WO2009043114A1 - Arsenoxide compound and method of use - Google Patents

Arsenoxide compound and method of use Download PDF

Info

Publication number
WO2009043114A1
WO2009043114A1 PCT/AU2008/001477 AU2008001477W WO2009043114A1 WO 2009043114 A1 WO2009043114 A1 WO 2009043114A1 AU 2008001477 W AU2008001477 W AU 2008001477W WO 2009043114 A1 WO2009043114 A1 WO 2009043114A1
Authority
WO
WIPO (PCT)
Prior art keywords
cysteine
serine
threonine
acid
hydrogen
Prior art date
Application number
PCT/AU2008/001477
Other languages
French (fr)
Inventor
Philip John Hogg
Pierre Dilda
Original Assignee
Newsouth Innovations Pty Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2007905434A external-priority patent/AU2007905434A0/en
Application filed by Newsouth Innovations Pty Limited filed Critical Newsouth Innovations Pty Limited
Publication of WO2009043114A1 publication Critical patent/WO2009043114A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/66—Arsenic compounds
    • C07F9/70—Organo-arsenic compounds
    • C07F9/74—Aromatic compounds
    • C07F9/78—Aromatic compounds containing amino groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
    • C07K5/06069—Ser-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides

Definitions

  • the present invention relates to organic arsenoxide compounds, pharmaceutical compositions comprising these compounds, and to their use in therapy, in particular, treatment of proliferative diseases.
  • Arsenical compounds have been used in the past as therapeutic agents for the treatment of disease. However, the inherent toxicities of arsenical compounds and their generally unfavourable therapeutic index have largely precluded their use as pharmaceutical agents.
  • Organic arsenoxide compounds are disclosed in WO 01/21628. Such compounds are described as having antiproliferative properties useful in the therapy of proliferative diseases.
  • WO 04/042079 discloses the use of organic arsenoxide compounds for inducing the mitochondrial permeability transmission (MPT) and also the use of such compounds for inducing apoptosis and necrosis, particularly in endothelial cells.
  • GSAO The compound 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid
  • GSAO is a synthetic tripeptide trivalent arsenical compound disclosed in WO 01/21628 and WO 04/042079.
  • GSAO perturbs the mitochondria in angiogenic endothelial cells, leading to proliferation arrest and cell death (WO 04/042079).
  • GSAO inactivates the mitochondrial inner membrane transporter, adenine nucleotide translocase (ANT), by cross-linking two of the three matrix facing thiols (WO 04/042079).
  • ANT adenine nucleotide translocase
  • ANT is a calcium receptor which undergoes a conformational change and a change in activity upon binding of calcium ions. GSAO binds to calcium-replete ANT, but binds minimally in the absence of calcium ions.
  • GSAO is a selective inhibitor of endothelial cell proliferation compared to tumour cells, with IC 5O values for proliferation arrest up to 30-fold higher in tumour cells. This is because endothelial cells are very poor at exporting GSAO, while the multidrug resistance-associated proteins (MRP) 1 and 2 in tumour and other cells may efficiently pump GSAO out the cytoplasm (Dilda et al., 2005b). In contrast, endothelial cells i o express very little MRP 1 /2.
  • MRP multidrug resistance-associated proteins
  • the present invention relates to organic arsenoxide compounds comprising a peptidic residue linked via an amide linking moiety to a phenylarsenoxide residue.
  • Organic arsenoxide compounds according to the present invention may be capable of crossing cell membranes, in particular, endothelial cell membranes and may be useful for the treatment of disease, in particular, treatment of proliferative disease.
  • the present invention relates to organic arsenoxide compounds of general formula (I):
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted Ci -3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy; m is an integer selected from 1, 2 and 3; 0 [AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; s [AA-3] (when present) is a ⁇ -glutamic acid residue; n is
  • the present invention relates to compounds of general formula io (I):
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted C 1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy; m is an integer selected from 1, 2 and 3; 0 [AA-I] is an amino acid residue selected from cysteine and serine;
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; 5 [AA-3] (when present) is a ⁇ -glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof. 0 In a third aspect the present invention relates to compounds of general formula (I):
  • R 1 is selected from hydrogen, Ci -3 alkyl and cyclopropyl
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted Ci -3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy; o m is an integer selected from 1, 2 and 3;
  • [AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and ⁇ -amino- ⁇ -hydroxy-isovaleric acid;
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine,s tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is O; and salts and hydrates thereof. 0
  • the present invention relates to compounds of general formula (I):
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted C 1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted Ci -3 alkoxy; m is an integer selected from 1, 2 and 3;
  • [AA-I] is an amino acid residue selected from cysteine and serine
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is 0; and salts and hydrates thereof.
  • the present invention relates to a pharmaceutical composition comprising a compound of formula (I) according to the first, second, third or fourth aspect of the invention, together with a pharmaceutically acceptable excipient, diluent or adjuvant.
  • the present invention relates to a method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
  • the present invention relates to a method of inhibiting angiogenesis in a vertebrate, comprising administering to the vertebrate an effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
  • the present invention relates to a method of inducing the Mitochondrial Permeability Transition (MPT) in a vertebrate comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
  • the compound of formula (I) may selectively induce the MPT in proliferating endothelial cells.
  • the present invention relates to a method of inducing apoptosis or necrosis in proliferating mammalian cells, comprising administering to the mammal an apoptosis- or necrosis-inducing amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
  • the cells may be proliferating endothelial cells.
  • the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for treating a proliferative disease in a vertebrate.
  • the present invention relates to the use of a compound of formula 5 (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inhibiting angiogenesis in a vertebrate.
  • the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inducing the MPT in a vertebrate.
  • the compound ofo formula (I) may selectively induce the MPT in proliferating endothelial cells.
  • the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inducing apoptosis or necrosis in proliferating mammalian cells.
  • the cells may be proliferating endothelial cells.
  • the present invention relates to a method of treating ⁇ -glutamyl transferase expressing tumours in a vertebrate, comprising administering to the vertebrate a compound of formula (I) according to the first, second, third or fourth aspect of the invention or a composition according to the fifth aspect of the invention.
  • the present invention relates to the use of a compound of formula0 (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for treating ⁇ -glutamyl transferase expressing tumours in a vertebrate.
  • C 1-3 alkyl group includes within its meaning monovalent (“alkyl”) and divalent (“alkylene”) straight chain or branched chain saturated aliphatic groups having from 1 to 3 carbon atoms.
  • alkyl monovalent
  • alkylene divalent
  • the term C 1-3 alkyl includes methyl, ethyl, 1 -propyl, and isopropyl.
  • C 2-3 alkenyl group includes within its meaning monovalent (“alkenyl”) and divalent (“alkenylene”) straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 3 carbon atoms and at least one double bond anywhere in the chain. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis or trans, or E or Z as appropriate. Examples of alkenyl groups include ethenyl, vinyl, allyl, 1-methylvinyl, 1-pro ⁇ enyl, and 2- ⁇ ro ⁇ enyl.
  • C 2-3 alkynyl group as used herein includes within its meaning monovalent (“alkynyl”) and divalent (“alkynylene”) unsaturated aliphatic hydrocarbon groups having from 2 to 3 carbon atoms and having at least one triple bond.
  • alkynyl groups include but are not limited to ethynyl, 1-propynyl.
  • alkoxy refers to straight chain or branched alkyloxy (i.e, O-alkyl) groups, wherein alkyl is as defined above.
  • alkoxy groups include methoxy, ethoxy, n-propoxy, and isopropoxy.
  • amino refers to groups of the form -NR a R b wherein R a and R b are individually selected from hydrogen, optionally substituted (C 1-4 )alkyl, optionally substituted (C 2-4 )alkenyl, optionally substituted (C 2-4 )alkynyl, optionally substituted (C 6- io)aryl and optionally substituted aralkyl groups, such as benzyl.
  • the amino group may be a primary, secondary or tertiary amino group.
  • amino acid as used herein includes naturally and non-naturally occurring amino acids, as well as substituted variants thereof.
  • amino acid therefore encompasses, for example, ⁇ , ⁇ , and ⁇ -amino acids. ⁇ -Amino acids are particularly preferred.
  • the (L) and (D) forms of amino acids are also included in the scope of the term “amino acid”. (L)-amino acids are a preferred form.
  • amino acid includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, ⁇ -amino- ⁇ - hydroxy-isovaleric acid and penicillamine.
  • the backbone of the amino acid residue may be substituted with one or more groups independently selected from (C 1-6 )alkyl, halogen, hydroxy, hydroxy(C 1-6 )alkyl, aryl, e.g., phenyl, aryl(C 1-3 )alkyl, e.g., benzyl, and (C 3- 6 )cycloalkyl.
  • C 6-10 aryl or variants such as “arylene” as used herein refers to monovalent (“aryl”) and divalent (“arylene”) single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • aromatic groups include phenyl, and naphthyl.
  • arylalkyl or variants such as “aralkyl” as used herein, includes within its meaning monovalent (“aryl”) and divalent (“arylene”), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight or branched chain alkylene radicals.
  • arylalkyl groups include benzyl.
  • C 3-8 heterocycloalkyl includes within its meaning monovalent (“heterocycloalkyl”) and divalent (“heterocycloalkylene”), saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 8 ring atoms, wherein from 1 to 5, or from 1 to 3, ring atoms are heteroatoms independently selected from O, N, NH, or S.
  • the heterocycloalkyl group may be C 3-6 heterocycloalkyl.
  • the heterocycloalkyl group may be C 3-5 heterocycloalkyl.
  • heterocycloalkyl groups include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.
  • C 5-10 heteroaromatic group and variants such as “heteroaryl” or “heteroarylene” as used herein, includes within its meaning monovalent (“heteroaryl”) and divalent (“heteroarylene”), single, polynuclear, conjugated and fused aromatic radicals having from 5 to 10 atoms, wherein 1 to 4 ring atoms, or 1 to 2 ring atoms are heteroatoms independently selected from O, N, NH and S.
  • the heteroaromatic group may be C 5-8 heteroaromatic.
  • heteroaromatic groups include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl, pyrrolyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, and the like.
  • halogen or variants such as “halide” or “halo” as used herein refers to fluorine, chlorine, bromine and iodine.
  • heteroatom or variants such as “hetero-” or “heterogroup” as used herein refers to O, N, NH and S.
  • optionally substituted means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, haloalkyl, haloalkynyl, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, NO 2 , NR a R b , nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino,
  • Preferred substituents include C 1-3 alkyl, Ci -3 alkoxy, -CH 2 -(Ci_ 3 )alkoxy, C 6-10 aryl, -CHa-phenyl, halo, hydroxyl, hydroxy(C 1-3 )alkyl (e.g., CH 2 OH), and halo(C 1-3 )alkyl (e.g, CF 3 , CH 2 CF 3 ).
  • substituents include C 1-3 alkyl, C 1-3 alkoxy, halo, hydroxyl, hydroxy(C 1-3 )alkyl (e.g, CH 2 OH), and halo(C 1-3 )alkyl (e.g, CF 3 , CH 2 CF 3 ).
  • administering and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
  • vertebrate includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian.
  • the vertebrate may be a human.
  • treatment refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • the term "effective amount” includes within itss meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide a desired effect.
  • the term “therapeutically effective amount” includes within its meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic effect.
  • the exact amount required will vary from subject to subject depending on factors such as the species being treated, the sex,0 age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. S
  • Figure 1 Inhibition of cell-surface ⁇ GT blunts cellular accumulation of GSAO and antiproliferative activity.
  • FIG. 1 Expression level of ⁇ GT positively correlates with sensitivity of cells to GSAO.
  • a and B c21/ ⁇ GT (high ⁇ GT) or c21 /basal (low ⁇ GT) melanoma cells were incubated with GSAO (0-1.8 mM) for 24 h (part A) or 72 h (part B) and cell viability was determined using WST-I. Results are expressed as percentage of untreated controls. Data points are the mean ⁇ SD of five determinations. Results are representative of two experiments.
  • C The potent ⁇ GT inhibitor, ABBA, blunts GSAO anti-proliferative activity in the melanoma cells.
  • c21/ ⁇ GT (high ⁇ GT) or c21/basal (low ⁇ GT) melanoma cells were incubated for 24 h in the absence or presence of 10 ⁇ M ABBA and a GSAO concentration representing the IC 50 for proliferation arrest (60 ⁇ M for high and 1250 ⁇ M for low ⁇ GT cells).
  • Cell viability was determined using WST-I and results expressed as percentage of untreated controls. Data points are the mean ⁇ SD of five determinations. Results are representative of two experiments. D. K; for inhibition of ⁇ GT by GSAO.
  • FIG. 3 The GSAO metabolite. GCAO. enters endothelial cells via the OATP. accumulates more rapidly in the cytosol and has greater anti-proliferative activity than GSAO.
  • BAE cells were incubated with 50 ⁇ M GSAO or GCAO for up to 4 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. The rates of accumulation of GSAO and GCAO are 0.03 and 0.23 nmol As atoms per 10 6 cells, respectively.
  • BAE cells were incubated for 24 h with 20 ⁇ M GCAO in the presence or absence of 200-500 ⁇ M of DIDS.
  • Cell viability was determined using MTT and results expressed as percentage of untreated control. Values are mean ⁇ SD of triplicate determinations. Results are representative of three experiments.
  • GCAO is secreted from endothelial cells by MRPl and is more efficient than GSAO at triggering the mitochondrial permeability transition.
  • BAE cells were incubated with 0.4-50 ⁇ M GCAO for 24 h in the absence or presence of 5 ⁇ M 4H10.
  • Cell viability was determined using MTT and results expressed as percentage of untreated control. Values are mean ⁇ SD of triplicate determinations. Results are representative of two experiments.
  • C Mitochondrial swelling induced by 0-300 ⁇ M GCAO as measured by decrease in light scattering at 520 nm over 60 min. The traces are representative of a minimum of two experiments performed in duplicate on two different mitochondrial preparations.
  • D Time for 25% maximum swelling as a function of GSAO or GCAO concentration. The data points are the average from two separate experiments.
  • FIG. 5 The GCAO metabolite, CAO, accumulates in the endothelial cells at a similar rate and has comparable anti-proliferative activity to GCAO.
  • A. Cleavage of GCAO by dipeptidase to produce CAO.
  • C. CAO and GCAO accumulate in the cytosol at a similar rate.
  • BAE cells were incubated with 50 ⁇ M GCAO or CAO for up to 4 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry.
  • the rates of accumulation of GCAO and CAO are 0.23 and 0.20 nmol As atoms per 10 6 cells, respectively.
  • CAO is secreted from endothelial cells by MRPl and is more efficient than GCAO at triggering the mitochondrial permeability transition.
  • FIG. 7 Metabolism of GSAO by endothelial cells.
  • the ⁇ -glutamyl residue of GSAO is cleaved at the cell surface by ⁇ -GT to produce GCAO, which is then transported across the plasma membrane by an OATP.
  • GCAO may be further processed to CAO before it reacts with ANT of the inner-mitochondrial membrane.
  • the present invention relates to organic arsenoxide compounds comprising a pendant group linked via an amide linking moiety to a phenylarsenoxide residue.
