WO2010102393A1 - Dérivés de 2-indolylimidazo[4,5-d]phénanthroline et leur utilisation pour inhiber l'angiogenèse - Google Patents

Dérivés de 2-indolylimidazo[4,5-d]phénanthroline et leur utilisation pour inhiber l'angiogenèse Download PDF

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WO2010102393A1
WO2010102393A1 PCT/CA2010/000337 CA2010000337W WO2010102393A1 WO 2010102393 A1 WO2010102393 A1 WO 2010102393A1 CA 2010000337 W CA2010000337 W CA 2010000337W WO 2010102393 A1 WO2010102393 A1 WO 2010102393A1
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compound
formula
alkyl
hydrogen
halogen
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Mario Huesca
Mei Wang
Aiping H. Young
Yoon Lee
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Lorus Therapeutics Inc.
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    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the im ention relates generalh to the field of anti-angiogenic therapeutics and in particular to the use of 2-indoh l imidazo[4,5-d]phenanthroline de ⁇ ati ⁇ es to inhibit angiogenesis
  • Angiogenesis is the fundamental process b ⁇ which new blood ⁇ essels are formed and is essential to a ⁇ ariet ⁇ of normal bod ⁇ actmties such as reproduction, de ⁇ elopment and w ound repair
  • the process is not completeh understood, it is behe ⁇ ed to im oh e a complex interplay of molecules which both stimulate and inhibit the growth of endothelial cells, the priman cells of the capillar ⁇ blood ⁇ essels
  • the angiogenic process im oh es not onh increased endothelial cell proliferation but also comprises a cascade of additional e ⁇ ents, including protease secretion b ⁇ endothelial cells.
  • Angiogenic stimuli for example, hy poxia or inflammaton cy tokines
  • VEGF ascular endothelial growth factor
  • FGF fibroblast growth factor
  • Angiogenesis is a "common denominator" shared b ⁇ man ⁇ diseases including cancer, cardio ⁇ ascular disease, blindness, arthritis, complications of AIDS, diabetes, Alzheimer ' s disease and others
  • cornea and cartilage are a ⁇ ascular in health ⁇ situations
  • se ⁇ eral diseases im ohing these tissues are complicated b ⁇ the mass ⁇ e arm al of new blood ⁇ essels
  • E ⁇ e angiogenic diseases include neo ⁇ ascular glaucoma, retrolental fibroplasia, macular degeneration and neo ⁇ ascula ⁇ zation of corneal grafts
  • new capillaries formed in the retina im ade the ⁇ itreous, bleed, and cause blindness Joint angiogenic diseases include rheumatoid arthritis and arthrosis Psoriasis also exhibits pen ascula ⁇ zation at the surface of the skin Regulation of cell replication and/or differentiation is important in the maintenance of existing blood ⁇ essels, for example,
  • VEGF vascular endothelial growth factor
  • FIt-I and Flk-1/KDR receptors also called VEGF receptor 1 (VEGFRl) and VEGF receptor 2 (VEGFR2)
  • VEGFRl and VEGFR2 VEGF receptor 1
  • VEGFR2 VEGF receptor 2
  • HIF-I is a heterodimeric basic-helix-loop-hehx-PAS transcription factor consisting of HIF-I alpha and HIF-I beta subunits HIF-I alpha expression and HIF-I transcriptional acti ⁇ it ⁇ increase exponential! ⁇ as cellular ox ⁇ gen concentration is decreased
  • HIF-I is considered a critical mediator of the cellular response to h ⁇ poxia through its regulation of genes that control angiogenesis HIFs regulate the transcription of h ⁇ poxia-inducible genes b ⁇ binding poxia response elements (HRE) found in the promoter and enhancer regions of inducible genes H ⁇ poxia response elements ha ⁇ e been found in the promoter regions of genes encoding VEGF, the VEGF receptor FIt-I and nitric oxide sy nthases (associated with ⁇ asodilatation) It is behe ⁇ ed that hy
  • Matrix metalloproteinases are also im oh ed in angiogenesis
  • MMPs are a famih of zinc proteinases that digest the extracellular matrix and are implicated in a ⁇ ariet ⁇ of pathological conditions, including earh stages of angiogenesis, cardio ⁇ ascular diseases, and cancer metastasis
  • MMPs including MMP-2 and MMP-9, are expressed in a ⁇ an et ⁇ of tissues
  • Compounds that inhibit MMP acti ⁇ it ⁇ are thought to be useful for the treatment or propr ⁇ laxis of conditions that im oh e tissue breakdown including rheumatoid arthritis, osteoarthritis, gastric ulceration and cancer metastasis, as w ell as management of angiogenesis-dependent diseases
  • Kruppel-hke factor KLF2 is a transcription factor that is expressed in lung, endothelial cells and h mphoc ⁇ tes and is essential for blood ⁇ essel lntegrit ⁇ and lung de ⁇ elopment KLF2 has been implicated in a number of processes including th ⁇ moc ⁇ te and T-cell migration (Carlson, C M .
  • KLF2 has also been shown to inhibit VEGF-A-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis
  • KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammator ⁇ factors in endothelial cells, and effect that is behe ⁇ ed to be due to inhibition of VEGFR2/KDR expression and promoter actmt ⁇ (Bhattachan a R, et al , J Biol Chem , 280 28848-51 (2005))
  • An object of the present invention is to provide 2-indolvl imidazo[4,5-d]phenanthroline derivatives and uses thereof in the inhibition of angiogenesis.
  • Rl, R2, R3 and R4 are independently selected from hydrogen, halogen, Cl - C4 alkyl, methoxy and phenyl;
  • R5 is hydrogen, Cl - C4 alkyl. -CH 2 -phenyl or cyclopentyl;
  • R6 is hydrogen, C l - C4 alkyl, phenyl optionally substituted with halogen or Cl - C4 alkyl, -CH 2 -heterocycle wherein the heterocycle is 6-membered ring comprising 1 or 2 heteroatoms selected from N and O and optionalh substituted with C l - C4 alk ⁇ 1.
  • Figure 1 depicts the ability of compounds 3 and 7 to inhibit tumour growth in a non- small cell lung carcinoma xenograft model (A) and in a colon adenocarcinoma xenograft model (B), the abiht ⁇ of compound 7 to inhibit tumour grow th in a non-small cell lung carcinoma xenograft model (C), and the abiht ⁇ of compounds 7, 63, 64, 69, 72, 73, 74, 18 and 78 to inhibit tumour cell growth in a non-small cell lung carcinoma xenograft model (D)
  • Figure 2 depicts the effect of compound 3 and compound 64 in the presence and absence of zinc on mRNA le ⁇ els in HT-29 cells in vitro for MTlA (A), MTF-I (B), C ⁇ din Dl (C) and KLF4 (D)
  • Figure 3 depicts the effects of compound 7 on ⁇ anous transcription factors.
  • A comparison of transcription factor actmt ⁇ in CCD- 18Co normal colon fibroblast cells and HT-29 colon carcinoma cells.
  • B transcription factor actmt ⁇ in CCD- 18Co normal colon fibroblast cells and HT-29 colon carcinoma cells treated with 1 ⁇ M compound 7;
  • C metallothionein 1 (MTlA) expression in HT-29 cells treated with 1 ⁇ M compound 7;
  • HSF-I heat shock factor 1
  • Figure 4 depicts the effect of compound 7 on niRNA levels of KLF4 (A) and (B), ⁇ - Catenin (C) and Cyclin Dl (D) in HT-29 colon cancer cells in vitro over time.
  • Figure 5 depicts the effect of compound 7 on niRNA levels of KLF4 (A), ⁇ -Catenin (B) and Cyclin Dl (C) in NSCLC H226 cancer cells in vitro over time.
  • Figure 6 depicts the effect of compound 7 on mRNA levels of KLF4 in vivo in tumours from a H226 mouse xenograft model over time.
  • Tumor samples w ere excised at different time point after Lv. injection of single dose of 15mg/kg compound 7.
  • Groups of 4 mice were injected and tumors were isolated at the time points indicated.
  • Gene expression levels of KLF-4 were measured in each tumor using RT-PCR.
