (fr)

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US5997912A (en) * 1996-09-03 1999-12-07 University Of Iowa Research Foundation Method for inhibiting growth of P. aeruginosa using gallium-containing compounds
EP1530720A2 (fr) * 2001-11-13 2005-05-18 David A. Sirbasku Programme d'eradication du cancer du sein
US7851512B2 (en) 2003-09-26 2010-12-14 Li Guogiao Composition containing artemisinin for treatment of malaria
US8038999B2 (en) 2000-05-10 2011-10-18 Sirbasku David A Breast cancer eradication program
WO2013113496A1 (fr) 2012-01-30 2013-08-08 Fresenius Kabi Deutschland Gmbh Amidon hydroxy-alkylé en combinaison avec des cytostatiques pour le traitement de cancers par réduction de taux de croissance tumorale
US8563249B2 (en) 2000-05-10 2013-10-22 Signe Biopharma Inc. Receptor gene screening for detecting or diagnosing cancer
EP2673257A2 (fr) * 2011-02-11 2013-12-18 Washington University Agents pet/spect pour applications en imagerie biomédicale
CN112638922A (zh) * 2018-05-03 2021-04-09 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法

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Cited By (16)

* Cited by examiner, † Cited by third party
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US6203822B1 (en) 1996-09-03 2001-03-20 University Of Iowa Research Foundation Gallium-containing compounds for the treatment of infections caused by intracellular pathogens and pathogens causing chronic pulmonary infection
US5997912A (en) * 1996-09-03 1999-12-07 University Of Iowa Research Foundation Method for inhibiting growth of P. aeruginosa using gallium-containing compounds
US8563249B2 (en) 2000-05-10 2013-10-22 Signe Biopharma Inc. Receptor gene screening for detecting or diagnosing cancer
US8038999B2 (en) 2000-05-10 2011-10-18 Sirbasku David A Breast cancer eradication program
EP1530720A2 (fr) * 2001-11-13 2005-05-18 David A. Sirbasku Programme d'eradication du cancer du sein
EP1530720A4 (fr) * 2001-11-13 2005-08-31 David A Sirbasku Programme d'eradication du cancer du sein
EP2306187A1 (fr) * 2001-11-13 2011-04-06 Signe BioPharma Inc. Programme d'éradication du cancer
US7851512B2 (en) 2003-09-26 2010-12-14 Li Guogiao Composition containing artemisinin for treatment of malaria
EP2673257A4 (fr) * 2011-02-11 2014-11-12 Univ Washington Agents pet/spect pour applications en imagerie biomédicale
EP2673257A2 (fr) * 2011-02-11 2013-12-18 Washington University Agents pet/spect pour applications en imagerie biomédicale
US9579408B2 (en) 2011-02-11 2017-02-28 Washington University PET/SPECT agents for applications in biomedical imaging
WO2013113765A1 (fr) 2012-01-30 2013-08-08 Fresenius Kabi Deutschland Gmbh Amidon hydroxyalkylé dans le traitement de cancers de la tête et du cou par réduction des taux de croissance tumorale
WO2013113747A1 (fr) 2012-01-30 2013-08-08 Fresenius Kabi Deutschland Gmbh Amidon hydroxyalkylé utilisé dans le traitement de cancers par réduction des taux de croissance tumorale
WO2013113496A1 (fr) 2012-01-30 2013-08-08 Fresenius Kabi Deutschland Gmbh Amidon hydroxy-alkylé en combinaison avec des cytostatiques pour le traitement de cancers par réduction de taux de croissance tumorale
CN112638922A (zh) * 2018-05-03 2021-04-09 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法
CN112638922B (zh) * 2018-05-03 2024-04-05 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法

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MX9709471A (es) 1998-02-28
EP0831806A1 (fr) 1998-04-01
CA2224115A1 (fr) 1996-12-19
AU6477196A (en) 1996-12-30
JPH11507641A (ja) 1999-07-06

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