  • Organic arsenoxide compounds according to the present invention may exhibit increased cellular uptake and/or increased efficacy and/or an improved therapeutic index relative to known arsenoxide compounds, such as, the compound 4-(N-(S-glutathionylacetyl)amino)- phenylarsinous acid ("GSAO").
  • the present invention relates to organic arsenoxide compounds of general formula (I): R 1 [AA-2] n
  • R 1 is selected from hydrogen, Ci -3 alkyl and cyclopropyl
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted C 1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy; m is an integer selected from 1 , 2 and 3; [AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and ⁇ -amino- ⁇ -hydroxy-isovaleric acid;
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine;
  • [AA-3] (when present) is a ⁇ -glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof.
  • the present invention further relates to organic arsenoxide compounds of general formula (I):
  • R 1 is selected from hydrogen, C 1-3 alkyl and cyclopropyl
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted C 1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy
  • m is an integer selected from 1, 2 and 3
  • [AA-I] is an amino acid residue selected from cysteine and serine
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine;
  • [AA-3] (when present) is a ⁇ - glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof.
  • the present invention further relates to organic arsenoxide compounds of general formula (I):
  • R 2 and R 3 may be the same or different and are independently selected from hydrogen, optionally substituted C 1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C 2-3 alkylene; and optionally substituted C 1-3 alkoxy; m is an integer selected from 1, 2 and 3; [AA-I] is an amino acid residue selected from cysteine serine, penicillamine, threonine and ⁇ -amino- ⁇ -hydroxy-isovaleric acid;
  • [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is O; and salts and hydrates thereof.
  • Optional substirueiits may be independently selected from C 1-3 alkyl, C 1-3 alkoxy,
  • (Ci -3 )alkyl e.g., CF 3 , CH 2 CF 3 .
  • the optional substituents are independently selected from hydroxyl, methoxy, halo, and (C 1-3 )alkyl. In one embodiment there are no optional substituents.
  • the As(OH) 2 group may be ortho- or para- to the N-atom on the phenyl ring. In one embodiment, the As(OH) 2 group is para- to the N-atom on the phenyl ring. In another embodiment the As(OH) 2 group is ortho- to the N-atom on the phenyl ring.
  • R 1 may be hydrogen, methyl or ethyl. In one embodiment R 1 is hydrogen.
  • R and R may be the same or different.
  • R and R may be independently selected from hydrogen, C 1-3 alkyl, C 2-3 alkenyl, C 1-3 alkoxy, halo- (C 1-3 )alkoxy, hydroxy(Ci -3 )alkyl and halo(Ci -3 )alkyl.
  • R 2 and R 3 may be independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, hydroxymethyl, CF 3 and OCF 3 .
  • R 2 and R 3 may be independently selected from hydrogen, methyl and ethyl.
  • R is methyl and R is hydrogen.
  • R 2 and R 3 are both methyl.
  • R 2 and R 3 are both hydrogen.
  • n is 1. In another embodiment m is 2.
  • the pendant group "[AA-l]-[AA-2]-[AA-3]" may comprise a single amino acid, a dipeptide, or tripeptide.
  • the pendant group is a single amino acid residue selected from cysteine and serine.
  • the pendant group is cysteine.
  • the pendant group is a dipeptide comprised of cysteine and a second amino acid residue. In a further embodiment the pendant group is a dipeptide residue comprised of serine and a second amino acid residue. In a further embodiment the pendant group is a dipeptide comprised of penicillamine and a second amino acid group. In a further embodiment the pendant group is a dipeptide comprised of threonine and a second amino acid group. In a further embodiment the pendant group is a dipeptide comprised of ⁇ -amino- ⁇ -hydroxy-isovaleric acid and a second amino acid group.
  • the second amino acid residue may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the pendant group is a tripeptide comprised of cysteine, a second amino acid residue and a ⁇ -glutamic acid residue. In a further embodiment the pendant group is a tripeptide comprised of serine, a second amino acid residue and a ⁇ -glutamic acid residue. In a further embodiment the pendant group is a tripeptide comprised of penicillamine, a second amino acid group and a ⁇ -glutamic acid residue.
  • the pendant group is a tripeptide comprised of threonine, a second amino acid group and a ⁇ -glutamic acid residue
  • the pendant group is a tripeptide comprised of ⁇ -amino- ⁇ -hydroxy-isovaleric acid, a second amino acid group and a ⁇ -glutamic acid residue.
  • the second amino acid residue may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the ⁇ -glutamic acid residue is attached via its ⁇ -COOH group.
  • [AA-2] and [AA-3] may be attached to the cysteine, serine, threonine, ⁇ -amino- ⁇ - hydroxy-isovaleric acid or penicillamine residue at the amino (NH 2 ) and/or acid (CO 2 H) grou ⁇ (s) of the cysteine, serine, threonine, ⁇ -amino- ⁇ -hydroxy-isovaleric acid or penicillamine residue.
  • the pendant group is a tripeptide
  • the arrangement of amino acids is -[AA-I] -[AA-2]- [AA-3] as shown below:
  • the arrangement of amino acids is [ AA-2]-[ AA- 1]-[ AA-3], where [AA-I] is attached to the (CR 2 R 3 ) m group, i.e., where [AA-I] is the central amino acid of the tripeptide.
  • the arrangement of amino acids is [AA-3]-
  • the [AA-I] amino acid is an (L) or (D) form of an amino acid.
  • [AA-I] is an (L)-amino acid.
  • the [AA-2] amino acid is in the (L) or (D) form of the amino acid.
  • the [AA-2] amino acid is an (L)-amino acid.
  • the [AA-3] amino acid is in the (L) or (D) form of the amino acid.
  • the [AA-3] amino acid is an (L)- amino acid.
  • Compounds of formula (I) according to the present invention having a terminal ⁇ - glutamyl residue may have the ⁇ -glutamyl residue cleaved from the compound by cell- surface ⁇ -glutamyltransferase ( ⁇ -GT) (also known as ⁇ -glutamyltranspeptidase; E.C 2.3.2.2).
  • ⁇ -GT cell- surface ⁇ -glutamyltransferase
  • the resultant compound i.e., without the ⁇ -glutamyl residue
  • the ⁇ -glutamyl residue of GSAO may be cleaved by cell-surface ⁇ -GT to give the compound 4-(N-(S- cysteinylglycylacetyl)amino)-phenylarsinous acid (GCAO), which is transported into the cell cytoplasm.
  • ⁇ -GT cell-surface ⁇ -GT
  • n is 1 and p is 1. In another embodiment, n is 1o and p is O. In a further embodiment n is 0 and p is 0.
  • R 1 , R 2 and R 3 are hydrogen; m is 1; n is 0; p is 0; [AA-I] is cysteine and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are independently hydrogen or methyl; m is 1s or 2; n is 1; p is 0; [AA-I] is cysteine, serine, threonine, ⁇ -amino- ⁇ -hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are independently hydrogen or methyl; m is 1 or 2; n is 1; p is 0; [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1 or 2; n is 1; p is 0;
  • [AA-I] is cysteine, serine, threonine, ⁇ -amino- ⁇ -hydroxy-isovaleric acid or penicillamine;
  • [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1 or 2; n is 1; p is 0;
  • [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA-5 1] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is threonine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA-
  • [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R , R and R are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA- 1] is threonine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R , R and R are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA- 1] is cysteine; [AA-2] is glycine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is glycine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is threonine; [AA-2] is glycine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; m is 1; n is 1; p is 0; [AA-
  • 5 1] is ⁇ -arnino- ⁇ -hydroxy-isovaleric acid; [AA-2] is glycine; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • the pendant group may be a dipeptide.
  • suitable dipeptide residues include cysteine-glycine; cysteine-alanine; cysteine-valine; cysteine-leucine; cysteine-serine; cysteine-threonine; cysteine-cysteine; io cysteine-aspartic acid; cysteine-glutamic acid; cysteine-lysine; and cysteine-arginine.
  • the dipeptide residue may be serine-glycine; serine-alanine; serine- valine; serine-leucine; serine-serine; serine-threonine; serine-cysteine; serine-aspartic acid; serine-glutamic acid; serine-lysine; and serine-arginine.
  • the dipeptide residue may be penicillamine-glycine; penicillamine-alanine; penicillamine-
  • the dipeptide residue may be threonine-glycine; threonine-alanine; threonine-valine; threonine-leucine; threonine-serine; threonine-threonine; threonine-cysteine; threonine-aspartic acid;
  • the dipeptide residue may be ⁇ -amino- ⁇ -hydroxy-isovaleric acid-glycine; ⁇ -amino- ⁇ - hydroxy-isovaleric acid-alanine; ⁇ -amino- ⁇ -hydroxy-isovaleric acid-valine; ⁇ -amino- ⁇ - hydroxy-isovaleric acid-leucine; ⁇ -amino- ⁇ -hydroxy-isovaleric acid-serine; ⁇ -amino- ⁇ - hydroxy-isovaleric acid-threonine; ⁇ -amino- ⁇ -hydroxy-isovaleric acid-cysteine; ⁇ -
  • the dipeptide residue may be cysteine- glycine; cysteine-alanine; cysteine-valine; cysteine-serine; cysteine-cysteine; cysteine- aspartic acid; cysteine-glutamic acid; or cysteine-lysine.
  • the 0 dipeptide residue may be serine-glycine; serine-alanine; serine-valine; serine-serine; serine-cysteine; serine-aspartic acid; serine-glutamic acid; or serine-lysine.
  • the dipeptide residue may be cysteine-glycine; cysteine-alanine; cysteine- serine; cysteine-aspartic acid; or cysteine-glutamic acid, hi other embodiments the dipeptide residue may be serine-glycine; serine-alanine; serine-serine; serine-aspartic s acid; or serine-glutamic acid, hi other embodiments the dipeptide residue may be cysteine-glycine; cysteine-alanine; cysteine-aspartic acid; or cysteine-glutamic acid.
  • the dipeptide residue may be serine-glycine; serine-alanine; serine- aspartic acid; or serine-glutamic acid. In other embodiments, the dipeptide residue may be cysteine-glycine or cysteine-alanine. In other embodiments, the dipeptide residue may
  • dipeptide residue may be cysteine-glycine or serine-glycine.
  • the pendant group may be a tripeptide, [AA- l]-[AA-2]-[AA-3], wherein the moiety [AA-l]-[AA-2] is any one of the foregoing dipeptide residues and [AA-3] is a ⁇ -glutamic acid residue.
  • the ⁇ -glutamic acid residueQ may be attached (via the ⁇ -COOH group) to either the [AA-I] or [AA-2] amino acid residue.
  • R 1 , R 2 and R 3 are independently hydrogen or methyl; n is 1, p is 1; [AA-I] is cysteine, serine, threonine, ⁇ -amino- ⁇ -hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine,s methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R , R and R are independently hydrogen or methyl; n is 1, p is 1; [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine,Q leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine,S cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is ⁇ -glutamic acid; and0 the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine;
  • [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] iss threonine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine,Q threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is ⁇ - glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the 5 phenyl ring.
  • R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is ⁇ - glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is ⁇ - glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] isS ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is serine; [AA-2] is glycine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is glycine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is glycine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • R 1 , R 2 and R 3 are each hydrogen; n is 1, p is 1; [AA-I] is ⁇ -amino- ⁇ -hydroxy-isovaleric acid; [AA-2] is glycine; [AA-3] is ⁇ -glutamic acid; and the As(OH) 2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
  • Cytoplasmic levels of compounds of formula (I) may be controlled by export from the cell by multidrug-resistance associated protein-1 (MRP-I).
  • MRP-I multidrug-resistance associated protein-1
  • cytoplasmic levels of GCAO are controlled by export from the cell via the MRP-I protein.
  • Cist 'trans (E/Z) isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
  • enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid.
  • a suitable optically active compound for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid.
  • the resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s)
  • Chiral compounds of the invention may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
  • chromatography typically HPLC
  • a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine.
  • Compounds of formula (I) in accordance with the present invention are capable of binding to cysteine residues of mitochondrial Adenine Nucleotide Translocator (ANT) in proliferating endothelial cells thereby inducing the Mitochondrial Permeability Transition (MPT). Accordingly, compounds of formula (I) according to the present invention may lead to proliferation arrest and cell death.
  • compounds of formula (I) may be selective inhibitors of endothelial cell proliferation.
  • compounds of formula (I) may be selective inhibitors of endothelial cell proliferation compared to tumour cells. Compounds of formula (I) therefore may be useful in the treatment of proliferative diseases.
  • Organic arsenic compounds according to the present invention may have one or more advantage(s) over known arsenical compounds, such as arsenic trioxide and the arsenoxide compounds disclosed in WO 01/21628 or WO 04/042079.
  • Compounds of formula (I) may exhibit increased cellular uptake relative to the known organic arsenoxide compound 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid ("GSAO").
  • GSAO organic arsenoxide compound 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid
  • compounds of formula (I) may accumulate in endothelial cells at a rate about 20-times, about 15-times, about 10-times, about 5-times, about 2- times faster than GSAO.
  • compounds of formula (I) accumulate in endothelial cells at a rate about 10-times faster than GSAO.
  • compounds of formula (I) accumulate in endothelial cells at a rate about 8-times faster than GSAO.
  • compounds of formula (I) accumulate in endothelial cells at a rate about 5-times faster than GSAO.
  • Compounds of formula (I), such as GCAO 3 may be more effective than known arsenoxide compounds, including arsenoxide compounds disclosed in WO 01/21628, such as GSAO, at inhibiting cellular proliferation (particularly proliferation of endothelial cells) and/or reducing the viability of endothelial cells, hi the context of this invention, "reducing the viability of endothelial cells” can include cell death, or progression towards cell death.
  • the IC 50 values of compounds of formula (I) as a measure of anti-proliferative activity may be about 10-times less than the IC 50 of GSAO.
  • the IC 50 of compounds of formula (I) may be about 7.5-times less than the IC 5 o of GSAO. In a further embodiment, the IC 50 of compounds of formula (I) may be about 5-times less than the IC 5O of GSAO. In another embodiment, the IC 5O of compounds of formula (I) may be about 2-times less than the IC 50 of GSAO.
  • compounds of formula (I), such as GCAO may be useful in the treatment of proliferative diseases.
  • another embodiment of the invention relates to a method of treating a cellular proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
  • the cells may be endothelial cells.
  • the vertebrate may be a mammal, such as a human.
  • the present invention relates to a method of inhibiting angiogenesis in a vertebrate, comprising administering to the vertebrate an effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
  • a further embodiment of the invention relates to a method of inducing the MPT in a vertebrate comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
  • Compounds of formula (I) according to the present invention may induce the MPT by binding to cysteine residues on mitochondrial Adenine Nucleotide Translocator (ANT).
  • ANT mitochondrial Adenine Nucleotide Translocator
  • the compound of formula (I) is from about 2 to about 20-times, about 2 to about 10-times, about 2 to about 5-times, e.g., about 4-times, more efficient at inducing the MPT than GSAO.
  • Another embodiment of the invention relates to a method of inducing apoptosis in proliferating mammalian cells, comprising administering to the mammal an apoptosis- inducing amount of a compound of formula (I) or a pharmaceutical composition thereof.
  • the invention relates to a method of treating ⁇ -glutamyl transferase expressing tumours in a vertebrate, comprising administering to the vertebrate a compound of formula (I) or a pharmaceutical composition thereof.
  • ⁇ -glutamyl transferase expressing tumours are tumours of the breast, prostate, colon, liver, ovary, lung, kidney, thyroid or pancreas.
  • chemotherapeutic agents include adriamycin, taxol, fluorouricil, melphalan, cisplatin, oxaliplatin, alpha interferon, vincristine, vinblastine, angioinhibins, TNP -470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-IOl, CM-101, Techgalan, thalidomide, SP-PG and the like.