  • Figure 7 (A-D) depicts the effect of compound 7 on mRNA levels of ⁇ -Catenin in vivo in tumours from the H226 mouse xenograft model described for Figure 6 over time.
  • Figure 8 depicts the effect of compound 7 on mRNA levels of Cyclin Dl in vivo in tumours from the H226 mouse xenograft model described for Figure 6 over time.
  • Figure 9 depicts the effect of compound 7 on mRNA levels of HIF-I ⁇ in H226 cancer cells in vitro over time.
  • Figure 10 depicts the ability of compound 7 to decrease tumour size in a human lung carcinoma H226 xenograft in CD-I nude mice. The tumor growth profile after single injection of 15mg/kg compound 7 is shown.
  • Figure 11 depicts the effect of compound 7 on mRNA levels of MTF-I (A-B), HIF- l ⁇
  • C-D C-D
  • MMP2 E
  • VEGFR2 F
  • Figure 12 depicts the results of immunohistochemistn anah sis demonstrating the effect of compound 7 (single treatment of 15 mg/kg) on micro ⁇ essel densit ⁇ in tumors (upper row of images) compared w ith a negat ⁇ e control group (low er row of images) Tumors w ere stained with anti-CD-31 antibodies to detect endothelial cells in sample sections
  • Figure 13 depicts the effect of compound 7 in the presence (A) and absence (C) of zinc on HUVEC cell growth in vitro, the effect of a negat ⁇ e control compound is shown in (B)
  • Figure 14 depicts the effect of compound 7 in the presence and absence of zinc on HUVEC tube formation in vitro HUVEC cells without (A-B) and with (C-D) ZnCl 2 DMSO control without ZnCl 2 (A). 0 5 ⁇ M of compound 7 without ZnCl 2 (B). DMSO control with ZnCl 2 (C). O 5 ⁇ M of compound 7 with ZnCl 2 (D). 5 ⁇ M of negat ⁇ e control treated cells (E)
  • Figure 15 depicts the abiht ⁇ of compound 7 to pre ⁇ ent tumor growth in human tumor xenograft models.
  • A model using H226 lung carcinoma cells and treatment with 15 mg/kg compound 7 or ⁇ ehicle control.
  • B model using HOP92 lung carcinoma cells and treatment with 10 mg/kg compound 7 or ⁇ ehicle control.
  • C model using H460 lung carcinoma cells and treatment with 7 5 mg/kg compound 7 or ⁇ ehicle control (1 C ⁇ cle).
  • D model using H226 lung carcinoma cells and treatment with 10 mg/kg compound 7 or ⁇ ehicle control All results w ere significant p ⁇ 0 05
  • the present inv ention relates to 2-indoh l imidazo[4,5-d]phenanthroline compounds of general Formula I, which, as demonstrated herein, are capable of inhibiting angiogenesis
  • pro ⁇ ides for methods and uses of compounds of Formula I for inhibiting angiogenesis Angiogenesis is a process in a number of diseases and disorders and another embodiment of the in ⁇ ention therefore pro ⁇ ides for methods and uses of compounds of Formula I to inhibit angiogenesis and thereb ⁇ facilitate the treatment of angiogenesis-associated diseases 01 disoideis
  • compounds of Formula I are capable of reducing expression of ⁇ ascular endothelial growth factor receptor 2 (VEGFR2)
  • VEGFR2 ⁇ ascular endothelial growth factor receptor 2
  • methods and uses of the compounds of Formula I for reducing expression of VEGFR2 m vivo or m vitro are pro ⁇ ided VEGF and its receptors (VEGFRl/Flt-1 and VEGFR2/F1L-1/KDR) are known to be im oh ed in regulation of angiogenesis in ⁇ anous disease contexts
  • Actu ation of VEGFR2 stimulates angiogenesis, while VEGFRl actu ation appears to pla> a dual function b ⁇ either stimulating angiogenesis or suppressing angiogenesis
  • the compounds of Formula I are capable of reducing the expression of Hy poxia-inducible factor- 1 (HIF- l ⁇ ) and the inv ention further pro ⁇ ides for methods and uses of the compounds of Formula I for reducing expression of HIF- l ⁇ m vivo or m vitro
  • the in ⁇ ention also contemplates application of the compounds of Formula I in ⁇ arious non- therapeutic situations in which inhibition of angiogenesis is required
  • halogen refers to fluorine, bromine, chlorine, and iodine atoms.
  • C 1 -C4 alkyl refers to a straight chain or branched alkyl group of one to four carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl. see- but ⁇ 1 and tert-butyl (t-butyl).
  • alkoxy refers to the group -OR, w here R is C1-C4 alkyl.
  • heterocycle refers to a saturated carbocyclic group having a single ring and including at least one hetero atom, such as N, O or S, within the ring.
  • cycloalkyl refers to a cyclic or poly cyclic alkyl group containing 3 to 15 carbon atoms.
  • therapy and treatment refer to an intervention performed w ith the intention of alleviating the symptoms associated w ith, preventing the development of, or altering the pathology of a disease, disorder or condition.
  • therapy and treatment are used in the broadest sense, and include the prevention (prophylaxis), moderation, management, reduction, or curing of a disease, disorder or condition at various stages. Prevention or reduction of the progression of a disease, disorder or condition is encompassed by these terms. Also encompassed by these terms is an intervention resulting in an alteration of physiology and/or biochemistry of a living subject.
  • Those in need of therapy/treatment include those already having the disease, disorder or condition as w ell as those prone to, or at risk of developing, the disease, disorder or condition and those in whom the disease, disorder or condition is to be prevented.
  • the therapeutic application of compounds of the invention therefore, refers to a therapy or treatment, as defined herein.
  • subject or "patient,” as used herein, refer to an animal in need of treatment, including humans and other mammals.
  • Administration of the compounds of Formula I "in combination with " one or more further therapeutic agents, is intended to include simultaneous (concurrent) administration and consecutive administration. Consecutive administration is intended to encompass various orders of administration of the therapeutic agent(s) and the compound(s) to the subject.
  • inhibitor. means to reduce, halt or hold in check, and thus inhibition may be complete or partial and may be of short or long term duration.
  • the term may be used in the context of inhibiting a process or action already begun or it may be used in the context of inhibiting initiation of a process or action.
  • the term "about" refers to approximately a +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • the present invention provides compounds of the general Formula I:
  • Rl, R2, R3, R4 are independently selected from hydrogen; halogen; C 1-C4 alkyl; C 1 -C4 alkoxy and phenyl;
  • R5 is hydrogen; C 1-C4 alkyl; C1-C4 alkyl substituted with phenyl; or cyclopentyl;
  • R6 is hydrogen; halogen; C1-C4 alkyl; C 1-C4 alkyl substituted with C5-C6 heterocycloalkyl wherein the heteroatom is N; phenyl; phenyl substituted with C l- C4 alkyl or halogen; C5 -C6 cycloalkyl; C5-C6 heterocycloalkyl; or adamantine.
  • Rl, R2, R3 and R4 are independently selected from hydrogen, halogen, Cl - C4 alkyl. methoxy and phenyl;
  • R5 is hydrogen, Cl - C4 alkyl. -CH 2 -phenyl or cyclopentyl;
  • R6 is hydrogen, C l - C4 alkyl, phenyl optionally substituted with halogen or Cl - C4 alkyl, -CFb-heterocycle therein the heterocycle is 6-membered ring comprising 1 or 2 heteroatoms selected from N and O and optionalh substituted with C 1 - C4 alkyl, C5 - C6 cycloalkyl or adamantane.
  • Rl is hydrogen, halogen, or C 1 - C4 alkyl; R3 is hydrogen; and
  • R2, R4, R5 and R6 are as defined abo ⁇ e.
  • Rl is hydrogen, halogen or C 1 - C4 alkyl
  • R2 and R4 are independently selected from hydrogen, halogen, Cl - C4 alkyl and methoxy;
  • R3 is hydrogen
  • R5 is hydrogen, Cl - C4 alkyl or cyclopentyl
  • R6 is hydrogen, C l - C4 alkyl, phem l optionalh substituted with halogen or Cl - C4 alkyl, -CFb-heterocycle wherein the heterocycle is 6-membered ring comprising a N heteroatom, cyclopentyl or adamantane.
  • Rl is hydrogen, halogen or C 1 - C4 alkyl
  • R2 and R4 are independently selected from hydrogen, halogen, Cl - C4 alkyl and methoxy; R3 is hydrogen;
  • R5 is hydrogen, Cl - C4 alkyl or cyclopentyl
  • R6 is hydrogen, C l - C4 alkyl, phenyl optionally substituted with halogen or Cl - C4 alkyl, -CFb-heterocycle therein the heterocycle is 6-membered ring comprising a N heteroatom, c ⁇ clopent ⁇ 1 or adamantine, and at least two of Rl, R2, R4, R5 and R6 are other than hydrogen.
  • Rl, R2, R3, R4 are independently hydrogen; halogen; or C 1-C4 alkyl; R5 is hydrogen; and R6 is Cl-C4 alkyl.
  • the compounds of general Formula I include a compound of Formula II :
  • R2 is halogen or methoxy; and R6 is Cl-C4 alkyl.
  • R2 is halogen; and R6 is Cl-C4 alkyl.
  • R2 is halogen; and R6 is methyl, isopropyl or t-butyl.
  • R2 is F or Br. and R6isCl-C4alk ⁇ l
  • R2 is F or Br.
  • R6 is meth ⁇ 1, isoprop ⁇ 1 or t-but ⁇ 1
  • the compound is compound 7 or a derivative thereof.
  • a derivative thereof in this context can be, for example, a compound of Formula I wherein: Rl, R3, R4, R5 and R7 are H; R2 is halogen; and R6 is Ci-C 4 alkyl.
  • the present invention includes various salts of the compounds defined by Formula I, including pharmaceutically acceptable salts.
  • Compounds according to the present invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with a number of organic and inorganic bases, and organic and inorganic acids, to form pharmaceutical! ⁇ acceptable salts.
  • salts refers to a salt that is substantialh non-toxic to living organisms.
  • Typical pharmaceutical! ⁇ acceptable salts include those salts prepared by reaction of the compound of the present invention with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts.
  • Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulphonic acid, methanesulphonic acid, oxalic acid.
  • Salts of amine groups may also comprise quarternary ammonium salts in which the amino nitrogen carries a suitable organic group such as an alkyl, lower alkenyl, substituted lower alkenyl, lower alkynyl, substituted lower alkynyl, or aralkyl moiety.
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
  • Bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
  • the particular counter ion forming a part of a salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counter ion does not contribute undesired qualities to the salt as a whole.
  • the compound of Formula I is provided as a pharmaceutically acceptable salt. In another embodiment, the compound of Formula I is provided in the form of a pharmaceutically acceptable hydrochloric salt.
  • the present invention further encompasses the pharmaceutically acceptable solvates of a compound of Formula I.
  • Man ⁇ of the compounds of Formula I can combine with solvents such as water, methanol, ethanol and acetonitrile to form pharmaceutically acceptable solvates such as the corresponding hydrate, methanolate, ethanolate and acetonitrilate.
  • the compounds of Formula I may have multiple asymmetric (chiral) centres. As a consequence of these chiral centres, the compounds occur as racemates, mixtures of enantiomers and as individual enantiomers, as well as diastereomers and mixtures of diastereomers. All asymmetric forms, individual isomers and combinations thereof, are within the scope of the present invention.
  • compounds of Formula I are prepared via solution or solid phase synthesis, by reacting a dione of Formula III with the aldehyde (IV) in the presence of ammonium acetate in acetic acid (see, for example, Krieg et a L,
  • the compounds of Formula (III) and (IV) are either commercially available or may be prepared using standard procedures known to a person skilled in the relevant art.
  • Compounds of Formula (III), for example, can be prepared by several general synthetic methods, such as those as described by: Fischer et. al (J. Am. Chem. Soc. 83:4208-4210 (1961)); Guijarro et al. (J. Am. Chem. Soc. 121 :4155-4157 (1999)); Chi et. al. (Synth. Comm. 24(15):2119-2122 (1994)); Armesto et. al. (Synthesis, 799-801 (1988)); Yamada et. al. (Bull. Soc Chem.
  • Compounds of Formula (IV) can be prepared by general synthetic methods described by Vilsmeier et. al. (Chem. Ber. 91:850-861 (1958) and Synthesis 8:641-645 (1985)).
  • compounds of formula (IV) can be prepared by reacting a compound of formula (V) w ith POCI3 in dimethylformamide (DMF) as shown below:
  • the separation and purification of the imidazo[4,5-d]phenanthroline products is generalh based on their property to form w ater-soluble salts
  • the impurities are extracted from the obtained solution with a nonpolar soh ent the aqueous la ⁇ er is basified and the separated imidazo[4,5- d]phenanthroline is filtered and recn stalhzed from a suitable soh ent
  • compounds of Formula I are capable of inhibiting angiogenesis
  • the compounds of Formula I are capable of reducing expression of VEGFR2
  • the compounds of Formula I are capable of reducing expression of HIF-I ⁇
  • the abiht ⁇ of a candidate compound of Formula I to inhibit angiogenesis m vitro and m vivo can be tested using standard techniques known in the art Similarh , the abilit ⁇ of the compounds to reduce expression of VEGFR2 and/or reduce expression of HIF- l ⁇ can be tested using standard techniques Exemplar ⁇ methods of testing candidate compounds of Formula I are pro ⁇ ided below and in the Examples included herein One skilled in the art will understand that other methods of testing the compounds are known in the art and are also suitable for testing candidate compounds
  • a In vitro Testing ⁇ ) Ability to Inhibit Angiogenesis A number of standard techniques are suitable for assessing the ability of candidate compounds of Formula I to inhibit angiogenesis in vitro.
  • a candidate compound of Formula I to inhibit proliferation of endothelial cells can be tested as follows. Cells of a specific test cell line are grown to a suitable density (e.g. approximately 1 x 10 4 ) and various concentrations of the candidate compound are added. After an appropriate incubation time (typically between about 48 to 74 hours), cell survival is assessed, for example, by assaying for tetrazolium salt (or modified tetrazolium salt) cleavage, or by using the resazurin reduction test (see Fields & Lancaster (1993) Am. Bwtechnol. Lab. 11 :48-50; O ' Brien et al., (2000) Eur J. Biochem.
  • Inhibition of cell proliferation is determined by comparison of cell survival in the treated culture with cell survival in one or more control cultures, for example, cultures not pre-treated with the candidate compound, those pre-treated with a control vehicle and/or those pre-treated with a control compound (typically a known therapeutic).
  • metabolic activity can also be used to assess the effect of candidate compounds on cell proliferation, given that proliferating cells tend to be metabolically more active than resting cells.
  • the migration and differentiation of endothelial cells during angiogenesis can be studied in vitro using, for example, the Matrigel tube formation assay (see Grant et al., J. Cell. Physiol. 153:614 (1992) and Kubota Y, et al., J. Cell Biol. 107: 1589-98 (1988)).
  • the Matrigel tube formation assay see Grant et al., J. Cell. Physiol. 153:614 (1992) and Kubota Y, et al., J. Cell Biol. 107: 1589-98 (1988)
  • cell culture plates are coated with a Matrigel solution and then incubated at 37 0 C to promote gelling.
  • HUVECs or other suitable cells are resuspended in growth media and added to each well.
  • the candidate compound, positive control compound(s) for example.
  • aFGF and/or bFGF aFGF and/or bFGF
  • media alone a second control
  • the plates are fixed and the length of the tubes/tube formation measured by microscopy, for example by differential interference contrast and/or fluorescence microscopy.
  • suitable cell lines to assess the anti-angiogenic properties of candidate compounds include, but are not limited to, human umbilical vein endothelial cells (HUVECs), bovine aortic endothelial cells (BAECs), human coronary artery endothelial cells (HCAECs) and vascular smooth muscle cells.
  • HUVECs can be isolated from umbilical cords using standard methods (see, for example, Jaffe et al. (1973) J. CIm. Invest. 52: 2745), or the ⁇ can be obtained from the ATCC or various commercial sources, as can other suitable endothelial cell lines.
  • the candidate compound can be introduced into a selected cell line and the amount of mRNA transcribed from the gene of interest can be measured by standard techniques such as Northern blot analysis, RT-PCR, and the like.