  • chemotherapeutic agents include alkylating agents such as nitrogen mustards including mechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureas including carmustine, lomustine, semustine and streptozocin; alkyl sulfonates including busulfan; triazines including dicarbazine; ethyenimines including thiotepa and hexamethylmelamine; folic acid analogues including methotrexate; pyrimidine analogues including 5-fluorouracil, cytosine arabinoside; purine analogues including 6-mercaptopurine and 6-thioguanine; antitumour antibiotics including actinomycin D; the anthracyclines including doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone antagonists including tamoxifen and cortiosteroids and mis
  • salts of the compounds of the present invention will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the inventive compounds or of the pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art.
  • compositions of formula I may be prepared by methods known to those skilled in the art, including for example, (i) by reacting a compound of formula (I) with the desired acid or base; (ii) by removing an acid- or base- labile protecting group from a suitable precursor of the compound of formula (I) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
  • the resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent.
  • the degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
  • suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid.
  • Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts.
  • S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methane
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolaniine and the like.
  • Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration.
  • the mode of administration is parenteral.
  • the mode of administration is oral.
  • the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound.
  • the compound also may be administered parenterally or intraperitoneally.
  • Dispersions of compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical compositions suitable for injection include sterile aqueous solutions
  • the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
  • the compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier.
  • the compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet.
  • the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • such compositions and preparations may contain at least 1% by weight of active compound.
  • the percentage of the compound(s) of formula (I) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit.
  • the amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.
  • pharmaceutically acceptable carrier is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions according to the present invention may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the carrier is an orally administrable carrier.
  • Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration. Also included in the scope of this invention are delayed release formulations.
  • prodrugs include esters, phosphonate esters etc, of the compound.
  • the compound of formula (I) may be administered by injection.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents.
  • Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum gragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules can be coated with shellac, sugar or both.
  • a syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the analogue can be incorporated into sustained-release preparations and formulations.
  • the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis.
  • Suitable buffer agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
  • Single or multiple administrations of the compounds and/or pharmaceutical compositions according to the invention may be carried out.
  • One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.
  • an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight.
  • an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.
  • an effective dosage may be up to about 500mg/m 2 .
  • an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , about 25 to about 350mg/m 2 , about 25 to about 300mg/m 2 , about 25 to about 250mg/m 2 , about 50 to about 250mg/m 2 , and about 75 to about 150mg/m 2 .
  • a compound of Formula (I) may be administered in an amount in the range from about 100 to about 1000 mg per day, for example, about 200 mg to about 750 mg per day, about 250 to about 500 mg per day, about 250 to about 300 mg per day, or about 270 mg to about 280 mg per day.
  • Compounds in accordance with the present invention may be administered as part of a therapeutic regimen with other drugs. It may be desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition. Accordingly, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound of formula (I) according to the present invention, may be combined in the form of a kit suitable for simultaneous or sequential administration of the compositions.
  • the invention will now be described in more detail, by way of illustration only, with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification.
  • Bovine aortic endothelial (BAE) cells were from Cell Application (San Diego, CA). BAE cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L- glutamine, and 5 units per mL penicillin and streptomycin (Gibco, Gaithersburg, MD). Melanoma cell clones expressing different ⁇ GT activity were produced as previously described (Franzini et al., 2006). The c21/basal and c21/ ⁇ GT clones express a ⁇ GT activity of 0.34 ⁇ 0.13 and 91 ⁇ 3.4 mU per mg of cellular protein, respectively.
  • Both clones were grown in RPMI 1640 medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine and 0.5 mg/ml G418 (Gibco). Cells were cultured at 37 °C in a 5% CO 2 , 95% air atmosphere.
  • Cell proliferation assay BAE cells were seeded in 96-well plates (5,000 cells per well) in 0.2 ml of culture medium. After 24 h of growth, the medium was replaced with fresh culture medium supplemented with GSAO, GCAO, CAO or 4H10 and cells were cultured for an additional 24, 48 or 72 h. Viable attached cells were determined using the tetrazolium salt MTT (Sigma, St. Louis, MO) according to the manufacturer's protocol. Results were expressed as percentage of untreated controls.
  • Melanoma cells were seeded in 96-well plates (4,000 cells per well) in 0.2 ml of culture medium. After 24 h of growth, the medium was replaced with fresh culture medium supplemented with GSAO and cells were cultured for an additional 24 or 72 h. Viable cells were determined using the WST-I reagent (Roche, Basel, Switzerland) according to the manufacturer's protocol. Results were expressed as percentage of untreated controls.
  • ⁇ GT inhibition was studied.
  • the ⁇ GT competitive inhibitor, L-2-amino-4-boronobutanoic acid (ABBA) was provided by Dr. R. E. London (Natl. Inst. Environ. Health Sci., NC, USA). Cells were exposed for 24 h to GSAO concentrations causing 50% of cell growth inhibition (IC 5 o) in the absence or presence of 10 ⁇ M ABBA. This concentration of ABBA alone had no effects on cell viability or proliferation (data not shown).
  • Ki for inhibition of ⁇ GT by GSAO Determination of ⁇ GT activity was performed according to Huseby and Str ⁇ mme
  • the formation of j5-nitroaniline as a function of time was monitored continuously by measuring the absorbance at 405 nm using a Molecular Devices M2 Microplate Reader (Palo Alto, CA).
  • the extinction coefficient used for p-nitroaniline was 9920 M -1 Cm "1 .
  • the apparent K; for inhibition of ⁇ GT by GSAO was estimated from the effect of fixed concentrations of GSAO on the initial velocity of hydrolysis of ⁇ -glutamyl- ⁇ -nitroanilide by ⁇ GT. The results were consistent with simple competitive inhibition.
  • the data points were globally fit by non-linear least squares regression using GraphPad (San Diego, CA) software.
  • GSAO was produced as described previously (WO 01/21628, the disclosure of which is incorporated herein by reference) to a purity >94% by HPLC.
  • a 50 mM solution of GSAO was made by dissolving solid in 20 mM Hepes, pH 7.0 buffer containing 0.14 M NaCl, 20 mM glycine and 1 mM EDTA.
  • 4-(N-(S- cysteinylglycylacetyl)amino)phenylarsinous acid (GCAO) was produced by cleaving the ⁇ -glutamyl group from GSAO with ovine kidney ⁇ -glutamyl transpeptidase type I (Sigma, product number G8040).
  • a lO mM solution of GSAO was incubated with 0.55 units per ml ⁇ GT in 15 mM Tris, pH 7.4 buffer containing 40 mM glycyl-glycine for 1 h at 3O 0 C.
  • the ⁇ GT was removed from the reaction by filtration using a YM3 Microcon membrane (Millipore, Billerica, MA).
  • CAO 4-(N-(S-cysteinylacetyl)amino)phenylarsinous acid
  • the concentrations of the metabolites were measured by titrating with dimercaptopropanol and calculating the remaining free thiols with 5,5'-dithiobis(2-nitrobenzoic acid) (Don et al, 2003).
  • the titrated solutions were sterile filtered and stored at 4 0 C in the dark until use. There was no significant loss in the active concentration of stock solutions of the arsenicals for at least a week when stored under these conditions.
  • GSAO and metabolites were characterized by HPLC (1200 Series; Agilent Technologies, Santa Clara, CA). Samples were resolved on a Zorbax Eclipse XDB-Cl 8 column (4.6 x 150 mm, 5 ⁇ m; Agilent Technologies) using a mobile phase of acetonitrile- water (25:75 vol/vol), flow rate of 0.5 ml.min "1 and detection by absorbance at 256 nrn. Purity of GSAO, GCAO and CAO by peak area was 94 ⁇ 1%, 69 ⁇ 3% and 84 ⁇ 4%, respectively.
  • Masses of the compounds were determined using ESI-MS. Spectra were acquired using an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City, CA). Samples ( ⁇ 1 pmol) were loaded into nanospray needles (Proxeon, Denmark) and the tip positioned ⁇ 10 mm from the orifice. Nitrogen was used as curtain gas and a potential of 900 V applied to the needle. A Tof MS scan was acquired (m/z 200- 2000, 1 s) and accumulated for ⁇ 1 min into a single file.
  • 1.6 xlO 6 or 7.5 xlO 5 BAE cells were seeded in petri dishes or 6-well-plates, respectively, and allowed to attach overnight. The medium was replaced and the cells were incubated for 30 min in the absence or presence of acivicin or 4H10. The cells were then incubated with 50 or 100 ⁇ M GSAO, GCAO or CAO. Cells were then washed twice with ice-cold PBS and lysed with 1 ml of 70% w/w nitric acid. Lysates were diluted 30-fold and analyzed for arsenic atoms using an Elan 6100 Inductively Coupled Plasma Spectrometer (Perkin Elmer Sciex Instruments, Shelton, CT).
  • Mitochondria were isolated from the livers of 250 g male Wistar rats using differential centrifugation as described previously (Dilda et al, 2005a). The final mitochondrial pellet was resuspended in 3 mM Hepes-KOH, pH 7.0 buffer containing 213 mM mannitol, 71 mM sucrose and 10 mM sodium succinate at a concentration of 30 mg of protein per mL.
  • Mitochondrial permeability transition induction was assessed spectrophotometrically by suspending the liver mitochondria at 0.5 mg of protein per mL at 37°C in 3 mM Hepes-KOH, pH 7.0 buffer containing 75 mM mannitol, 250 mM sucrose, 10 mM sodium succinate, and 2 ⁇ M rotenone (Dilda et al., 2005a). Swelling was measured by monitoring the associated decrease in light scattering at 520 nm using a Molecular Devices M2 Microplate Reader (Palo Alto, CA).
  • Results are presented as means ⁇ SE. All tests of statistical significance were two- sided and P values ⁇ 0.05 were considered statistically significant.
  • Example 2 Results
  • Inhibition of ⁇ GT by GSAO was measured from the effect of fixed concentrations of GSAO on the initial velocity of hydrolysis of ⁇ -glutamyl-p-nitroanilide by ⁇ GT.
  • the apparent K; for competitive inhibition of ⁇ GT by GSAO was estimated to be 1.6 ⁇ 0.2 mM, which is similar to the apparent K m for hydrolysis of the chromogenic substrate, ⁇ - glutamyl-p-nitroanilide (2.4 ⁇ 0.4 mM) (Fig. 2D). This finding implies that the phenylarsinous acid moiety does not denionstrably affect access of ⁇ GT to the glutathione pendant of GSAO.
  • the OATP family is involved in endothelial cell uptake of the product of GSAO cleavage by ⁇ GT, GCAO.
  • GCAO was produced by cleaving the ⁇ -glutamyl group from GSAO with porcine kidney ⁇ -glutamyl transpeptidase. The enzyme was removed from the reactions by size- exclusion filtration and GCAO analyzed by HPLC. GSAO and GCAO have retention times of 3.03 and 3.38 min respectively (Fig. 3A).
  • the organic anion transporting polypeptide (OATP) family of transporters function independently of ATP and sodium gradients and were originally characterized as uptake transporters (Hagenbuch et al., 2003). OATP family members have been implicated in transport of glutathione-S- conjugates (Kobayashi et al., 2003), so they were candidate transporters for GCAO.
  • OATP organic anion transporting polypeptide
  • DIDS 4,4'- Diisothiocyanostilbene-2,2'-disulfonic acid
  • GCAO accumulates more rapidly in endothelial cells and has greater anti-proliferative activity than GSAO.
  • GCAO accumulated in BAE cells at a 8.7-fold faster rate than GSAO (Fig. 3B).
  • the rate of accumulation of GCAO was 3.8 ⁇ 0.1 pmol As atoms per 10 6 cells per min, compared to 0.4 ⁇ 0.1 pmol As atoms per 10 6 cells per min for GSAO.
  • the faster rate of accumulation of GCAO corresponded to a ⁇ 5-fold increased anti-proliferative activity.
  • the IC 50 for GSAO and GCAO in a 24 h BAE cell proliferation assay was 97 ⁇ 5 ⁇ M and 18 ⁇ 3 ⁇ M, respectively (Fig. 3C).
  • GCAO is secreted from endothelial cells by MRPl.
  • GCAO accumulation in cells is controlled by rate of export by MRPl and MRP2 (Dilda et al., 2005b).
  • MRPl rate of export
  • MRP2 MRP2
  • the effect of the MRPl inhibitor 4H10 on accumulation in endothelial cells was measured.
  • Cellular accumulation of GCAO was increased 3 -fold when MRPl was inhibited (Fig. 4A), which correlated with more potent anti-proliferative effect (Fig. 4B). The inhibitor alone had no effect on BAE cell proliferation (data not shown).
  • GCAO triggers the mitochondrial permeability transition more rapidly than GSAO.
  • GSAO inactivates the mitochondrial inner membrane transporter adenine nucleotide translocase (ANT), which leads to proliferation arrest and cell death (Don et al., 2003).
  • GCAO also induces the mitochondrial permeability transition (Fig. 4C).
  • Comparison of the time for 25% maximal swelling as a function GSAO or GCAO concentration indicates that GCAO is approximately twice as efficacious as GSAO at triggering the permeability transition (Fig. 4D). This finding indicates that an intact glutathione pendant is not necessary for GSAO to inactivate ANT.
  • GCAO and its metabolite, CAO accumulate in endothelial cells at the same rate and have comparable anti-proliferative activity.
  • CAO was produced by cleaving the glycine amino acid from GCAO with porcine kidney aminopeptidase N (Fig. 5A). The enzyme was removed from the reactions by size- exclusion filtration and the product of the reaction was analyzed by HPLC (Fig. 5B).
  • CAO has a retention time of 3.64 min.
  • the rate of accumulation of CAO in BAE cells was 3.3 ⁇ 0.1 pmol As atoms per
  • Fig. 5C The similar rate of accumulation of the two compounds corresponded to a comparable anti-proliferative activity.
  • the IC 50 for GCAO and CAO in a 48 h BAE cell proliferation assay was 9 ⁇ 2 ⁇ M and 11 ⁇ 2 ⁇ M, respectively (Fig. 5D).
  • CAO is secreted from endothelial cells by MRPl.
  • CAO triggers the mitochondrial permeability transition more rapidly than GCAO.
  • CAO Like GSAO and GCAO, CAO also induces the mitochondrial permeability transition (Fig. 6B). Comparison of the time for 25% maximal swelling as a function GCAO or CAO concentration indicates that CAO is approximately twice as efficacious as GCAO at triggering the permeability transition (Fig. 6C).
  • GSAO is a substrate for ⁇ GT ⁇ GT is present on the outer surface of the plasma membrane (Horiuchi et al., 1978) of virtually all cells.
  • the enzyme catalyses hydrolysis of the bond linking the glutamate and cysteine residues of extracellular glutathione and glutathione-S-conjugates (Enoiu et al., 2002).
  • GSAO is effectively a glutathione-S-conjugate of aminophenylarsonous acid. It has been demonstrated in several ways that GSAO is a substrate for ⁇ GT. First, GSAO is an efficient substrate for isolated ⁇ GT.
  • glycine-cysteine peptide bond of GCAO is readily cleaved by dipeptidases to produce
  • CAO accumulated in endothelial cells at the same rate as GCAO and had comparable anti-proliferative activity. Like GSAO and GCAO, CAO was also a substrate for MRPl in endothelial cells.
  • Fig. 2 The findings shown in Fig. 2 indicate that there is a degree of association between the susceptibility of cells to proliferation arrest by GSAO and the expression level of ⁇ GT. Metabolism of GSAO by tumor cell and/or tumor endothelium ⁇ GT would produce high local concentrations of GCAO that will then block tumor angiogenesis and tumor growth. Notably, tumors of the breast, prostate, colon, liver, ovary, lung, kidney, thyroid and pancreas express ⁇ GT (Hannigan et al., 1996).
  • Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20, 7579-7587.