  • the amount of protein expressed from the target gene in the cell can be measured by standard techniques such as Western blot analysis.
  • the amount of mRNA or protein produced in a cell treated with the candidate compound can then be compared with the amount produced in control cells and will provide an indication of how successfully the compound has inhibited expression of the target gene.
  • Suitable control cells include, for example, untreated cells and/or cells treated with a control compound.
  • the candidate compounds can be screened for their ability to modulate expression of the target gene(s) in a selected cell line using standard methods for screening expression of multiple genes ("expression profiling " ).
  • expression profiling Such methods are well known in the art and include, for example, microarray analysis, such as high density microarray assays containing 10-fold more (for example, 19,000) human genes to identify suitable functional clusters of genes whose expression is affected by the compound.
  • microarrays of short DNA sequences or oligonucleotides.
  • Methods of constructing microarrays are well known in the art [see, for example, Ausubel. et a L, Current Protocols m Molecular Biology, John Wile ⁇ & Sons, Inc, NY. (1989 and updates)].
  • microarrays can be custom made.
  • Pre-made microarrays are also commercially available for man ⁇ organisms including, for example, GeneChip* (Affimetrix, Santa Clara, CA), AtlasTM (BD Biosciences-CLONTECH.
  • RNA is isolated from cells treated with the candidate compound and from control cells. If necessary, the RNA can be amplified by conventional techniques to ensure a sufficient quantity for anal ⁇ sis.
  • RNA is then hybridised to the microarray under suitable conditions and a routine anah sis of the microarray by commercially available scanners and software is conducted to identify genes whose expression is altered in the treated cells relative to the control cells.
  • Suitable hybridization conditions can readily be determined by one skilled in the art using standard techniques. Following the identification of such other genes, mRNA quantitation and respective protein levels can also be evaluated to determine the extent of the effect of the compound on the genes under investigation.
  • a number of assays are know n in the art for testing the ability of candidate compounds of Formula I to inhibit angiogenesis m vivo.
  • the ability of the candidate compounds to inhibit endothelial cell migration can be determined using the chick chorioallantoic membrane (CAM) assay.
  • CAM chick chorioallantoic membrane
  • Matrigel plug assay and/or corneal micropocket assay are know n in the art for testing the ability of candidate compounds of Formula I to inhibit angiogenesis m vivo.
  • the CAM assay is a standard assay that is used to evaluate the ability of a test compound to inhibit the growth of blood vessels into various tissues, i.e. both angiogenesis and neovascularization (see Brooks et ai, in Methods m Molecular Biology, VoI 129, pp 257-269 (2000), ed A R Hewlett Humana Press Inc , Totem a. NJ. Ausprunk et al , (1975) Am J Pathol , 79 597-618.
  • the Mat ⁇ gel plug assa ⁇ is also a standard method for e ⁇ supering the anti-angiogenic properties of compounds m vivo (see, for example, Passaniti, et al ,(1992) Lab Invest 67 519-528)
  • a test compound is introduced into cold liquid Mat ⁇ gel which, after subcutaneous miction into a suitable animal model, solidifies and permits penetration b ⁇ host cells and the formation of new blood ⁇ essels After a suitable period of time, the animal is sacrificed and the Mat ⁇ gel plug is reco ⁇ ered.
  • the corneal micropocket assa ⁇ is usualh conducted in mice, rats or rabbits and has been described in detail b ⁇ others (see D'Amato, et al , (1994) Proc Natl, Acad Sci USA, 91 4082-4085. Koch et al , (1991) Agents Actions, 34 350-7.
  • pellets for implantation are prepared from sterile r ⁇ dron poh mer containing a suitable amount of the test compound
  • the pellets are surgicalh implanted into corneal stromal micropockets created at an appropriate distance medial to the lateral corneal limbus of the animal
  • Angiogenesis can be quantitated at ⁇ a ⁇ ous times after pellet implantation through the use of stereomicroscop ⁇ T ⁇ picalh , the length of neo ⁇ essels generated from the limbal ⁇ essel ring tow ard the centre of the cornea and the width of the neo ⁇ essels are measured
  • Candidate compounds of Formula I can also be tested for their effect on angiogenesis during tumor formation using standard xenograft models.
  • mice bearing established tumors can be treated with the compound and. after a suitable time, sacrificed and the tumors removed. Density of microvessels in the excised tumors can be assessed using standard immunohistochemical techniques and compared to that of untreated tumors and/or tumors treated with a known anti-angiogenic agent.
  • the compounds of Formula I can also be submitted to toxicity testing if desired. Toxicity tests for potential drugs are well-known in the art (see, for example, Hayes, A. W., ed., (1994), Principles and Methods of Toxicology, 3 rd ed.. Raven Press, NY; Maines, M., ed.. Current Protocols in Toxicology, John Wile ⁇ ' & Sons, Inc., NY).
  • In vitro acute toxicity testing of a compound of Formula I can be performed using mammalian cell lines (see, for example, Ekw all, B., Ann. K Y. Acad. Sa., (1983) 407:64-77). Selection of an appropriate cell line is dependent on the potential application of the candidate compound and can be readily determined by one skilled in the art. For example, these tests include the treatment of human primary fibroblasts in vitro with the compounds of Formula I in the presence of a commercial carrier. Cells are then tested at different time points following treatment for their viability using a standard viability assay, such as the tn pan-blue exclusion assay, XTT or MTT assays.
  • a standard viability assay such as the tn pan-blue exclusion assay, XTT or MTT assays.
  • Cells can also be assayed for their ability to synthesize DNA, for example, using a thymidine incorporation assay, and for changes in cell cycle dynamics, for example, using a standard cell sorting assay in conjunction with a fluoroc ⁇ tometer cell sorter (FACS).
  • FACS fluoroc ⁇ tometer cell sorter
  • In vivo toxicity testing can be performed by standard methodology, for example, by injecting van ing concentrations of the candidate compound into an appropriate animal model. The compound can be injected once, or administration can be repeated over several days. The toxic effects of the compound can be evaluated over an appropriate time period by monitoring the mortality, changes in behavior, appearance, and bod ⁇ w eight of the animals. After the completion of the period of assessment, the animals can be sacrificed and the appearance and w eight of the relevant organs determined. If necessary, additional assessments of, for example, hematological profiles, histology, and liver enzyme analysis may be performed. An indication of the toxicity of a compound can also be obtained during the in vivo testing of
  • the genotoxicity of compounds of Formula I can be assessed in vitro if necessary using standard techniques such as the Ames Assay to screen for mutagenic activity, the mouse lymphoma assay to determine the ability of a test article to induce gene mutation in a mammalian cell line, m vitro chromosomal aberration assays using, for example, Chinese hamster ovary cells (CHO) to determine any DNA rearrangements or damage induced by the test article.
  • Other assays include the sister chromatid assay, which determines any exchange between the arms of a chromosome induced by the test article and m vitro mouse micronucleus assays to determine any damage to chromosomes or to the mitotic spindle.
  • the genotoxicity of compounds of Formula I can also be assessed m vivo if necessary using the m vivo sister chromatid exchange assay, m vivo micronucleus assay, or the m vivo chromosomal abberation assay. Protocols for these and other standard assays are known in the art, for example, see OECD Guidelines for the Testing of Chemicals and protocols developed by the ISO.
  • compositions comprising the compounds of Formula I will need to meet certain criteria in order to be suitable for human use and to meet regulatory requirements.