Abstract

The present invention relates to organic arsenoxide compounds, pharmaceutical compositions comprising these compounds, and to their use in therapy, in particular treatment of proliferative diseases.

Description

Arsenoxide Compound and Method of Use
Technical Field
The present invention relates to organic arsenoxide compounds, pharmaceutical compositions comprising these compounds, and to their use in therapy, in particular, treatment of proliferative diseases.
Background
Arsenical compounds have been used in the past as therapeutic agents for the treatment of disease. However, the inherent toxicities of arsenical compounds and their generally unfavourable therapeutic index have largely precluded their use as pharmaceutical agents. Organic arsenoxide compounds are disclosed in WO 01/21628. Such compounds are described as having antiproliferative properties useful in the therapy of proliferative diseases. WO 04/042079 discloses the use of organic arsenoxide compounds for inducing the mitochondrial permeability transmission (MPT) and also the use of such compounds for inducing apoptosis and necrosis, particularly in endothelial cells.
The compound 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid ("GSAO") is a synthetic tripeptide trivalent arsenical compound disclosed in WO 01/21628 and WO 04/042079. GSAO perturbs the mitochondria in angiogenic endothelial cells, leading to proliferation arrest and cell death (WO 04/042079). GSAO inactivates the mitochondrial inner membrane transporter, adenine nucleotide translocase (ANT), by cross-linking two of the three matrix facing thiols (WO 04/042079). ANT exchanges matrix ATP for cytosolic ADP across the inner-mitochondrial membrane and is the key component of the mitochondrial permeability transition pore (Belzacq et al., 2001; Larochette et al., 1999).
Inactivation of ANT by GSAO causes an increase in superoxide levels, proliferation arrest, ATP depletion, mitochondrial depolarization and apoptosis in endothelial cells (WO 04/042079). The strong selectivity of GSAO for proliferating endothelial cells is a consequence of the higher mitochondrial calcium levels in proliferating cells (Don et al., 2003). ANT is a calcium receptor which undergoes a conformational change and a change in activity upon binding of calcium ions. GSAO binds to calcium-replete ANT, but binds minimally in the absence of calcium ions. Calcium content within the mitochondrial matrix increases several-fold in proliferating cells, and it is believed to be the higher mitochondrial calcium concentration that sensitises proliferating cells to GSAO-induced pore formation. Tumour growth and metastasis is dependent on tumour blood vessel formation, or angiogenesis. Proper functioning of ANT is essential for cell viability, so targeting this protein in angiogenic endothelial cells may be a powerful means of inhibiting angiogenesis.
5 GSAO is a selective inhibitor of endothelial cell proliferation compared to tumour cells, with IC5O values for proliferation arrest up to 30-fold higher in tumour cells. This is because endothelial cells are very poor at exporting GSAO, while the multidrug resistance-associated proteins (MRP) 1 and 2 in tumour and other cells may efficiently pump GSAO out the cytoplasm (Dilda et al., 2005b). In contrast, endothelial cells i o express very little MRP 1 /2.
There is a need for alternative therapies for treating proliferative diseases, such as cancer, and related conditions.
The present invention relates to organic arsenoxide compounds comprising a peptidic residue linked via an amide linking moiety to a phenylarsenoxide residue. is Organic arsenoxide compounds according to the present invention may be capable of crossing cell membranes, in particular, endothelial cell membranes and may be useful for the treatment of disease, in particular, treatment of proliferative disease.
Summary
20 In a first aspect the present invention relates to organic arsenoxide compounds of general formula (I):
R1 [AA-2]n
[AA-3]p
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; s R1 is selected from hydrogen, Ci_3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted Ci-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3; 0 [AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and α-amino-β-hydroxy-isovaleric acid; [AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; s [AA-3] (when present) is a γ-glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof.
In a second aspect the present invention relates to compounds of general formula io (I):
Figure imgf000004_0001
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; I5 R1 is selected from hydrogen, C1-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3; 0 [AA-I] is an amino acid residue selected from cysteine and serine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; 5 [AA-3] (when present) is a γ-glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof. 0 In a third aspect the present invention relates to compounds of general formula (I):
Figure imgf000005_0001
(I) wherein s the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring;
R1 is selected from hydrogen, Ci-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted Ci-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; o m is an integer selected from 1, 2 and 3;
[AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and α-amino-β-hydroxy-isovaleric acid;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine,s tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is O; and salts and hydrates thereof. 0 In a fourth aspect the present invention relates to compounds of general formula (I):
Figure imgf000005_0002
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring;S R1 is selected from hydrogen, Ci-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted Ci-3 alkoxy; m is an integer selected from 1, 2 and 3;
[AA-I] is an amino acid residue selected from cysteine and serine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is 0; and salts and hydrates thereof. In a fifth aspect the present invention relates to a pharmaceutical composition comprising a compound of formula (I) according to the first, second, third or fourth aspect of the invention, together with a pharmaceutically acceptable excipient, diluent or adjuvant.
In another aspect the present invention relates to a method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
In a further aspect the present invention relates to a method of inhibiting angiogenesis in a vertebrate, comprising administering to the vertebrate an effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention.
In another aspect the present invention relates to a method of inducing the Mitochondrial Permeability Transition (MPT) in a vertebrate comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention. The compound of formula (I) may selectively induce the MPT in proliferating endothelial cells.
In a further aspect the present invention relates to a method of inducing apoptosis or necrosis in proliferating mammalian cells, comprising administering to the mammal an apoptosis- or necrosis-inducing amount of a compound of formula (I) according to the first, second, third or fourth aspect of the invention, or a composition according to the fifth aspect of the invention. The cells may be proliferating endothelial cells. In a further aspect the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for treating a proliferative disease in a vertebrate.
In another aspect the present invention relates to the use of a compound of formula 5 (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inhibiting angiogenesis in a vertebrate.
In yet another aspect the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inducing the MPT in a vertebrate. The compound ofo formula (I) may selectively induce the MPT in proliferating endothelial cells.
In a further aspect the present invention relates to the use of a compound of formula (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for inducing apoptosis or necrosis in proliferating mammalian cells. The cells may be proliferating endothelial cells. s In a further aspect the present invention relates to a method of treating γ-glutamyl transferase expressing tumours in a vertebrate, comprising administering to the vertebrate a compound of formula (I) according to the first, second, third or fourth aspect of the invention or a composition according to the fifth aspect of the invention.
In a further aspect the present invention relates to the use of a compound of formula0 (I) according to the first, second, third or fourth aspect of the invention in the manufacture of a medicament for treating γ-glutamyl transferase expressing tumours in a vertebrate.
Abbreviations
GSAO, 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid s ANT, Adenine nucleotide translocase
MPT, Mitochondrial permeability transmission
MRP, Multidrug resistance-associated proteins
BAE, Bovine aortic endothelial
GCAO, 4-(N-(S-cysteinylglycylacetyl)amino)phenylarsinous acid 0 CAO, 4-(N-(S-cysteinylacetyl)amino)phenylarsinous acid
OATP, Organic anion transporting polypeptide
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
ABBA, L-2-amino-4-boronobutanoic acid γGT, γ-glutamyl transferase S DMEM, Dulbecco's Modified Eagle's Medium MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Definitions
The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
As used herein, the term "C1-3 alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 3 carbon atoms. Thus, for example, the term C1-3 alkyl includes methyl, ethyl, 1 -propyl, and isopropyl.
The term "C2-3 alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 3 carbon atoms and at least one double bond anywhere in the chain. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis or trans, or E or Z as appropriate. Examples of alkenyl groups include ethenyl, vinyl, allyl, 1-methylvinyl, 1-proρenyl, and 2-ρroρenyl.
The term "C2-3 alkynyl group" as used herein includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") unsaturated aliphatic hydrocarbon groups having from 2 to 3 carbon atoms and having at least one triple bond. Examples of alkynyl groups include but are not limited to ethynyl, 1-propynyl.
The term "alkoxy" as used herein refers to straight chain or branched alkyloxy (i.e, O-alkyl) groups, wherein alkyl is as defined above. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, and isopropoxy.
The term "amino" as used herein refers to groups of the form -NRaRb wherein Ra and Rb are individually selected from hydrogen, optionally substituted (C1-4)alkyl, optionally substituted (C2-4)alkenyl, optionally substituted (C2-4)alkynyl, optionally substituted (C6-io)aryl and optionally substituted aralkyl groups, such as benzyl. The amino group may be a primary, secondary or tertiary amino group.
The term "amino acid" as used herein includes naturally and non-naturally occurring amino acids, as well as substituted variants thereof. The term "amino acid" therefore encompasses, for example, α, β, and γ-amino acids. α-Amino acids are particularly preferred. The (L) and (D) forms of amino acids are also included in the scope of the term "amino acid". (L)-amino acids are a preferred form. For example, the term "amino acid" includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, α-amino-β- hydroxy-isovaleric acid and penicillamine. The backbone of the amino acid residue may be substituted with one or more groups independently selected from (C1-6)alkyl, halogen, hydroxy, hydroxy(C1-6)alkyl, aryl, e.g., phenyl, aryl(C1-3)alkyl, e.g., benzyl, and (C3- 6)cycloalkyl.
The term "C6-10 aryl" or variants such as "arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of aromatic groups include phenyl, and naphthyl.
The term "arylalkyl" or variants such as "aralkyl" as used herein, includes within its meaning monovalent ("aryl") and divalent ("arylene"), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight or branched chain alkylene radicals. Examples of arylalkyl groups include benzyl.
In the context of this specification the term "arsenoxide" is synonymous with "arsinous acid" and refers to the moiety As(OH)2, which may also be represented as As=O.
The term "C3-8 heterocycloalkyl" as used herein, includes within its meaning monovalent ("heterocycloalkyl") and divalent ("heterocycloalkylene"), saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 8 ring atoms, wherein from 1 to 5, or from 1 to 3, ring atoms are heteroatoms independently selected from O, N, NH, or S. The heterocycloalkyl group may be C3-6 heterocycloalkyl. The heterocycloalkyl group may be C3-5 heterocycloalkyl. Examples of heterocycloalkyl groups include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.
The term "C5-10 heteroaromatic group" and variants such as "heteroaryl" or "heteroarylene" as used herein, includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), single, polynuclear, conjugated and fused aromatic radicals having from 5 to 10 atoms, wherein 1 to 4 ring atoms, or 1 to 2 ring atoms are heteroatoms independently selected from O, N, NH and S. The heteroaromatic group may be C5-8 heteroaromatic. Examples of heteroaromatic groups include pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl, pyrrolyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, and the like.
The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine.
The term "heteroatom" or variants such as "hetero-" or "heterogroup" as used herein refers to O, N, NH and S. The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, haloalkyl, haloalkynyl, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, NO2, NRaRb, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl, alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylaryl, aralkyl, alkylheteroaryl, cyano, cyanate, isocyanate, CO2H, C02alkyl, C(O)NH2, -C(O)NH(alkyl), and -C(O)N(alkyl)2. Preferred substituents include C1-3 alkyl, Ci-3 alkoxy, -CH2-(Ci_3)alkoxy, C6-10 aryl, -CHa-phenyl, halo, hydroxyl, hydroxy(C1-3)alkyl (e.g., CH2OH), and halo(C1-3)alkyl (e.g, CF3, CH2CF3). Particularly preferred substituents include C1-3 alkyl, C1-3 alkoxy, halo, hydroxyl, hydroxy(C1-3)alkyl (e.g, CH2OH), and halo(C1-3)alkyl (e.g, CF3, CH2CF3). In the context of this specification the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
5 In the context of this specification, the term "vertebrate" includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian. The vertebrate may be a human. o In the context of this specification, the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
In the context of this specification the term "effective amount" includes within itss meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide a desired effect. Thus, the term "therapeutically effective amount" includes within its meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the sex,0 age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation. S
Brief Description of the Figures
Figure 1. Inhibition of cell-surface γGT blunts cellular accumulation of GSAO and antiproliferative activity. A. Structure of GSAO and the product of γGT cleavage, GCAO. γGT catalyses the hydrolysis of the bond between GSAO' s glutamate and cysteine0 residues. B. Inhibition of GSAO accumulation in endothelial cells by reduced glutathione. BAE cells were incubated without or with 250 μM of reduced glutathione and 100 μM GSAO for 4 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. Values are the mean ± SD of three determinations. The results are representative of three experiments. C. Inhibition of GSAO accumulation in endothelialS cells by acivicin. BAE cells were incubated without or with 500 μM acivicin for 30 min prior to addition of 100 μM GSAO for 4 h. Cytosolic arsenic was measured by inductively coupled plasma spectrometry. Values are the mean ± SD of three determinations. The results are representative of three experiments. D. The γGT inhibitor, acivicin, blunts GSAO anti-proliferative activity in endothelial cells. BAE cells were incubated with 0.3 μM acivicin for 30 min and then with 40 μM GSAO for 24 h. Cell viability was determined using MTT and results expressed as percentage of untreated control. Values are mean ± SD of triplicate determinations. Results are representative of three experiments. ***: pO.OOl, **: pO.Ol, *: ρ<0.05.