  • standard in vitro and in vivo tests can be conducted to determine information about the metabolism and pharmacokinetic (PK) of the compositions and combinations (including data on drug-drug interactions w here appropriate) which can be used to design human clinical trials
  • Phase I Phase II Studies are conducted to inv estigate the safet ⁇ , tolerabilit ⁇ and PK of the compounds and to help design Phase II studies, for example, in terms of appropriate doses, routes of administration, administration protocols
  • Phase I studies allow the selection of safe dose le ⁇ els for Phase II studies
  • An important factor in the protocol design of the Phase II studies is the adequate recruitment of the patient population to be studied based on stringent selection criteria defining the demographics (age, race and sex) of the stud ⁇ and the pre ⁇ ious medical histon of the patient
  • a protocol for Phase II studies t ⁇ picalh specifies baseline data that can be used to characterise the population, to e ⁇ sine the success of randomization in achie ⁇ ing balance of important prognostic factors, and to allow for consideration of ad) usted anah ses
  • Clinical biomarkers can be defined as follow s (Atkinson A et al CIm Pharmacol Ther 69, 89-95 (2001) Biological marker (biomarker) a characteristic that is ob)ecti ⁇ eh measured and e ⁇ supered as an indicator of normal biological process, pathogenic process, or pharmacological response to a therapeutic inten ention
  • Clinical endpomt a characteristic or ⁇ anable that reflects how a patient feels or functions, or how long a patient sun i ⁇ es
  • Surrogate endpomt biomarker intended to substitute for a clinical endpoint A clinical im estigator uses epidemiological, therapeutic, pathophy siological, or other scientific e ⁇ idence to select a surrogate endpoint that is expected to predict benefit, harm or the lack of benefit or harm
  • the FDA defines a surrogate endpoint. or marker, as a laboraton measurement or sical sign that is used in therapeutic trials as a substitute for a clinicalh meaningful endpoint that is a direct measure of how a patient feels, functions or sur ⁇ i ⁇ e and is expected to predict the effect of therap ⁇
  • Phase III trials focus on determining how the compound compares to the standard, or most wideh accepted, treatment
  • patients are randomh assigned to one of tw o or more "arms "
  • tw o arms for example, one arm will rece ⁇ e the standard treatment (control group) and the other arm will be treated with the test compound (in ⁇ estigational group)
  • Phase IV trials can be used to further e ⁇ sinumeric dosage of the compound Phase IV trials are less common than Phase I, II and III trials and w ould take place after the compound has been appro ⁇ ed for standard use
  • the in ⁇ ention pro ⁇ ides for methods of using and uses of compounds of Formula I to inhibit angiogenesis, to treat angiogenesis associated diseases or disorders, inhibit VEGFR2 expression and/or inhibit HIF- l ⁇ expression
  • the compounds of Formula I can be used to inhibit angiogenesis m vitro or m vivo
  • angiogenesis-associated diseases and disorders are terms used generalh to refer to a disease or disorder which occurs as a consequence of, or which results in, increased ⁇ ascula ⁇ zation in a tissue
  • Angiogenesis maj be an actual cause of the disease/disorder or it maj be a condition resulting from the disease/disorder
  • angiogenesis-associated diseases and disorders include, but are not limited to tumor-growth and/or metastasis, including solid tumors, Kaposi sarcoma, angiofibromas, retrolental fibroplasia, and hemangiomas.
  • ocular diseases and disorders including keratoplasty angiogenesis, angiogenic glaucoma, macular degeneration (for example
  • ALD diabetic retinopathy
  • retinopathy of prematurity angiogenic corneal disorder
  • pten gium retinal degeneration
  • retrolental fibroplasias trachoma.
  • corneal neo ⁇ ascula ⁇ zation corneal neo ⁇ ascula ⁇ zation
  • choroidal neo ⁇ ascula ⁇ zation ⁇ clitis
  • renal diseases including glomerulonephritis, diabetic nephropathy , malignant nephrosclerosis, thrombotic microangiopathic sy ndrome, transplant rejections and glomerulopathy .
  • cerebro ⁇ ascular diseases including multiple sclerosis, experimental allergic encephalomy elitis, bacterial meningitis, ischemia, brain edema.
  • fibrotic diseases including cirrhosis of the h ⁇ er. and mesangial cell proliferate e diseases.
  • senile keratosis examples include senile keratosis, endometriosis, osteoporosis h mphangiogenesis, Sturge-Weber S ⁇ ndrome, neurofibromatosis tuberous sclerosis and asthma
  • the in ⁇ ention pro ⁇ ides for the use of the compounds of Formula I to inhibit tumor-associated angiogenesis Angiogenesis is critical to the growth and metastasis of solid tumors Inhibition of angiogenesis in a cancer context, therefore, helps to stabilise the disease b ⁇ reducing or halting the grow th and/or spread of the tumor Accordingly , one embodiment of the in ⁇ ention pro ⁇ ides for the use of the compounds of Formula I to stabilise a cancer in a patient b ⁇ inhibiting tumor-associated angiogenesis Another embodiment pro ⁇ ides for the use of the compounds of Formula I to inhibit metastasis of a solid tumor in a patient, and potentialh contain the neoplastic growth at the priman site, b ⁇ inhibiting tumor-associated angiogenesis A further embodiment pro ⁇ ides for the use of the compounds of Formula I to increase progression-free sur ⁇ i ⁇ al in a patient b ⁇ inhibiting tumor-associated angiogenesis
  • pro ⁇ ides for the use of the compounds of Formula I to reduce the recurrence of a solid tumor such as in ⁇ asi ⁇ e breast carcinoma
  • Anti-angiogenic agents are known to increase the effica ⁇ of traditional cancer therapies, including chemotherapy and radiation therap ⁇
  • pro ⁇ ides for the use of the compounds of Formula I as an antiangiogenic component of a combination therap ⁇ for the treatment of cancer Another embodiment pro ⁇ ides for the use of the compounds of Formula I in combination with one or more chemotherapeutic to treat cancer Examples of suitable chemotherapeutics include, but are not limited to, carboplatin, cisplatin, c ⁇ clophosphamide, danorubicin, docetaxel, doxorubicin,
  • Anti-angiogenic agents are parti cularh useful in combination with metronomic chemotherapy
  • Metronomic chemotherapy in ⁇ oh es the frequent administration of one or more chemotherapeutic agents at doses significanth below the maximum tolerated dose (MTD) with no prolonged drug-free breaks
  • the main targets of metronomic chemotherapy are the endothelium or tumor stroma rather than the tumor itself and the efficac ⁇ of metronomic chemotherapy can be enhanced b ⁇ combining the chemotherapy w ith an anti-angiogenic agent Accordingly , one embodiment of the in ⁇ ention pro ⁇ ides for the use of compounds of Formula 1 in combination with metronomic chemotherapy for the treatment of cancer
  • the in ⁇ ention pro ⁇ ides for the use of the compounds of Formula I in the treatment of non-cancerous angiogenesis associated disorders such as angiogenesis-associated ocular diseases and disorders, angiogenesis-associated immune disorders, angiogenesis-associated cardio ⁇ ascular diseases, angiogenesis-associated skin disorders, angiogenesis-associated chronic inflammaton disease, angiogenesis- associated renal diseases, angiogenesis-associated cerebro ⁇ ascular diseases, angiogenesis-associated fibrotic diseases, mesangial cell proliferate e diseases, senile keratosis, endometriosis, osteoporosis h mphangiogenesis. Sturge-Weber sy ndrome.
  • the invention provides for the use of the compounds of Formula I in the treatment of ocular diseases characterized by undesired neovascularization, for example, age-related macular degeneration (AMD), diabetic retinopathy, corneal neovascularization, choroidal neovascularization, cyclitis. Hippei- Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization. Purrs cher ' s retinopathy, renal vein occlusion and the like.
  • AMD age-related macular degeneration
  • diabetic retinopathy diabetic retinopathy
  • corneal neovascularization corneal neovascularization
  • choroidal neovascularization cyclitis.
  • Hippei- Lindau Disease retinopathy of prematurity, pterygium, histoplasmos
  • compounds of Formula I are capable of reducing expression of vascular endothelial growth factor receptor 2 (VEGFR2). Accordingly, in one embodiment, the use of the compounds of Formula I for reducing expression of VEGFR2 in vivo or in vitro is provided.
  • Inhibitors of VEGF and its receptors have been shown to be effective in controlling angiogenesis in various disease contexts, including for example, solid tumors (such as colorectal, breast, lung and kidney tumors), AMD, diabetic retinopathy and renal vein occlusion.
  • the invention provides for the use of the compounds of Formula I in the treatment of angiogenesis associated diseases and disorders for which treatment with VEGF inhibitors is known to be effective, such as growth or metastasis of solid tumors (including colorectal, breast, lung and kidney). AMD, diabetic retinopathy and retinal vein occlusion.
  • the invention provides for the use of the compounds of Formula I for reducing expression of HIF-l ⁇ m vivo or m vitro.