Figure 2. Expression level of γGT positively correlates with sensitivity of cells to GSAO. A and B. c21/γGT (high γGT) or c21 /basal (low γGT) melanoma cells were incubated with GSAO (0-1.8 mM) for 24 h (part A) or 72 h (part B) and cell viability was determined using WST-I. Results are expressed as percentage of untreated controls. Data points are the mean ± SD of five determinations. Results are representative of two experiments. C. The potent γGT inhibitor, ABBA, blunts GSAO anti-proliferative activity in the melanoma cells. c21/γGT (high γGT) or c21/basal (low γGT) melanoma cells were incubated for 24 h in the absence or presence of 10 μM ABBA and a GSAO concentration representing the IC50 for proliferation arrest (60 μM for high and 1250 μM for low γGT cells). Cell viability was determined using WST-I and results expressed as percentage of untreated controls. Data points are the mean ± SD of five determinations. Results are representative of two experiments. D. K; for inhibition of γGT by GSAO. Inhibition of γGT by GSAO was measured from the effect of fixed concentrations of GSAO (0-4 mM) on the initial velocity of hydrolysis of γ-glutamyl-p-nitroanilide (0-2.5 mM) by γGT (10 Units per mL). Glycyl-glycine concentration was 40 mM. The solid lines represent the best global fit of the data to simple competitive inhibition, with apparent Km, Vmax and K; values of 2.4 ± 0.4 mM, 4.5 ± 0.5 μM per s and 1.6 ± 0.2 mM, respectively. Results are representative of two experiments. ***: p<0.001
Figure 3. The GSAO metabolite. GCAO. enters endothelial cells via the OATP. accumulates more rapidly in the cytosol and has greater anti-proliferative activity than GSAO. A. Cl 8 reverse phase HPLC analysis of 5 nmol of GSAO and GCAO. B. GCAO accumulates in cells at a much faster rate than GSAO. BAE cells were incubated with 50 μM GSAO or GCAO for up to 4 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. The rates of accumulation of GSAO and GCAO are 0.03 and 0.23 nmol As atoms per 106 cells, respectively. Data points are the mean ± SD of three determinations and the solid line is the linear least squares fit of the data. The results are representative of two experiments. C. GSAO and GCAO IC5O values for proliferation arrest of endothelial cells. BAE cells were incubated with 0.8-100 μM GSAO or GCAO for 24, 48 or 72h. Cell viability determined using MTT and results expressed as percentage of untreated control. Values are mean ± SD of triplicate determinations. Results are representative of three to five experiments. ***: p<0.001. D An OATP blocker blunts GCAO anti-proliferative activity in BAE cells. BAE cells were incubated for 24 h with 20 μM GCAO in the presence or absence of 200-500 μM of DIDS. Cell viability was determined using MTT and results expressed as percentage of untreated control. Values are mean ± SD of triplicate determinations. Results are representative of three experiments.
Figure 4. GCAO is secreted from endothelial cells by MRPl and is more efficient than GSAO at triggering the mitochondrial permeability transition. A. GCAO is secreted from cells by MRPl. BAE cells pretreated for 30 min with 10 μM 4H10 were incubated with 50 μM GCAO for 2 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. Data points are the mean ± SD of three determinations. The results are representative of two experiments. ***: p<0.001. B. Inhibition of MRPl enhances GCAO's antiproliferative activity. BAE cells were incubated with 0.4-50 μM GCAO for 24 h in the absence or presence of 5 μM 4H10. Cell viability was determined using MTT and results expressed as percentage of untreated control. Values are mean ± SD of triplicate determinations. Results are representative of two experiments. C. Mitochondrial swelling induced by 0-300 μM GCAO as measured by decrease in light scattering at 520 nm over 60 min. The traces are representative of a minimum of two experiments performed in duplicate on two different mitochondrial preparations. D. Time for 25% maximum swelling as a function of GSAO or GCAO concentration. The data points are the average from two separate experiments.
Figure 5. The GCAO metabolite, CAO, accumulates in the endothelial cells at a similar rate and has comparable anti-proliferative activity to GCAO. A. Cleavage of GCAO by dipeptidase to produce CAO. B. Cl 8 reverse phase HPLC analysis of 5 nmol of GCAO and CAO. C. CAO and GCAO accumulate in the cytosol at a similar rate. BAE cells were incubated with 50 μM GCAO or CAO for up to 4 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. The rates of accumulation of GCAO and CAO are 0.23 and 0.20 nmol As atoms per 106 cells, respectively. Data points are the mean ± SD of three determinations and the solid line is the linear least squares fit of the data. The results are representative of two experiments. D. GCAO and CAO IC50 values for proliferation arrest of endothelial cells. BAE cells were incubated with 0.8-100 μM GCAO or CAO for 24, 48 or 72h. Cell viability determined using MTT and results expressed as percentage of untreated control. Values are mean ± SD of triplicate determinations. Results are representative of three to five experiments. ***: p<0.001
Figure 6. CAO is secreted from endothelial cells by MRPl and is more efficient than GCAO at triggering the mitochondrial permeability transition. A. CAO is secreted from cells by MRPl. BAE cells pretreated for 30 min with 10 μM 4H10 were incubated with 50 μM CAO for 2 h and cytosolic arsenic was measured by inductively coupled plasma spectrometry. The values are the mean ± SD of three determinations. The results are representative of two experiments. B. Mitochondrial swelling induced by 0-300 μM CAO as measured by decrease in light scattering at 520 ran over 60 min. The traces are representative of a minimum of two experiments performed in duplicate on two different mitochondrial preparations. C. Time for 25% maximum swelling as a function of GCAO or CAO concentration. The data points are the average from two separate experiments.
Figure 7. Metabolism of GSAO by endothelial cells. The γ-glutamyl residue of GSAO is cleaved at the cell surface by γ-GT to produce GCAO, which is then transported across the plasma membrane by an OATP. GCAO may be further processed to CAO before it reacts with ANT of the inner-mitochondrial membrane.
Detailed Description
The present invention relates to organic arsenoxide compounds comprising a pendant group linked via an amide linking moiety to a phenylarsenoxide residue. Organic arsenoxide compounds according to the present invention may exhibit increased cellular uptake and/or increased efficacy and/or an improved therapeutic index relative to known arsenoxide compounds, such as, the compound 4-(N-(S-glutathionylacetyl)amino)- phenylarsinous acid ("GSAO").
In particular, the present invention relates to organic arsenoxide compounds of general formula (I): R1 [AA-2]n
[AA-3]p
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R1 is selected from hydrogen, Ci-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1 , 2 and 3; [AA-I] is an amino acid residue selected from cysteine, serine, penicillamine, threonine and α-amino-β-hydroxy-isovaleric acid;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine;
[AA-3] (when present) is a γ-glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof. The present invention further relates to organic arsenoxide compounds of general formula (I):
Figure imgf000015_0001
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring;
R1 is selected from hydrogen, C1-3 alkyl and cyclopropyl; R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3; [AA-I] is an amino acid residue selected from cysteine and serine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; [AA-3] (when present) is a γ- glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof.
The present invention further relates to organic arsenoxide compounds of general formula (I):
Figure imgf000016_0001
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R1 is selected from hydrogen, C1-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3; [AA-I] is an amino acid residue selected from cysteine serine, penicillamine, threonine and α-amino-β-hydroxy-isovaleric acid;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; n is 0 or 1 ; p is O; and salts and hydrates thereof.
Embodiments of the compounds of general formula (I) are described below. It should be understood that any one or more of the embodiment(s) may be combined with any other embodiment(s) as appropriate.
Optional substirueiits may be independently selected from C1-3 alkyl, C1-3 alkoxy,
-CH2-(C1-3)alkoxy, C6-10 aryl, -CH2-phenyl, halo, hydroxyl, hydroxy(C1-3)alkyl, and halo-
(Ci-3)alkyl, e.g., CF3, CH2CF3. In one embodiment the optional substituents are independently selected from hydroxyl, methoxy, halo, and (C1-3)alkyl. In one embodiment there are no optional substituents.
The As(OH)2 group may be ortho- or para- to the N-atom on the phenyl ring. In one embodiment, the As(OH)2 group is para- to the N-atom on the phenyl ring. In another embodiment the As(OH)2 group is ortho- to the N-atom on the phenyl ring.
R1 may be hydrogen, methyl or ethyl. In one embodiment R1 is hydrogen.
0 1 0 1 R and R may be the same or different. In one embodiment R and R may be independently selected from hydrogen, C1-3 alkyl, C2-3 alkenyl, C1-3 alkoxy, halo- (C1-3)alkoxy, hydroxy(Ci-3)alkyl and halo(Ci-3)alkyl. In another embodiment R2 and R3 may be independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, hydroxymethyl, CF3 and OCF3. In another embodiment R2 and R3 may be independently selected from hydrogen, methyl and ethyl. In another embodiment R is methyl and R is hydrogen. In a further embodiment R2 and R3 are both methyl. In another embodiment R2 and R3 are both hydrogen.
In one embodiment m is 1. In another embodiment m is 2.
The pendant group "[AA-l]-[AA-2]-[AA-3]" may comprise a single amino acid, a dipeptide, or tripeptide. In one embodiment the pendant group is a single amino acid residue selected from cysteine and serine. In another embodiment the pendant group is cysteine.
In another embodiment the pendant group is a dipeptide comprised of cysteine and a second amino acid residue. In a further embodiment the pendant group is a dipeptide residue comprised of serine and a second amino acid residue. In a further embodiment the pendant group is a dipeptide comprised of penicillamine and a second amino acid group. In a further embodiment the pendant group is a dipeptide comprised of threonine and a second amino acid group. In a further embodiment the pendant group is a dipeptide comprised of α-amino-β-hydroxy-isovaleric acid and a second amino acid group. The second amino acid residue may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine.
In another embodiment the pendant group is a tripeptide comprised of cysteine, a second amino acid residue and a γ-glutamic acid residue. In a further embodiment the pendant group is a tripeptide comprised of serine, a second amino acid residue and a γ-glutamic acid residue. In a further embodiment the pendant group is a tripeptide comprised of penicillamine, a second amino acid group and a γ-glutamic acid residue. In a further embodiment the pendant group is a tripeptide comprised of threonine, a second amino acid group and a γ-glutamic acid residue, hi a further embodiment the pendant group is a tripeptide comprised of α-amino-β-hydroxy-isovaleric acid, a second amino acid group and a γ-glutamic acid residue. The second amino acid residue may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine. The γ-glutamic acid residue is attached via its γ-COOH group.
[AA-2] and [AA-3] may be attached to the cysteine, serine, threonine, α-amino-β- hydroxy-isovaleric acid or penicillamine residue at the amino (NH2) and/or acid (CO2H) grouρ(s) of the cysteine, serine, threonine, α-amino-β-hydroxy-isovaleric acid or penicillamine residue. In one embodiment, when the pendant group is a tripeptide, the arrangement of amino acids is -[AA-I] -[AA-2]- [AA-3] as shown below:
Figure imgf000018_0001
In another embodiment the arrangement of amino acids is [ AA-2]-[ AA- 1]-[ AA-3], where [AA-I] is attached to the (CR2R3)m group, i.e., where [AA-I] is the central amino acid of the tripeptide. In another embodiment, the arrangement of amino acids is [AA-3]-
[AA-I H AA-2], where [AA-I] is attached to the (CR2R3)ra group, i.e., where AA-I is the central amino of the tripeptide.
In one embodiment the [AA-I] amino acid is an (L) or (D) form of an amino acid.
In another embodiment [AA-I] is an (L)-amino acid. In one embodiment the [AA-2] amino acid is in the (L) or (D) form of the amino acid. In another embodiment the [AA-2] amino acid is an (L)-amino acid. In one embodiment the [AA-3] amino acid is in the (L) or (D) form of the amino acid. In another embodiment the [AA-3] amino acid is an (L)- amino acid. Compounds of formula (I) according to the present invention having a terminal γ- glutamyl residue may have the γ-glutamyl residue cleaved from the compound by cell- surface γ-glutamyltransferase (γ-GT) (also known as γ-glutamyltranspeptidase; E.C 2.3.2.2). The resultant compound (i.e., without the γ-glutamyl residue) may then be taken 5 up by cells. In a specific embodiment of the invention, the γ-glutamyl residue of GSAO may be cleaved by cell-surface γ-GT to give the compound 4-(N-(S- cysteinylglycylacetyl)amino)-phenylarsinous acid (GCAO), which is transported into the cell cytoplasm.
In one embodiment of the invention n is 1 and p is 1. In another embodiment, n is 1o and p is O. In a further embodiment n is 0 and p is 0.
In one embodiment R1, R2 and R3 are hydrogen; m is 1; n is 0; p is 0; [AA-I] is cysteine and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are independently hydrogen or methyl; m is 1s or 2; n is 1; p is 0; [AA-I] is cysteine, serine, threonine, α-amino-β -hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. 0 In another embodiment R1, R2 and R3 are independently hydrogen or methyl; m is 1 or 2; n is 1; p is 0; [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. 5 In another embodiment R1, R2 and R3 are each hydrogen; m is 1 or 2; n is 1; p is 0;
[AA-I] is cysteine, serine, threonine, α-amino-β-hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. o In another embodiment R1, R2 and R3 are each hydrogen; m is 1 or 2; n is 1; p is 0;
[AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA-5 1] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is threonine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is α-amino-β-hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA-
1] is cysteine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R , R and R are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA- 1] is threonine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is α-amino-β-hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R , R and R are each hydrogen; m is 1 ; n is 1 ; p is 0; [AA- 1] is cysteine; [AA-2] is glycine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is penicillamine; [AA-2] is glycine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA- 1] is threonine; [AA-2] is glycine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment R1, R2 and R3 are each hydrogen; m is 1; n is 1; p is 0; [AA-
5 1] is α-arnino-β-hydroxy-isovaleric acid; [AA-2] is glycine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In various embodiments of the invention the pendant group may be a dipeptide. Examples of suitable dipeptide residues include cysteine-glycine; cysteine-alanine; cysteine-valine; cysteine-leucine; cysteine-serine; cysteine-threonine; cysteine-cysteine; io cysteine-aspartic acid; cysteine-glutamic acid; cysteine-lysine; and cysteine-arginine. In other embodiments, the dipeptide residue may be serine-glycine; serine-alanine; serine- valine; serine-leucine; serine-serine; serine-threonine; serine-cysteine; serine-aspartic acid; serine-glutamic acid; serine-lysine; and serine-arginine. In other embodiments, the dipeptide residue may be penicillamine-glycine; penicillamine-alanine; penicillamine-
I5 valine; penicillamine-leucine; penicillamine-serine; penicillamine-threonine; penicillamine-cysteine; penicillamine-aspartic acid; penicillamine-glutamic acid; penicillamine-lysine; and penicillamine-arginine. In other embodiments, the dipeptide residue may be threonine-glycine; threonine-alanine; threonine-valine; threonine-leucine; threonine-serine; threonine-threonine; threonine-cysteine; threonine-aspartic acid;
20 threonine-glutamic acid; threonine-lysine; and threonine-arginine. hi other embodiments, the dipeptide residue may be α-amino-β-hydroxy-isovaleric acid-glycine; α-amino-β- hydroxy-isovaleric acid-alanine; α-amino-β-hydroxy-isovaleric acid-valine; α-amino-β- hydroxy-isovaleric acid-leucine; α-amino-β-hydroxy-isovaleric acid-serine; α-amino-β- hydroxy-isovaleric acid-threonine; α-amino-β-hydroxy-isovaleric acid-cysteine; α-
25 amino-β-hydroxy-isovaleric acid-aspartic acid; α-amino-β-hydroxy-isovaleric acid- glutamic acid; α-amino-β-hydroxy-isovaleric acid-lysine; and α-amino-β-hydroxy- isovaleric acid-arginine. In other embodiments, the dipeptide residue may be cysteine- glycine; cysteine-alanine; cysteine-valine; cysteine-serine; cysteine-cysteine; cysteine- aspartic acid; cysteine-glutamic acid; or cysteine-lysine. In other embodiments the 0 dipeptide residue may be serine-glycine; serine-alanine; serine-valine; serine-serine; serine-cysteine; serine-aspartic acid; serine-glutamic acid; or serine-lysine. In other embodiments the dipeptide residue may be cysteine-glycine; cysteine-alanine; cysteine- serine; cysteine-aspartic acid; or cysteine-glutamic acid, hi other embodiments the dipeptide residue may be serine-glycine; serine-alanine; serine-serine; serine-aspartic s acid; or serine-glutamic acid, hi other embodiments the dipeptide residue may be cysteine-glycine; cysteine-alanine; cysteine-aspartic acid; or cysteine-glutamic acid. In other embodiments the dipeptide residue may be serine-glycine; serine-alanine; serine- aspartic acid; or serine-glutamic acid. In other embodiments, the dipeptide residue may be cysteine-glycine or cysteine-alanine. In other embodiments, the dipeptide residue may
5 be serine-glycine or serine-alanine. In other embodiments the dipeptide residue may be cysteine-glycine or serine-glycine.
In other embodiments of the invention the pendant group may be a tripeptide, [AA- l]-[AA-2]-[AA-3], wherein the moiety [AA-l]-[AA-2] is any one of the foregoing dipeptide residues and [AA-3] is a γ-glutamic acid residue. The γ-glutamic acid residueQ may be attached (via the γ-COOH group) to either the [AA-I] or [AA-2] amino acid residue.
In another embodiment, R1, R2 and R3 are independently hydrogen or methyl; n is 1, p is 1; [AA-I] is cysteine, serine, threonine, α-amino-β-hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine,s methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R , R and R are independently hydrogen or methyl; n is 1, p is 1; [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine,Q leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine,S cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is γ-glutamic acid; and0 the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is α-amino-β-hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine;
[AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom
5 attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. Q In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] iss threonine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is α-amino-β-hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, valine,Q threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is γ- glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the5 phenyl ring.
In another embodiment, R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is γ- glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
1 0 1 0 In another embodiment, R , R and R are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is γ- glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] isS α-amino-β-hydroxy-isovaleric acid; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is serine; [AA-2] is glycine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is penicillamine; [AA-2] is glycine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is threonine; [AA-2] is glycine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
In another embodiment, R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is α-amino-β-hydroxy-isovaleric acid; [AA-2] is glycine; [AA-3] is γ-glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring. Cytoplasmic levels of compounds of formula (I) may be controlled by export from the cell by multidrug-resistance associated protein-1 (MRP-I). In one embodiment, cytoplasmic levels of GCAO (similar to the compound GSAO) are controlled by export from the cell via the MRP-I protein.
Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of formula (I), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. Also included are acid addition or base salts wherein the counterion is optically active, for example, J-lactate or /-lysine, or racemic, for example, ^/-tartrate or c?/-arginine.
Cist 'trans (E/Z) isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (I) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
Therapeutic Application(s)
Compounds of formula (I) in accordance with the present invention, such as GCAO, and pharmaceutically acceptable salts and hydrates thereof, are capable of binding to cysteine residues of mitochondrial Adenine Nucleotide Translocator (ANT) in proliferating endothelial cells thereby inducing the Mitochondrial Permeability Transition (MPT). Accordingly, compounds of formula (I) according to the present invention may lead to proliferation arrest and cell death. Advantageously, compounds of formula (I) may be selective inhibitors of endothelial cell proliferation. For example, compounds of formula (I) may be selective inhibitors of endothelial cell proliferation compared to tumour cells. Compounds of formula (I) therefore may be useful in the treatment of proliferative diseases. Organic arsenic compounds according to the present invention may have one or more advantage(s) over known arsenical compounds, such as arsenic trioxide and the arsenoxide compounds disclosed in WO 01/21628 or WO 04/042079.
Compounds of formula (I) may exhibit increased cellular uptake relative to the known organic arsenoxide compound 4-(N-(S-glutathionylacetyl)amino)phenylarsinous acid ("GSAO"). For example, compounds of formula (I) may accumulate in endothelial cells at a rate about 20-times, about 15-times, about 10-times, about 5-times, about 2- times faster than GSAO. In an embodiment, compounds of formula (I) accumulate in endothelial cells at a rate about 10-times faster than GSAO. In another embodiment, compounds of formula (I) accumulate in endothelial cells at a rate about 8-times faster than GSAO. In a further embodiment, compounds of formula (I) accumulate in endothelial cells at a rate about 5-times faster than GSAO.
Compounds of formula (I), such as GCAO3 may be more effective than known arsenoxide compounds, including arsenoxide compounds disclosed in WO 01/21628, such as GSAO, at inhibiting cellular proliferation (particularly proliferation of endothelial cells) and/or reducing the viability of endothelial cells, hi the context of this invention, "reducing the viability of endothelial cells" can include cell death, or progression towards cell death. For example, the IC50 values of compounds of formula (I) as a measure of anti-proliferative activity may be about 10-times less than the IC50 of GSAO. In another embodiment, the IC50 of compounds of formula (I) may be about 7.5-times less than the IC5o of GSAO. In a further embodiment, the IC50 of compounds of formula (I) may be about 5-times less than the IC5O of GSAO. In another embodiment, the IC5O of compounds of formula (I) may be about 2-times less than the IC50 of GSAO.
Therefore, in other aspects of the invention compounds of formula (I), such as GCAO, may be useful in the treatment of proliferative diseases. Accordingly, another embodiment of the invention relates to a method of treating a cellular proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
The cells may be endothelial cells. The vertebrate may be a mammal, such as a human.
In another embodiment the present invention relates to a method of inhibiting angiogenesis in a vertebrate, comprising administering to the vertebrate an effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
A further embodiment of the invention relates to a method of inducing the MPT in a vertebrate comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) or a pharmaceutical composition thereof. Compounds of formula (I) according to the present invention may induce the MPT by binding to cysteine residues on mitochondrial Adenine Nucleotide Translocator (ANT). In one embodiment, the compound of formula (I) is from about 2 to about 20-times, about 2 to about 10-times, about 2 to about 5-times, e.g., about 4-times, more efficient at inducing the MPT than GSAO. Another embodiment of the invention relates to a method of inducing apoptosis in proliferating mammalian cells, comprising administering to the mammal an apoptosis- inducing amount of a compound of formula (I) or a pharmaceutical composition thereof.
In a further embodiment the invention relates to a method of treating γ-glutamyl transferase expressing tumours in a vertebrate, comprising administering to the vertebrate a compound of formula (I) or a pharmaceutical composition thereof. In one embodiment the γ-glutamyl transferase expressing tumours are tumours of the breast, prostate, colon, liver, ovary, lung, kidney, thyroid or pancreas.
Therapeutic advantages may be realised through combination regimens. In combination therapy the respective agents may be administered simultaneously, or sequentially in any order. Accordingly, methods of treatment according to the present invention may be applied in conjunction with conventional therapy, such as radiotherapy, chemotherapy, surgery, or other forms of medical intervention. Examples of chemotherapeutic agents include adriamycin, taxol, fluorouricil, melphalan, cisplatin, oxaliplatin, alpha interferon, vincristine, vinblastine, angioinhibins, TNP -470, pentosan polysulfate, platelet factor 4, angiostatin, LM-609, SU-IOl, CM-101, Techgalan, thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents such as nitrogen mustards including mechloethamine, melphan, chlorambucil, cyclophosphamide and ifosfamide, nitrosoureas including carmustine, lomustine, semustine and streptozocin; alkyl sulfonates including busulfan; triazines including dicarbazine; ethyenimines including thiotepa and hexamethylmelamine; folic acid analogues including methotrexate; pyrimidine analogues including 5-fluorouracil, cytosine arabinoside; purine analogues including 6-mercaptopurine and 6-thioguanine; antitumour antibiotics including actinomycin D; the anthracyclines including doxorubicin, bleomycin, mitomycin C and methramycin; hormones and hormone antagonists including tamoxifen and cortiosteroids and miscellaneous agents including cisplatin and brequinar, and regimens such as COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), and PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine).
Pharmaceutical and/or Therapeutic Formulations
Typically, for medical use, salts of the compounds of the present invention will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the inventive compounds or of the pharmaceutically acceptable salt thereof. By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art.
Pharmaceutically acceptable salts of compounds of formula I may be prepared by methods known to those skilled in the art, including for example, (i) by reacting a compound of formula (I) with the desired acid or base; (ii) by removing an acid- or base- labile protecting group from a suitable precursor of the compound of formula (I) or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound of formula (I) to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
Thus, for instance, suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences, 1977, 66:\-\9. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolaniine and the like.
Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. In one embodiment, the mode of administration is parenteral. In another embodiment, the mode of administration is oral. Depending on the route of administration, the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The compound also may be administered parenterally or intraperitoneally.
Dispersions of compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
The compound(s) of the invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Suitably, such compositions and preparations may contain at least 1% by weight of active compound. The percentage of the compound(s) of formula (I) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit. The amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained. The language "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
In one embodiment, the carrier is an orally administrable carrier. Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration. Also included in the scope of this invention are delayed release formulations.
Compounds of formula (I) according to the invention also may be administered in the form of a "prodrug". Suitable prodrugs include esters, phosphonate esters etc, of the compound.
In one embodiment, the compound of formula (I) may be administered by injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or anti-fungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations.
The pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof. Single or multiple administrations of the compounds and/or pharmaceutical compositions according to the invention may be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.
Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests. Generally, an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight. Alternatively, an effective dosage may be up to about 500mg/m2. For example, generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m2, about 25 to about 350mg/m2, about 25 to about 300mg/m2, about 25 to about 250mg/m2, about 50 to about 250mg/m2, and about 75 to about 150mg/m2.
In another embodiment, a compound of Formula (I) may be administered in an amount in the range from about 100 to about 1000 mg per day, for example, about 200 mg to about 750 mg per day, about 250 to about 500 mg per day, about 250 to about 300 mg per day, or about 270 mg to about 280 mg per day.
Compounds in accordance with the present invention may be administered as part of a therapeutic regimen with other drugs. It may be desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition. Accordingly, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound of formula (I) according to the present invention, may be combined in the form of a kit suitable for simultaneous or sequential administration of the compositions. The invention will now be described in more detail, by way of illustration only, with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification.
Examples
Example 1 : Experimental Procedures Cell culture
Bovine aortic endothelial (BAE) cells were from Cell Application (San Diego, CA). BAE cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mM L- glutamine, and 5 units per mL penicillin and streptomycin (Gibco, Gaithersburg, MD). Melanoma cell clones expressing different γGT activity were produced as previously described (Franzini et al., 2006). The c21/basal and c21/γGT clones express a γGT activity of 0.34 ± 0.13 and 91 ± 3.4 mU per mg of cellular protein, respectively. Both clones were grown in RPMI 1640 medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine and 0.5 mg/ml G418 (Gibco). Cells were cultured at 37 °C in a 5% CO2, 95% air atmosphere.
Cell proliferation assay BAE cells were seeded in 96-well plates (5,000 cells per well) in 0.2 ml of culture medium. After 24 h of growth, the medium was replaced with fresh culture medium supplemented with GSAO, GCAO, CAO or 4H10 and cells were cultured for an additional 24, 48 or 72 h. Viable attached cells were determined using the tetrazolium salt MTT (Sigma, St. Louis, MO) according to the manufacturer's protocol. Results were expressed as percentage of untreated controls.
Melanoma cells were seeded in 96-well plates (4,000 cells per well) in 0.2 ml of culture medium. After 24 h of growth, the medium was replaced with fresh culture medium supplemented with GSAO and cells were cultured for an additional 24 or 72 h. Viable cells were determined using the WST-I reagent (Roche, Basel, Switzerland) according to the manufacturer's protocol. Results were expressed as percentage of untreated controls.
In separate sets of experiments the effect of γGT inhibition on GSAO toxicity was studied. The γGT competitive inhibitor, L-2-amino-4-boronobutanoic acid (ABBA), was provided by Dr. R. E. London (Natl. Inst. Environ. Health Sci., NC, USA). Cells were exposed for 24 h to GSAO concentrations causing 50% of cell growth inhibition (IC5o) in the absence or presence of 10 μM ABBA. This concentration of ABBA alone had no effects on cell viability or proliferation (data not shown).
Ki for inhibition of γGT by GSAO Determination of γGT activity was performed according to Huseby and Strømme
(Huseby and Stromme, 1974) using γ-glutamyl-p-nitroanilide as substrate and glycyl- glycine as transpeptidation acceptor. γGT, γ-glutamyl-p-nitroanilide, glycyl-glycine and GSAO in 15 mM Tris, pH 9.0 buffer were incubated at 250C in Linbro/Titertek E.I.A. microtitration plate wells. The reactions were started by the addition of γGT to a final volume of 200 μl. Reactant concentrations are found in the figure legend of Fig. 2. The formation of j5-nitroaniline as a function of time was monitored continuously by measuring the absorbance at 405 nm using a Molecular Devices M2 Microplate Reader (Palo Alto, CA). The extinction coefficient used for p-nitroaniline was 9920 M-1Cm"1. The apparent K; for inhibition of γGT by GSAO was estimated from the effect of fixed concentrations of GSAO on the initial velocity of hydrolysis of γ-glutamyl-^-nitroanilide by γGT. The results were consistent with simple competitive inhibition. The data points were globally fit by non-linear least squares regression using GraphPad (San Diego, CA) software.
Production of GSAO metabolites