  • the compounds of the present invention are typically formulated prior to administration.
  • the present invention thus provides pharmaceutical compositions comprising one or more compounds of Formula I and a pharmaceutically acceptable carrier, diluent, or excipient.
  • the pharmaceutical compositions are prepared by known procedures using well-known and readily available ingredients.
  • Compounds of the general Formula I or pharmaceutical compositions comprising the compounds may be administered orally, topically, parenteral! ⁇ , by inhalation or spray. or rectalh in dosage unit formulations containing conv entional non-toxic pharmaceutical! ⁇ acceptable carriers, ad)u ⁇ ants and ⁇ ehicles
  • the act ⁇ e compound is incorporated into an acceptable ⁇ ehicle to form a composition for topical administration to the affected area, such as hy drophobic or h ⁇ drophihc creams or lotions, or into a form suitable for oral, rectal or parenteral administration, such as s ⁇ nips, elixirs, tablets, troches, lozenges, hard or soft capsules, pills, suppositories, oih or aqueous suspensions, dispersible pow ders or granules, emulsions, in)ectables, or solutions
  • the present im ention also pro ⁇ ides for pharmaceutical compositions comprising one or more of the compounds of Formula I and a ⁇ ehicle, such as an artificial membrane ⁇ esicle (including a liposome, lipid micelle and the like), microparticle or microcapsule
  • a ⁇ ehicle such as an artificial membrane ⁇ esicle (including a liposome, lipid micelle and the like), microparticle or microcapsule
  • compositions intended for oral use ma ⁇ be prepared in either solid or fluid unit dosage forms
  • Fluid unit dosage form can be prepared according to procedures known in the art for the manufacture of pharmaceutical compositions and such compositions ma ⁇ contain one or more agents selected from the group consisting of sw eetening agents, fla ⁇ ou ⁇ ng agents, colouring agents and preserv ing agents in order to pro ⁇ ide pharmaceuticalh elegant and palatable preparations
  • An elixir is prepared b ⁇ using a h ⁇ droalcoholic (e g , ethanol) ⁇ ehicle with suitable sw eeteners such as sugar and saccharin, together w ith an aromatic fla ⁇ o ⁇ ng agent
  • Suspensions can be prepared w ith an aqueous ⁇ ehicle with the aid of a suspending agent such as acacia, tragacanth, meth ⁇ lcellulose and the like
  • Solid formulations such as tablets contain the act ⁇ e ingredient in admixture with nontoxic pharmaceuticalh acceptable excipients that are suitable for the manufacture of tablets
  • excipients ma ⁇ be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate granulating and disintegrating agents for example, corn starch, or alginic acid binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc and other com entional ingredients such as dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose. meth ⁇ lcellulose, and functionally similar materials.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate granulating and disintegrating agents for example, corn starch, or alginic acid binding agents, for example
  • the tablets may be uncoated or the ⁇ may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with w ater or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Soft gelatin capsules are prepared by machine encapsulation of a slum of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
  • Aqueous suspensions contain active materials in admixture w ith excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxylmethylcellulose, methyl cellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example poh oxy ethylene stearate, or condensation products of ethylene oxide w ith long chain aliphatic alcohols, for example hepta-decaeth ⁇ leneox ⁇ cetanol, or condensation products of ethylene oxide w ith partial esters derived from fatly acids and a hexitol such as polyox ⁇ ethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or ⁇ -propyl- p-hs droxy benzoate, one or more colouring agents, one or more flavouring agents or one or more sweetening a • *g£>e v nts, such as sucrose or saccharin.
  • preservatives for example ethyl, or ⁇ -propyl- p-hs droxy benzoate
  • colouring agents for example ethyl, or ⁇ -propyl- p-hs droxy benzoate
  • flavouring agents for example sucrose or saccharin.
  • sweetening a • *g£>e v nts such as sucrose or saccharin.
  • suspensions may be formulated by suspending the active ingredients in a vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oil ⁇ suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavouring agents may be added to provide palatable oral preparations.
  • These compositions may be preserv ed by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of w ater provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preserv atives.
  • a dispersing or wetting agent, suspending agent and one or more preserv atives are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.
  • compositions of the invention may also be in the form of oil-in- water emulsions.
  • the oil phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhy drides, for example sorbitan monooleate, and condensation products of the said partial esters w ith ethylene oxide, for example poly oxy ethy lene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or a suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1.3-butanediol.
  • the acceptable vehicles and solvents that may be employ ed are water. Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventional! ⁇ employed as a solvent or suspending medium.
  • any bland fixed oil may be employ ed including sy nthetic mono- or digh cerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Adjuvants such as local anaesthetics, preservatives and buffering agents can also be included in the injectable solution or suspension.
  • the compound of the general Formula I may be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and w ill therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and w ill therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • the compounds can be formulated into solutions, suspensions, and ointments appropriate for use in the eye (see, for example, Mitra (ed.), (1993) Ophthalmic Drug Delivery Systems, Marcel Dekker, Inc., New York, N.Y.; Havener, (1983) Ocular Pharmacology, CV. Mosby Co., St. Louis).
  • compounds of Formula I that show poor solubility can be formulated with appropriate hydrophilic carriers, solubility enhancers and/or surfactants.
  • hydrophilic carriers, solubility enhancers and surfactants include, but are not limited to, polyvinylpyrrolidone, polyethylene glycols (PEGs), poh propylene glycol (PPG), Plasdone-S630, carboxymethyl cellulose, hydroxypropyl cellulose, guar gum. xanthan gum, sodium alginate, methyl cellulose, HPMC, dextrin, cyclodextrins, galactomannan, Tween-80, Docusate sodium.
  • Compounds of Formula I may be administered to a subject by a variety of routes depending on the disease or disorder to be treated, for example, the compounds may be administered orally, topically, parenteral! ⁇ , by inhalation or spray, or rectally in dosage unit formulations.
  • parenteral as used herein includes subcutaneous injections. intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal, intrathecal injection or infusion techniques.
  • the present invention contemplates the administration of one or more compounds of Formula I either alone or in combination with other therapies.
  • other therapies For example, with radiation or chemotherapy when administered to cancer patients, or with other standard therapeutics used in the treatment of the disease or disorder being treated.
  • the dosage to be administered is not subject to defined limits, but it will usually be an effective amount. It will usually be the equivalent, on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active free drug to achieve its desired pharmacological and physiological effects.
  • the compositions may be formulated in a unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Examples of ranges for the compound(s) in each dosage unit are from about 0.05 to about 100 mg, or more usually, from about 1.0 to about 50 m 1 gB.-
  • Daily dosages of the compounds of the present invention w ill typically fall within the range of about 0.01 to about 100 mg/kg of bod ⁇ weight, in single or divided dose.
  • the actual amount of the compound(s) to be administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the above dosage range is given by w ay of example only and is not intended to limit the scope of the invention in any wa ⁇ .
  • dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing harmful side effects, for example, by first dividing the larger dose into several smaller doses for administration throughout the day.
  • it ma> be desirable to administer the compound at a dosage low er than the MTD For example, in those embodiments relating to the use of the compounds of Formula I in combination with metronomic chemotherap ⁇ , the compound ma ⁇ be administered at the MTD, shghth below the MTD or significant! ⁇ below the MTD
  • kits containing one or more compounds of Formula I The contents of the kit can be 1 ⁇ ophihzed and the kit can additional! ⁇ contain a suitable soh ent for reconstitution of the 1 ⁇ ophihzed components
  • Indmdual components of the kit w ould be packaged in separate containers and. associated with such containers, can be a notice in the form prescribed b ⁇ a go ⁇ ernmental agenc ⁇ regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects appro ⁇ al b ⁇ the agenc ⁇ of manufacture, for use or sale for human or animal administration
  • the liquid solution can be an aqueous solution, for example a sterile aqueous solution
  • the compounds ma ⁇ be formulated into a pharmaceutical! ⁇ acceptable s ⁇ ⁇ ngeable composition
  • the container means ma ⁇ itself be an inhalant, s ⁇ ⁇ nge, pipette, e ⁇ e dropper, or other such like apparatus, from which the formulation ma ⁇ be applied to an infected area of the sub)ect. such as the lungs, iniected into an sub)ect. or e ⁇ en applied to and mixed with the other components of the kit
  • kits or packs comprising one or more compound of the present im ention in combination with one or more standard therapeutic for combination therap ⁇ applications are also contemplated b ⁇ the present in ⁇ ention
  • HT-29 colon carcinoma cells used in this example and subsequent examples w ere maintained as a monolayer in a growth medium; McCoy's 5A modified medium (Sigma. St. Louis, MO), supplemented with 2 mM L-glutamine (Gibco, Grand Island. NY), 10% fetal bovine serum (FBS) (Multicell, WISENT Inc. St-Bruno, QC), antibiotic-antimycotic (Multicell), at 37 0 C in a 5% CO 2 - humidified incubator. Cells w ere transferred onto 150mm tissue culture plates and grown until sub-confluency (70-80%) prior to their use.