GSAO was produced as described previously (WO 01/21628, the disclosure of which is incorporated herein by reference) to a purity >94% by HPLC. A 50 mM solution of GSAO was made by dissolving solid in 20 mM Hepes, pH 7.0 buffer containing 0.14 M NaCl, 20 mM glycine and 1 mM EDTA. 4-(N-(S- cysteinylglycylacetyl)amino)phenylarsinous acid (GCAO) was produced by cleaving the γ-glutamyl group from GSAO with ovine kidney γ-glutamyl transpeptidase type I (Sigma, product number G8040). A lO mM solution of GSAO was incubated with 0.55 units per ml γGT in 15 mM Tris, pH 7.4 buffer containing 40 mM glycyl-glycine for 1 h at 3O0C. The γGT was removed from the reaction by filtration using a YM3 Microcon membrane (Millipore, Billerica, MA).
4-(N-(S-cysteinylacetyl)amino)phenylarsinous acid (CAO) was produced by cleaving the glycine amino acid from GCAO with porcine kidney aminopeptidase N (Type IV-S, Sigma, product number L5006). The GCAO filtrate was incubated with 2 units per ml aminopeptidase N for 1 h at 37°C The aminopeptidase N was removed from the reaction by filtration using a YM3 Microcon membrane (Millipore). The concentrations of the metabolites were measured by titrating with dimercaptopropanol and calculating the remaining free thiols with 5,5'-dithiobis(2-nitrobenzoic acid) (Don et al, 2003). The titrated solutions were sterile filtered and stored at 40C in the dark until use. There was no significant loss in the active concentration of stock solutions of the arsenicals for at least a week when stored under these conditions.
HPLC and ESI-MS analysis of the organoarsenicals
GSAO and metabolites were characterized by HPLC (1200 Series; Agilent Technologies, Santa Clara, CA). Samples were resolved on a Zorbax Eclipse XDB-Cl 8 column (4.6 x 150 mm, 5μm; Agilent Technologies) using a mobile phase of acetonitrile- water (25:75 vol/vol), flow rate of 0.5 ml.min"1 and detection by absorbance at 256 nrn. Purity of GSAO, GCAO and CAO by peak area was 94 ± 1%, 69 ± 3% and 84 ± 4%, respectively.
Masses of the compounds were determined using ESI-MS. Spectra were acquired using an API QStar Pulsar i hybrid tandem mass spectrometer (Applied Biosystems, Foster City, CA). Samples (~1 pmol) were loaded into nanospray needles (Proxeon, Denmark) and the tip positioned ~10 mm from the orifice. Nitrogen was used as curtain gas and a potential of 900 V applied to the needle. A Tof MS scan was acquired (m/z 200- 2000, 1 s) and accumulated for ~1 min into a single file. The mass spectrum of GCAO gave molecular ions at 420.0 m/z [GCAOH-H]+, 402.0 m/z [GCAO+H-H2O]+ and 383.9 m/z [GCAO+H-2H2θ]+ confirming the cleavage of the γ-glutamyl group from GSAO. The mass spectrum of CAO gave molecular ions at 362.9 m/z [CAO+H]+, 384.9 m/z [CAO+Na]4" and 344.9 m/z [CAOH-H-H2O]+ confirming the cleavage of the glycine from GCAO.
Accumulation of GSAO and metabolites in BAE cells
Depending on the type of experiments, 1.6 xlO6 or 7.5 xlO5 BAE cells were seeded in petri dishes or 6-well-plates, respectively, and allowed to attach overnight. The medium was replaced and the cells were incubated for 30 min in the absence or presence of acivicin or 4H10. The cells were then incubated with 50 or 100 μM GSAO, GCAO or CAO. Cells were then washed twice with ice-cold PBS and lysed with 1 ml of 70% w/w nitric acid. Lysates were diluted 30-fold and analyzed for arsenic atoms using an Elan 6100 Inductively Coupled Plasma Spectrometer (Perkin Elmer Sciex Instruments, Shelton, CT).
Mitochondrial swelling assay.
Mitochondria were isolated from the livers of 250 g male Wistar rats using differential centrifugation as described previously (Dilda et al, 2005a). The final mitochondrial pellet was resuspended in 3 mM Hepes-KOH, pH 7.0 buffer containing 213 mM mannitol, 71 mM sucrose and 10 mM sodium succinate at a concentration of 30 mg of protein per mL. Mitochondrial permeability transition induction was assessed spectrophotometrically by suspending the liver mitochondria at 0.5 mg of protein per mL at 37°C in 3 mM Hepes-KOH, pH 7.0 buffer containing 75 mM mannitol, 250 mM sucrose, 10 mM sodium succinate, and 2 μM rotenone (Dilda et al., 2005a). Swelling was measured by monitoring the associated decrease in light scattering at 520 nm using a Molecular Devices M2 Microplate Reader (Palo Alto, CA).
Statisical analyses
Results are presented as means ± SE. All tests of statistical significance were two- sided and P values < 0.05 were considered statistically significant. Example 2: Results
Inhibition of cell-surface γGT blunts cellular accumulation of GSAO and antiproliferative activity.
Both the γGT substrate, reduced glutathione, and the γGT inhibitor, acivicin, significantly reduced accumulation of GSAO in endothelial cells (Fig. IB and C). Acivicin also blunted GSAO' s anti-proliferative activity (Fig. ID). These findings implied that GSAO' s effect on endothelial cells was modified by cell-surface γGT. To test this conclusion, the effect of varying cell-surface expression of γGT on GSAO' s antiproliferative activity was assessed. Previously well characterized c21 melanoma cell clones expressing low (0.34 mU per mg cell protein) or high (91 mU per mg cell protein) levels of γGT activity were employed (Franzini et al., 2006).
Expression level of y GT positively correlates with sensitivity of cells to GSAO.
Melanoma c21/basal and c21/γGT clones expressing low (0.34 mU per mg cell protein) or high (91 mU per mg cell protein) levels of γGT activity were tested for sensitivity to GSAO in 24 or 72 h assays. The 24 h GSAO IC50 (concentration of compound that inhibits 50%) for proliferation arrest was ~60 μM and ~1250 μM for high and low γGT cells, respectively (Fig. 2A). These IC50 values fell to ~20 μM and — 115 μM, respectively, in 72 h assays (Fig. 2B). The anti-proliferative effects of GSAO were negated in both high and low expressing cells with the γGT inhibitor, ABBA (Fig. 2C). Treatment with ABBA alone at the concentration used had no effect on cell proliferation or viability (data not shown).
These findings indicate that there is a degree of association between the susceptibility of cells to proliferation arrest by GSAO and the expression level of γGT. The time dependence of the anti-proliferative effect suggests that GSAO is being metabolized by γGT and that the metabolite(s) have an involvement in inhibiting cell proliferation. To test whether GSAO is a substrate for γGT, the apparent Kj for binding of GSAO to γGT was measured.
Ki for binding of γGT to GSAO
Inhibition of γGT by GSAO was measured from the effect of fixed concentrations of GSAO on the initial velocity of hydrolysis of γ-glutamyl-p-nitroanilide by γGT. The apparent K; for competitive inhibition of γGT by GSAO was estimated to be 1.6 ± 0.2 mM, which is similar to the apparent Km for hydrolysis of the chromogenic substrate, γ- glutamyl-p-nitroanilide (2.4 ± 0.4 mM) (Fig. 2D). This finding implies that the phenylarsinous acid moiety does not denionstrably affect access of γGT to the glutathione pendant of GSAO.
The OATP family is involved in endothelial cell uptake of the product of GSAO cleavage by γGT, GCAO.
GCAO was produced by cleaving the γ-glutamyl group from GSAO with porcine kidney γ-glutamyl transpeptidase. The enzyme was removed from the reactions by size- exclusion filtration and GCAO analyzed by HPLC. GSAO and GCAO have retention times of 3.03 and 3.38 min respectively (Fig. 3A). The organic anion transporting polypeptide (OATP) family of transporters function independently of ATP and sodium gradients and were originally characterized as uptake transporters (Hagenbuch et al., 2003). OATP family members have been implicated in transport of glutathione-S- conjugates (Kobayashi et al., 2003), so they were candidate transporters for GCAO. 4,4'- Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) is an inhibitor of the plasma membrane OATP family of transporters. Accumulation of GCAO in endothelial cells (data not shown) and anti-proliferative activity was reduced by DIDS (Fig 3D), which implies that this family of transporters may be involved in GCAO translocation across the plasma membrane.
GCAO accumulates more rapidly in endothelial cells and has greater anti-proliferative activity than GSAO.
GCAO accumulated in BAE cells at a 8.7-fold faster rate than GSAO (Fig. 3B). The rate of accumulation of GCAO was 3.8 ± 0.1 pmol As atoms per 106 cells per min, compared to 0.4 ± 0.1 pmol As atoms per 106 cells per min for GSAO. The faster rate of accumulation of GCAO corresponded to a ~5-fold increased anti-proliferative activity. The IC50 for GSAO and GCAO in a 24 h BAE cell proliferation assay was 97 ± 5 μM and 18 ± 3 μM, respectively (Fig. 3C). It is apparent from the results that the IC5O for GSAO markedly decreases with time of incubation and much less so for GCAO. For example, the 72 h GSAO IC50 is similar to the 24 h IC50 for GCAO. These results are consistent with GSAO being cleaved at the cell-surface by γGT and the resultant GCAO entering the cell and playing a role in proliferation arrest.
GCAO is secreted from endothelial cells by MRPl.
Cellular accumulation of GCAO is a balance between rate of uptake and rate of export from the cell. GSAO accumulation in cells is controlled by rate of export by MRPl and MRP2 (Dilda et al., 2005b). To test whether GCAO is also exported by MRPl, the effect of the MRPl inhibitor 4H10 on accumulation in endothelial cells was measured. Cellular accumulation of GCAO was increased 3 -fold when MRPl was inhibited (Fig. 4A), which correlated with more potent anti-proliferative effect (Fig. 4B). The inhibitor alone had no effect on BAE cell proliferation (data not shown).
GCAO triggers the mitochondrial permeability transition more rapidly than GSAO.
GSAO inactivates the mitochondrial inner membrane transporter adenine nucleotide translocase (ANT), which leads to proliferation arrest and cell death (Don et al., 2003). GCAO also induces the mitochondrial permeability transition (Fig. 4C). Comparison of the time for 25% maximal swelling as a function GSAO or GCAO concentration indicates that GCAO is approximately twice as efficacious as GSAO at triggering the permeability transition (Fig. 4D). This finding indicates that an intact glutathione pendant is not necessary for GSAO to inactivate ANT.
GCAO and its metabolite, CAO, accumulate in endothelial cells at the same rate and have comparable anti-proliferative activity.
CAO was produced by cleaving the glycine amino acid from GCAO with porcine kidney aminopeptidase N (Fig. 5A). The enzyme was removed from the reactions by size- exclusion filtration and the product of the reaction was analyzed by HPLC (Fig. 5B).
CAO has a retention time of 3.64 min.
The rate of accumulation of CAO in BAE cells was 3.3 ± 0.1 pmol As atoms per
IO6 cells per min, compared to 3.8 ± 0.1 pmol As atoms per 106 cells per min for GCAO
(Fig. 5C). The similar rate of accumulation of the two compounds corresponded to a comparable anti-proliferative activity. The IC50 for GCAO and CAO in a 48 h BAE cell proliferation assay was 9 ± 2 μM and 11 ± 2 μM, respectively (Fig. 5D).
CAO is secreted from endothelial cells by MRPl.
Similar to the result for GCAO (Fig. 4C), cellular accumulation of CAO was increased 2.2-fold when MRPl was inhibited (Fig. 6A). The inhibitor alone had no effect on BAE cell proliferation (data not shown).
CAO triggers the mitochondrial permeability transition more rapidly than GCAO.
Like GSAO and GCAO, CAO also induces the mitochondrial permeability transition (Fig. 6B). Comparison of the time for 25% maximal swelling as a function GCAO or CAO concentration indicates that CAO is approximately twice as efficacious as GCAO at triggering the permeability transition (Fig. 6C).
GSAO is a substrate for γGT γGT is present on the outer surface of the plasma membrane (Horiuchi et al., 1978) of virtually all cells. The enzyme catalyses hydrolysis of the bond linking the glutamate and cysteine residues of extracellular glutathione and glutathione-S-conjugates (Enoiu et al., 2002). GSAO is effectively a glutathione-S-conjugate of aminophenylarsonous acid. It has been demonstrated in several ways that GSAO is a substrate for γGT. First, GSAO is an efficient substrate for isolated γGT. Second, endothelial cell accumulation and anti-proliferative activity of GSAO was blunted by extracellular glutathione and an active site inhibitor of γGT. The glutathione likely acted as a competitive substrate inhibitor of γGT in this experiment. Third, the level of cell surface γGT correlated strongly with the sensitivity of model cells to GSAO.
There was a marked time dependence of the anti-proliferative effect of GSAO on endothelial cells.
The longer the incubation of GSAO with γGT-expressing cells the lower the IC50 for proliferation arrest. This time dependence is consistent with a mechanism where GSAO is cleaved by γGT and the product of this reaction is inhibiting cell proliferation. This mechanism was tested by making the product of γGT cleavage, GCAO, and examining how endothelial cells respond to this compound. GCAO accumulated much more rapidly in endothelial cells than GSAO and had greater anti-proliferative activity.
The glycine-cysteine peptide bond of GCAO is readily cleaved by dipeptidases to produce
CAO.
A number of dipeptidases are found on the plasma membrane (Cannon et al., 2000) and in the cytosol (Josch et al., 1998). It is likely that GCAO is cleaved on or in the cell by one or more dipeptidases. CAO accumulated in endothelial cells at the same rate as GCAO and had comparable anti-proliferative activity. Like GSAO and GCAO, CAO was also a substrate for MRPl in endothelial cells.
GSAO and yGT-positive tumors.
The findings shown in Fig. 2 indicate that there is a degree of association between the susceptibility of cells to proliferation arrest by GSAO and the expression level of γGT. Metabolism of GSAO by tumor cell and/or tumor endothelium γGT would produce high local concentrations of GCAO that will then block tumor angiogenesis and tumor growth. Notably, tumors of the breast, prostate, colon, liver, ovary, lung, kidney, thyroid and pancreas express γGT (Hannigan et al., 1996).
References
Belzacq, A. S., El Hamel, C, Vieira, H. L., Cohen, L, Haouzi, D., Metivier, D., Marchetti, P., Brenner, C, and Kroemer, G. (2001). Adenine nucleotide translocator mediates the mitochondrial membrane permeabilization induced by lonidamine, arsenite and CD437. Oncogene 20, 7579-7587.
Dilda, P. J., Decollogne, S., Rossiter-Thornton, M., and Hogg, P. J. (2005a). Para to ortho repositioning of the arsenical moiety of the angiogenesis inhibitor 4-(N-(S- glutathionylacetyl)amino)phenylarsenoxide results in a markedly increased cellular accumulation and antiproliferative activity. Cancer Res 65, 11729-11734. Dilda, P. J., Don, A. S., Tanabe, K. M., Higgins, V. J., Allen, J. D., Dawes, I. W., and Hogg, P. J. (2005b). Mechanism of selectivity of an angiogenesis inhibitor from screening a genome-wide set of Saccharomyces cerevisiae deletion strains. J Natl Cancer Inst 97, 1539-1547. Don, A. S., Kisker, O., Dilda, P., Donoghue, N., Zhao, X., Decollogne, S., Creighton, B., Flynn, E., Folkman, J., and Hogg, P. J. (2003). A peptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells. Cancer Cell 3, 497-509.
Donoghue, N., Yam, P. T., Jiang, X. M., and Hogg, P. J. (2000). Presence of closely spaced protein thiols on the surface of mammalian cells. Protein Sci 9, 2436-2445. Franzini, M., Corti, A., Lorenzini, E., Paolicchi, A., Pompella, A., De Cesare, M., Perego, P., Gatti, L., Leone, R., Apostoli, P., and Zunino, F. (2006). Modulation of cell growth and cisplatin sensitivity by membrane gamma-glutamyltransferase in melanoma cells. Eur J Cancer 42, 2623-2630. Huseby, N. E., and Stromme, J. H. (1974). Practical points regarding routine determination of gamma-glutamyl transferase (gamma-GT) in serum with a kinetic method at 37 degrees C. Scand J Clin Lab Invest 34, 357-363.
Larochette, N., Decaudin, D., Jacotot, E., Brenner, C, Marzo, L, Susin, S. A., Zamzami, N., Xie, Z., Reed, J., and Kroemer, G. (1999). Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Exp Cell Res 249, 413-421. McStay, G. P., Clarke, S. J., and Halestrap, A. P. (2002). Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem J 367, 541-548.
Pebay-Peyroula, E., Dahout-Gonzalez, C, Kahn, R., Trezeguet, V., Lauquin, G. J., and Brandolin, G. (2003). Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside. Nature 426, 39-44.
Hagenbuch, B., and Meier, P. J. (2003) Biochim. Biophys. Acta 1609, 1-18.
Kobayashi, D., Nozawa, T., Imai, K., Nezu, J., Tsuji, A., and Tamai, I. (2003) J.
Pharmacol. Exp. Ther. 306, 703-708. Horiuchi, S., Inoue, M., and Morino, Y. (1978) Eur. J. Biochem. 87, 429-437,
Enoiu, M., Herber, R., Wennig, R., Marson, C, Bodaud, H., Leroy, P., Mitrea, N., Siest,
G., and Wellman, M. (2002) Arch. Biochem. Biophys. 397, 18-27.
Cannon, V. T., Barfuss, D. W., and Zalups, R. K. (2000) J. Am. Soc. Nephrol. 11, 394-
402. Josch, C, Sies, H., and Akerboom, T. P. (1998) Biochem. Pharmacol. 56, 763-771.
Hanigan, M. H., and Frierson, H. F., Jr. (1996) J. Histochem. Cytochem. 44, 1101-1108.

Claims

CLAIMS:
1. A compound of formula (I)
Figure imgf000042_0001
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R1 is selected from hydrogen, C1-3 alkyl and cyclopropyl;
R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3;
[AA-I] is an amino acid residue selected from cysteine, serine, threonine, α-amino- β-hydroxy-isovaleric acid and penicillamine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine;
[AA-3] (when present) is a γ-glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof.
2. A compound of formula (I)
Figure imgf000042_0002
(I) wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R1 is selected from hydrogen, C1-3 alkyl and cyclopropyl; R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted C1-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted C1-3 alkoxy; m is an integer selected from 1, 2 and 3; s [AA-I] is an amino acid residue selected from cysteine and serine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; o [AA-3] (when present) is a γ-glutamic acid residue; n is 0 or 1 ; p is 0 or 1 ; and salts and hydrates thereof. 3. A compound of formula (I)
Figure imgf000043_0001
5
(D wherein the As(OH)2 group may be ortho-, meta- or para- to the N-atom on the phenyl ring; R1 is selected from hydrogen, Ci-3 alkyl and cyclopropyl; 0 R2 and R3 may be the same or different and are independently selected from hydrogen, optionally substituted Ci-3 alkyl, optionally substituted cyclopropyl, optionally substituted C2-3 alkylene; and optionally substituted Ci-3 alkoxy; m is an integer selected from 1, 2 and 3;
[AA-I] is an amino acid residue selected from cysteine, serine, threonine, α-amino-5 β-hydroxy-isovaleric acid and penicillamine;
[AA-2] is an amino acid residue selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine; 0 n is 0 or 1 ; p is 0; and salts and hydrates thereof.
4. A compound according to any one of claims 1 to 3, wherein the As(OH)2 group is para- to the N-atom on the phenyl ring.
5. A compound according to any one of claims 1 or 3, wherein the As(OH)2 group is ortho- to the N-atom on the phenyl ring.
6. A compound according to any one of claims 1 to 5, wherein R1 is hydrogen, methyl or ethyl.
7. A compound according to claim 6, wherein R1 is hydrogen.
8. A compound according to any one of claims 1 to 7, wherein R and R are the same or different and are independently selected from hydrogen, C1-3 alkyl, C2-3 alkenyl,
Ci-3 alkoxy, halo-(C1-3)alkoxy, hydroxy(C1-3)alkyl and halo(C1-3)alkyl.
9. A compound according to any one of claims 1 to 7, wherein R and R are independently selected from hydrogen, methyl, ethyl, methoxy, vinyl, hydroxymethyl, CF3 and OCF3.
10. A compound according to any one of claims 1 to 7, wherein R2 and R3 are independently selected from hydrogen, methyl and ethyl.
11. A compound according to any one of claims 1 to 7, wherein R2 and R3 are both hydrogen.
12. A compound according to any one of claims 1 to 11 , wherein m is 1.
13. A compound according to any one of claims 1 to 12, wherein n is 0, p is 0 and
[AA-I] is selected from cysteine and serine.
14. A compound according to any one of claims 1 to 13, wherein R1, R2 and R3 are hydrogen; n is 0, p is 0; [AA-I] is cysteine and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
15. A compound according to any one of claims 1 to 12, wherein R1, R2 and R3 are independently hydrogen or methyl; n is 1, p is 0; [AA-I] is cysteine, serine, threonine, α-amino-β-hydroxy-isovaleric acid or penicillamine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
16. A compound according to any one of claims 1 to 12, wherein R , R and R are each hydrogen; n is 1, p is 0; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
17. A compound according to any one of claims 1 to 12, wherein R1, R2 and R3 are each hydrogen; n is 1, p is 0; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, and glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
18. A compound according to any one of claims 1 to 12, wherein R , R and R are each hydrogen; n is 1, p is 0; AA-I is cysteine; AA-2 is glycine; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
19. A compound according to any one of claims 1, 2 or 4 to 12, wherein R , R and R3 are independently selected from hydrogen and methyl; n is 1, p is 1; [AA-I] is cysteine or serine; [AA-2] is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, serine, threonine, cysteine, tyrosine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
20. A compound according to any one of claims 1, 2 or 4 to 12, wherein R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, leucine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, and arginine; [AA-3] is glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
21. A compound according to any one of claims 1, 2 or 4 to 12, wherein R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, valine, threonine, cysteine, aspartic acid, glutamic acid, and arginine; [AA-3] is glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
22. A compound according to any one of claims 1, 2 or 4 to 12, wherein R1, R2 and R3 are each hydrogen; n is 1, p is 1; [AA-I] is cysteine; [AA-2] is selected from glycine, alanine, and glutamic acid; [AA-3] is glutamic acid; and the As(OH)2 group is ortho or para to the nitrogen atom attached to the phenyl ring.
23. A compound according to any one of claims 1 to 12, wherein n is 1; p is 0 or 1; and [AA-l]-[AA-2] is selected from cysteine-glycine; cysteine-alanine; cysteine- valine; cysteine-leucine; cysteine-serine; cysteine-threonine; cysteine-cysteine; cysteine- aspartic acid; cysteine-glutamic acid; cysteine-lysine; and cysteine-arginine, serine- glycine; serine-alanine; serine-valine; serine-leucine; serine-serine; serine-threonine; serine-cysteine; serine-aspartic acid; serine-glutamic acid; serine-lysine; and serine- arginine.
24. A compound according to any one of claims 1 or 3 to 12, wherein n is 1; p is 0 or 1; and [AA-l]-[AA-2] is selected from penicillamine-glycine; penicillamine-alanine; penicillamine-valine; penicillamine-leucine; penicillamine-serine; penicillamine- threonine; penicillamine-cysteine; penicillamine-aspartic acid; penicillamine-glutamic acid; penicillamine-lysine; penicillamine-arginine; threonine-glycine; threonine-alanine; threonine-valine; threonine-leucine; threonine-serine; threonine-threonine; threonine- cysteine; threonine-aspartic acid; threonine-glutamic acid; threonine-lysine; threonine- arginine; α-amino-β-hydroxy-isovaleric acid-glycine; α-amino-β-hydroxy-isovaleric acid-alanine; α-amino-β-hydroxy-isovaleric acid-valine; α-amino-β-hydroxy-isovaleric acid-leucine; α-amino-β-hydroxy-isovaleric acid-serine; α-amino-β-hydroxy-isovaleric acid-threonine; α-amino-β-hydroxy-isovaleric acid-cysteine; α-amino-β-hydroxy- isovaleric acid-aspartic acid; α-amino-β-hydroxy-isovaleric acid-glutamic acid; α-amino- β-hydroxy-isovaleric acid-lysine; and α-amino-β-hydroxy-isovaleric acid-arginine.
25. A pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 24 or a salt or hydrate thereof, together with a pharmaceutically acceptable excipient, diluent or adjuvant.
26. A method of treating a proliferative disease in a vertebrate, the method comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 24, or a composition according to claim 25.
27. A method of inhibiting angiogenesis in a vertebrate, comprising administering to the vertebrate an effective amount of a compound of formula (I) according to any one of claims 1 to 24, or a composition according to claim 25.
28. A method of inducing the Mitochondrial Permeability Transition (MPT) in a vertebrate comprising administering to the vertebrate a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 24, or a composition according to claim 25.
29. A method of inducing apoptosis or necrosis in proliferating mammalian cells, comprising administering to the mammal an apoptosis- or necrosis-inducing amount of a compound of formula (I) according to any one of claims 1 to 24, or a composition according to claim 25.
30. The method according to claim 29, wherein the cells are proliferating endothelial cells.
31. A method of treating γ-glutamyl transferase expressing tumours in a vertebrate, comprising administering to the vertebrate a compound of formula (I) according to any one of claims 1 to 24, or a composition according to claim 25.
32. The method according to claim 31, wherein the γ-glutamyl transferase expressing tumours are tumours of the breast, prostate, colon, liver, ovary, lung, kidney, thyroid or pancreas.
lOi
I5
20 5 0 5
PCT/AU2008/001477 2007-10-04 2008-10-03 Arsenoxide compound and method of use WO2009043114A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2007905434 2007-10-04
AU2007905434A AU2007905434A0 (en) 2007-10-04 Arsenoxide compound and method of use

Publications (1)

Publication Number Publication Date
WO2009043114A1 true WO2009043114A1 (en) 2009-04-09

Family

ID=40525779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2008/001477 WO2009043114A1 (en) 2007-10-04 2008-10-03 Arsenoxide compound and method of use

Country Status (1)

Country Link
WO (1) WO2009043114A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069371A1 (en) * 2006-11-01 2009-06-17 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021628A1 (en) * 1999-09-20 2001-03-29 Unisearch Limited A substantially cell membrane impermeable compound and use thereof
WO2002074305A1 (en) * 2001-03-19 2002-09-26 Unisearch Limited Use of a substantially cell membrane impermeable compound for treating arthritis
WO2003003011A1 (en) * 2001-06-28 2003-01-09 Unisearch Limited Modification of angiogenesis by targeting protein tyrosine phosphatases
WO2003039564A1 (en) * 2001-11-08 2003-05-15 Unisearch Limited Selective targeting of apoptotic cells
WO2004042079A1 (en) * 2002-11-07 2004-05-21 Unisearch Limited Induction of the mitochondrial permeability transition
WO2008052279A1 (en) * 2006-11-01 2008-05-08 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021628A1 (en) * 1999-09-20 2001-03-29 Unisearch Limited A substantially cell membrane impermeable compound and use thereof
WO2002074305A1 (en) * 2001-03-19 2002-09-26 Unisearch Limited Use of a substantially cell membrane impermeable compound for treating arthritis
WO2003003011A1 (en) * 2001-06-28 2003-01-09 Unisearch Limited Modification of angiogenesis by targeting protein tyrosine phosphatases
WO2003039564A1 (en) * 2001-11-08 2003-05-15 Unisearch Limited Selective targeting of apoptotic cells
WO2004042079A1 (en) * 2002-11-07 2004-05-21 Unisearch Limited Induction of the mitochondrial permeability transition
WO2008052279A1 (en) * 2006-11-01 2008-05-08 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DILDA, P. J. ET AL.: "Mechanism of selectivity of an angiogenesis inhibitor from screening a genome-wide set of Saccharomyces cerevisiae deletion strains", J. NAT. CANCER INST., vol. 97, no. 20, 2005, pages 1539 - 1547 *
DILDA, P. J. ET AL.: "Para to ortho repositioning of the arsenical moiety of the angiogenesis inhibitor 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide results in a markedly increased cellular accumulation and antiproliferative activity", CANCER RES., vol. 65, no. 24, 2005, pages 11729 - 11734 *
DON, A. S. ET AL.: "A peptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells", CANCER CELL, vol. 3, no. 5, 2003, pages 497 - 509, XP008051811, DOI: doi:10.1016/S1535-6108(03)00109-0 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2069371A1 (en) * 2006-11-01 2009-06-17 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof
EP2069371A4 (en) * 2006-11-01 2010-02-10 Newsouth Innovations Pty Ltd Organo-arsenoxide compounds and use thereof
US8268883B2 (en) 2006-11-01 2012-09-18 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof
US9765101B2 (en) 2006-11-01 2017-09-19 Newsouth Innovations Pty Limited Organo-arsenoxide compounds and use thereof

Similar Documents

Publication Publication Date Title
US20220257563A1 (en) Nitrile-Containing Antiviral Compounds
AU2016245864C1 (en) Substituted quinazoline compounds and methods of use thereof
CN110691779B (en) Method for treating hematological malignancies and ewing&#39;s sarcoma
US9156824B2 (en) CDC7 inhibitors
KR20210083286A (en) 2-(2-acryloyl-2,6-diazaspiro[3.4]octan-6-yl)-6-(1H-indazol-4-yl as inhibitor of G12C mutant KRAS protein to inhibit tumor metastasis )-benzonitrile derivatives and related compounds
EP2332917B1 (en) Compounds for PIM kinase inhibition and for treating malignancy
CA3041563C (en) Pyrimidin-2-amine derivatives and pharmaceutical compositions thereof useful as inhibitors of cyclin-dependent kinase 12 (cdk12)
WO2018140600A1 (en) Fused hetero-hetero bicyclic compounds and methods of use thereof
EP3630745A2 (en) Covalent inhibitors of kras
EP3573964A1 (en) Benzothiophene and benzothiazole compounds and methods of use thereof
EP3573954A1 (en) Fused bicyclic benzoheteroaromatic compounds and methods of use thereof
EP3377481A1 (en) 2-substituted quinazoline compounds comprising a substituted heterocyclic group and methods of use thereof
WO2017058792A1 (en) Inhibitors of kras g12c mutant proteins
WO2017058805A1 (en) Inhibitors of kras g12c mutant proteins
WO2016168540A1 (en) Fused-tricyclic inhibitors of kras and methods of use thereof
EP3193851A1 (en) Combination therapies for treatment of cancer
US9765101B2 (en) Organo-arsenoxide compounds and use thereof
US20060142352A1 (en) Compounds useful for the treatment of cancer, compositions thereof and methods therewith
US11465983B2 (en) USP7 inhibitors for treating multiple myeloma
WO2009043114A1 (en) Arsenoxide compound and method of use
WO2022086986A1 (en) Treatment of hematological malignancies with inhibitors of menin
CA3159167A1 (en) Masp-2 inhibitors and methods of use
US20200199081A1 (en) Stk19 inhibitors for treatment of cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08800112

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08800112

Country of ref document: EP

Kind code of ref document: A1