  • mice Groups of 3 ICR female mice were injected with one intra-peritoneal (i.p.) injection of
  • the compounds to be tested were prepared to provide enough for one week at concentrations of 4.0 mg/ml and 2.0 mg/ml. Briefly, 50 mg of each compound w as dissolved in 1.25 ml of 100 % DMSO and diluted with 6.25 ml of Lutrol (30 % in water) and 5 ml water, to prepare a solution of 4mg/ml compound in 15% Lutrol in water. Next, 4 ml of each prepared solution w as further diluted with 4 ml of 15% Lutrol in water to provide the 2.0 mg/ml solution of each compound. For the vehicle control solution, 8 ml of 15% Lutrol in water w as prepared. Compounds at concentrations of 4.0 mg/ml and 2.0 mg/ml and the vehicle control were administered as described above. Toxicity w as evaluated based on mortality, changes in behaviour, appearance and weight and is shown in Table 2.
  • EXAMPLE 12 IN VITRO ANTIPROLIFERATIVE ACTIVITY OF COMPOUNDS OF FORMULA I IN HT-29 COLON CARCINOMA CELLS
  • EXAMPLE 13 IN VITRO ABILITY OF COMPOUNDS OF FORMULA I TO MODULATE CELL GROWTH INHIBITION IN HUMAN CANCER CELLS LINES
  • IC50 Cell growth inhibition IC50 ( ⁇ M) by compounds of Formula I w as tested on various cancer cell types by XTT cell proliferation assay. Low or sub-micromolar IC50 was observed in different types of cancer as shown in Table 4. The growth inhibitor ⁇ effects of the compounds of Formula I as shown below for the melanoma cell line SK-MEL-2 w ere reproducible by Lorus Therapeutics Inc.
  • IC50 values for compounds of Formula I in the breast cancer cell line MDA-MB- 435 were between 0.2 ⁇ M and 0.6 ⁇ M as shown below in Table 4.
  • Table 4 Cell Growth Inhibition IG;o ( ⁇ M) by Compounds of Formula I in Different Cancer Cell Types
  • EXAMPLE 14 IN VIVO EFFICACY OF COMPOUNDS OF FORMULA I IN LARGE-CELL LUNG CARCINOMA AND COLON CARCINOMA XENOGRAFT MODELS
  • Tumor growth inhibition b ⁇ compounds of Formula I in mouse xenograft models is shown in Figure 1 Route of administration and schedule studies for non-small cell lung carcinoma (H460) ( Figure IA) and colon adenocarcinoma (HT-29) ( Figure IB), minimal effect ⁇ e dose for non-small cell lung carcinoma (H460) ( Figure 1C), and efficac ⁇ of optimized compounds of Formula I in non-small cell lung carcinoma (H460) ( Figure ID)
  • EXAMPLE 15 IN VITRO ABILITY OF COMPOUND 64 TO MODULATE GENE EXPRESSION OF MTlA, MTF-I, CYCLIN Dl AND KLF4 IN HT-29 CELLS
  • EXAMPLE 16 DIFFERENTIAL TRANSCRIPTION ACTIVITY IN NORMAL AND CANCER CELLS IN VITRO UPON TREATMENT WITH COMPOUND 7
  • KLF4 is known to act as a transcriptional activator of several cell cycle inhibitors, as well as a transcriptional repressor of several genes encoding promoters of the cell cycle.
  • the transcriptional activity of 50 transcription factors was investigated in colon carcinoma cells treated with 1 ⁇ M Compound 7 for between 2 and 16 hours. DNA-binding activities of the transcription factors were measured by Multiplex Transcription Factor Testing Service (Marligen Biosciences, Ijamsville, MD).
  • Figure 3 A shows the differential transcriptional profile of colon carcinoma HT-29 cells and normal colon fibroblast CCD- 18Co cells after treatment w ith Compound 7. Altered activity of several transcription factors w as observed in colon cancer cells with no significant effect on normal cells; particularly the activation of transcription factors involved in tumor suppression and tumor promoters (Figure 3B).
  • HSF-I Heat shock factor 1
  • Figure 3 A & D Heat shock factor 1
  • HSF-I is a known transcriptional regulator of KLF4 in response to stress, and can be negatively regulated by forming a complex w ith MTF-I.
  • EXAMPLE 17 IN VITRO ABILITY OF COMPOUND 7 TO MODULATE GENE EXPRESSION OF KLF4, ⁇ -CATENIN AND CYCLIN Dl IN HT-29 CELLS
  • HT-29 cells 5 X 10 5 cells in 5 niL volume of growth medium w ere seeded in 6-cm dishes and incubated overnight at 37 0 C.
  • the medium w as removed and replaced with growth medium containing 0.1% DMSO vehicle control or 1 ⁇ M of Compound 7. After incubation for 4, 7, 16, or 24 hours, the cells w ere detached by trypsinization, collected by centrifugation and w ashed once with PBS.
  • RNA w as extracted from cells by using TRIZOL (Invitrogen, Life Technologies, Carlsbad. CA), following manufacturer ' s instruction, and the level of KLF4 mRNA, ⁇ -catenin mRNA, and Cyclin Dl mRNA w as measured by real time PCR using the comparative CT method as follow s.
  • First strand cDNA w as synthesized from 200 ng total RNA in Biometra Tpersonal Thermal Cycler (Abgene, UK), using pd(N)6 random hexamer (Amersham Biosciences, Piscataw ay, NJ) and SuperscriptTM II Reverse Transcriptase kit (Invitrogen) by following manufacturer ' s protocol.
  • EXAMPLE 18 IN VITRO ABILITY OF COMPOUND 7 TO MODULATE GENE EXPRESSION OF KLF4, ⁇ -CATENIN, AND CYCLIN Dl IN H226 CELLS
  • H226 cells 5 X K 5 cells in 5 mL volume of growth medium were seeded in 6-cm dishes and incubated overnight at 37 0 C. The medium was removed and replaced with growth medium containing 0.1% DMSO vehicle control or 1 ⁇ M of Compound 7. After incubation for 4, 7, 16, or 24 hours, the cells were detached by trypsinization, collected by centrifugation and washed once with PBS. Total RNA w as extracted from cells by using TRIZOL (Invitrogen, Life Technologies, Carlsbad. CA), following manufacturer ' s instruction, and the level of KLF4 mRNA, ⁇ -catenin mRNA, and Cyclin Dl mRNA w as measured by real time PCR using the comparative CT method as described in Example 17.
  • TRIZOL Invitrogen, Life Technologies, Carlsbad. CA
  • EXAMPLE 19 IN VIVO ABILITY OF COMPOUND 7 TO MODULATE GENE EXPRESSION OF KLF4, ⁇ -CATENIN, AND CYCLIN Dl IN A NSCLC CANCER XENOGRAFT MODEL
  • Cyclin Dl is a downstream target of ⁇ -catenin, whose transcription is regulated by KLF4. As such, the involvement of the Wnt/ ⁇ -catenin signaling pathway in the mode of action of compound 7 w as investigated.
  • ⁇ -catenin, cyclin D and KLF4 gene expression w as analyzed by RT-PCR in samples extracted from HT-29 colon cancer cells treated with Compound 7 in vitro, and from HT-29 xenograft tumors from treated CD-I nude mice.
  • EXAMPLE 20 IN VITRO ABILITY OF COMPOUND 7 TO MODULATE GENE EXPRESSION OF HIF-l ⁇ IN H226 CELLS
  • the ability of Compound 7 to modulate the mRNA level of HIF-I ⁇ genes in human non-small cell lung cancer H226 cells was determined in vitro as follows. H226 cells, 5 X 10 5 cells in 5 niL volume of growth medium were seeded in 6-cm dishes and incubated overnight at 37 0 C. The medium w as removed and replaced w ith growth medium containing 0.1% DMSO vehicle control or 1 ⁇ M of Compound 7. After incubation for 4, 7.
  • RNA w as extracted from cells by using TRIZOL (Invitrogen, Life Technologies, Carlsbad. CA), following manufacturer ' s instruction, and the level of HIF- l ⁇ niRNA was measured by real time PCR using the comparative CT method as described in Example 17.
  • Tumor growth profile after single injection of 15mg/kg of Compound 7 in human lung carcinoma H226 xenograft in CD-I nude mice is shown in Figure 10.
  • EXAMPLE 22 IN VIVO ABILITY OF COMPOUND 7 TO MODULATE GENE EXPRESSION OF MTF-I, HIF- l ⁇ , MMP2 AND VEGFR2 IN A NSLC CANCER XENOGRAFT MODEL
  • RNA w as extracted from 30 mg of tumour tissue using Rneasy Mini kit (QIAGEN, Valencia, CA).
  • VMD microvessel density
  • EXAMPLE 24 IN VITRO ABILITY OF COMPOUND 7 TO INHIBIT CELL GROWTH IN HUVEC CELLS
  • HAVEC Human Umbilical Vein Endothelial Cells
  • cell viability w as quantified using the XTT (sodium 3 ⁇ -[l-(phenylamino-carbonyl)-3,4-tetrazolium]-bis (4-methox ⁇ -6-nitro) benzene sulfonic acid hydrate) colorimetric assay (Roche Applied Science, Penzberg, German ⁇ " ) according to manufacturer ' s instructions.
  • the plates were further incubated 15 at 37 0 C for 2 hours and the absorbance of each well w as measured by a multi-well spectrophotometer (BioTek Instruments Inc., Winooski, VT) at 460 nm. The data were adjusted relative to the blank and were expressed as a percentage of cell growth compared to the DMSO control.
  • HUVEC cells (6 x 10 4 /well) were seeded in 6-w ell plates overnight before being treated with 0.5 ⁇ M of Compound 7 or 5 ⁇ M of the structurally related negative control compound from Example 79 with or without the presence of 100 ⁇ M of ZnCb in a total volume of 2 mL growth medium. After incubation of the cells at 37 0 C for 24 hours, the cells w ere trypsinized and resuspended in growth media to 3 x ICrVmL. One hundred micro litres of cell suspension (3 x 10 4 /well) were plated onto BD GFR Matrigel-coated 96-well plates and incubated for 2 hours. The coating procedure was performed according to the manufacturer ' s Thin Gel Method (BD Biosciences, Bedford. MA). The tube formation w as assessed using an inverted light microscope at 4OX magnification.
  • MTF-I metal transcription factor 1
  • EXAMPLE 16 PREVENTION OF TUMOR GROWTH IN HUMAN XENOGRAFT MODELS WITH COMPOUND 7
  • Human lung carcinoma H226, H460 and HOP92, and human colon carcinoma HT-29 cells were employed. Cancer cells were injected into CD-I athymic mice (Charles River Laboratories) at the flank. When the tumor size reached 70-100 mm the mice w ere treated with Compound 7 or vehicle control administered intravenously for 2 cycles of 5 consecutive days with a 5-10 day break between cycles.
  • Figure 15A shows the decrease in tumor growth in the model using H226 lung carcinoma cells and treatment with 15 mg/kg Compound 7 or vehicle control.
  • Figure 15B shows the decrease in tumor growth in the model using HOP92 lung carcinoma cells and treatment with 10 mg/kg Compound 7 or vehicle control.
  • Figure 15C shows the decrease in tumor growth in the model using H460 lung carcinoma cells and treatment with 7.5 mg/kg Compound 7 or vehicle control (1 cycle).
  • Figure 15D shows the decrease in tumor growth in the model using H226 lung carcinoma cells and treatment with 10 mg/kg Compound 7 or vehicle control. All results were significant p ⁇ 0.05.

Abstract

L'invention porte sur des composés 2-indolylimidazo[4,5-d]phénanthroline de formule (I) capables d'inhiber l'angiogenèse. L'invention porte également sur des procédés d'inhibition de l'angiogenèse utilisant des composés de formule (I), ainsi que sur des procédés et des utilisations des composés de formule (I) dans le traitement de maladies et troubles associés à l'angiogenèse tels que le cancer, la dégénérescence maculaire liée à l'âge (DMA), la rétinopathie diabétique et l'occlusion veineuse rétinienne.
PCT/CA2010/000337 2009-03-12 2010-03-12 Dérivés de 2-indolylimidazo[4,5-d]phénanthroline et leur utilisation pour inhiber l'angiogenèse WO2010102393A1 (fr)

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CN102276607A (zh) * 2011-05-26 2011-12-14 山西大学 一种咪唑[4,5-f]并1,10-邻菲罗啉衍生物及其制备方法和用途
WO2015051304A1 (fr) * 2013-10-04 2015-04-09 Aptose Biosciences Inc. Compositions, biomarqueurs et leur utilisation dans le traitement du cancer
US10080739B2 (en) 2003-11-14 2018-09-25 Aptose Biosciences Inc. Aryl imidazoles and their use as anti-cancer agents
CN110028437A (zh) * 2019-01-30 2019-07-19 浙江农林大学暨阳学院 一种微波促进制备2-苯基-3-醛基吲哚类化合物的方法
US11149047B2 (en) 2017-10-30 2021-10-19 Aptose Biosciences, Inc. Aryl imidazoles for treatment of cancer

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WO2005047266A1 (fr) * 2003-11-14 2005-05-26 Lorus Therapeutics Inc. Imidazoles d'aryle et leur utilisation comme agents anticancereux
WO2006126177A2 (fr) * 2005-05-25 2006-11-30 Genesense Technologies Inc. Derives de 2-indolyl imidazo[4,5-d]phenanthroline et utilisation dans le traitement du cancer

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WO2005047266A1 (fr) * 2003-11-14 2005-05-26 Lorus Therapeutics Inc. Imidazoles d'aryle et leur utilisation comme agents anticancereux
WO2006126177A2 (fr) * 2005-05-25 2006-11-30 Genesense Technologies Inc. Derives de 2-indolyl imidazo[4,5-d]phenanthroline et utilisation dans le traitement du cancer

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10080739B2 (en) 2003-11-14 2018-09-25 Aptose Biosciences Inc. Aryl imidazoles and their use as anti-cancer agents
CN102276607A (zh) * 2011-05-26 2011-12-14 山西大学 一种咪唑[4,5-f]并1,10-邻菲罗啉衍生物及其制备方法和用途
CN102276607B (zh) * 2011-05-26 2012-10-17 山西大学 一种咪唑[4,5-f]并1,10-邻菲罗啉衍生物及其制备方法和用途
WO2015051304A1 (fr) * 2013-10-04 2015-04-09 Aptose Biosciences Inc. Compositions, biomarqueurs et leur utilisation dans le traitement du cancer
WO2015051302A1 (fr) * 2013-10-04 2015-04-09 Aptose Biosciences Inc. Compositions et procédés de traitement de cancers
US9567643B2 (en) 2013-10-04 2017-02-14 Aptose Biosciences Inc. Compositions and methods for treating cancers
EP3650023A1 (fr) * 2013-10-04 2020-05-13 Aptose Biosciences Inc. Compositions pour le traitement de cancers
US11104957B2 (en) 2013-10-04 2021-08-31 Aptose Biosciences, Inc. Compositions and methods for treating cancers
US11149047B2 (en) 2017-10-30 2021-10-19 Aptose Biosciences, Inc. Aryl imidazoles for treatment of cancer
CN110028437A (zh) * 2019-01-30 2019-07-19 浙江农林大学暨阳学院 一种微波促进制备2-苯基-3-醛基吲哚类化合物的方法

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