WO2009108704A1 - O-alkylated pi-3 kinase inhibitor prodrug combination - Google Patents

O-alkylated pi-3 kinase inhibitor prodrug combination Download PDF

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Publication number
WO2009108704A1
WO2009108704A1 PCT/US2009/035148 US2009035148W WO2009108704A1 WO 2009108704 A1 WO2009108704 A1 WO 2009108704A1 US 2009035148 W US2009035148 W US 2009035148W WO 2009108704 A1 WO2009108704 A1 WO 2009108704A1
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cancer
compound
agent
group
salt
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PCT/US2009/035148
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French (fr)
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Joseph R. Garlich
Donald L Durden
Mary Patterson
Jingdon Su
Robert G. Suhr
Xiaodong Peng
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Semafore Pharmaceuticals, Inc.
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Publication of WO2009108704A1 publication Critical patent/WO2009108704A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/386—Polyphosphonic acids containing hydroxy substituents in the hydrocarbon radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/3873—Polyphosphonic acids containing nitrogen substituent, e.g. N.....H or N-hydrocarbon group which can be substituted by halogen or nitro(so), N.....O, N.....S, N.....C(=X)- (X =O, S), N.....N, N...C(=X)...N (X =O, S)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6524—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having four or more nitrogen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • 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
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to prodrugs of O-alkylated PI-3 kinase inhibitors, combinations thereof, and methods of using these prodrugs and combinations.
  • PI-3 kinases are a large family of lipid kinases that phosphorylate phosphatidylinositol in the D3 position to generate an important second messenger, phosphatidylinositol 3 '-phosphate.
  • Members of the PI-3 kinase family are divided into 3 classes based on sequence homology and the product formed by enzyme catalysis.
  • the class I PI-3 kinases are composed of 2 subunits: a 110 kd catalytic subunit and an 85 kd regulatory subunit.
  • Class I PI-3 kinases are involved in important signal transduction events downstream of cytokines, integrins, growth factors and immunoreceptors, which suggests that control of this pathway may lead to important therapeutic effects.
  • LY294002 (2-(4-morpholinyl)-8- phenyl-4H-l-benzopyran-4-one) (Compound 1) is a well known specific inhibitor of class I PI-3 kinases and has been demonstrated to possess anti-cancer properties.
  • LY294002 has no tissue specific properties and has been demonstrated to be rapidly metabolized in animals. Because of these factors, LY294002 would need to be administered at frequent intervals and thus has the potential to also inhibit PI-3 kinases in normal cells thereby leading to undesirable side effects.
  • PI3K PI-3 kinase
  • O-alkylated PI-3 kinase inhibitors that are useful alone or in combination with other agent(s) or treatment(s) for therapeutic use including for treating cancer.
  • Ring A is benzo;
  • Z 1 is S or O;
  • Z 2 is S or O
  • R 1 and R 2 independently are H, optionally substituted Ci_ 24 aliphatic, optionally substituted aryl, hydroxyl, halogen, Ci_ 24 alkoxy, C 3 _ 12 heterocycle, cyano, amino, or, are taken together to form an optionally substituted C 3 _ 12 cycloaliphatic or optionally substituted aryl;
  • R 3 represents H, optionally substituted Ci- ⁇ aliphatic, and optionally substituted aryl
  • R 4 and R 5 independently are H, optionally substituted Ci_ 12 aliphatic, optionally substituted aryl, C 3 _ 12 heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted C 3 _ 12 heterocycle or optionally substituted heteroaryl;
  • Re represents H, optionally substituted Ci_ 24 aliphatic, optionally substituted aryl, alkoxy, carboxy, amino, C 3 _ 12 heterocycle, aryloxy, any of which may be optionally substituted with a targeting agent, selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate; and
  • a targeting agent selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate; and
  • L represents a linker group selected from oxygen, sulphur, -NH-, -CH 2 -, -C(O)-, - C(O)NH- or saturated or unsaturated aliphatic group of up to 6 carbon atoms wherein one or two saturated carbons of the chain are optionally replaced by -C(O) , C(O)C(O) , -CONH-, - CONHNH-, -C(O)O-, -OC(O)-, -NHCO 2 -, -0-, -NHCONH-, -OC(O)NH-, -NHNH-, -NHCO-, - S-, -SO-, -SO 2 -, -NH-, -SO 2 NH- or NHS02-; wherein the bond between Z 1 and L of the compound is hydrolyzable.
  • the compound may have the formula:
  • Z 3 and Z 4 independently are S or O;
  • R 7 represents -CH 2 -, -CH(CH 3 )-, -CH(Ph)-, -C(CH 3 )(COOH)- or CH(CH(CH 3 ) 2 ).
  • the R 1 -RnIgA-R 2 of the compound may have a structure may have a formula selected from the group consisting of:
  • R4-N-R 5 are selected from the group consisting of:
  • R 6 is selected from the group consisting of:
  • the compound may have the formula:
  • the compound may also have the formula:
  • T is selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate.
  • the R 6 -T of the compound may have a structure selected from the group consisting of:
  • the compound may also have the formula:
  • T of the compound may be a vitamin, which may be folate or vitamin C.
  • T may also be a peptide, which may be an RGD-containing peptide selected from the group consisting of RGDs, c(RGDfK), vitronectin, fibronectin, somato statin-receptor agonists and somatostatin-receptor antagonists.
  • T may also be a bone-seeking agent, which may be selected from the group consisting of a phosphonate, phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate, and hydroxyapatite-binding polypeptides.
  • the bone-seeking agent may also be EDTMP, DOTMP, ABDTMP, BAD, MTX-BP, CF-BP, (Asp)6, (Glu)6, alendronate, pamidronate, 4 aminobutylphosphonic acid, l-hydroxyethane-l,l-diphosphonic acid, aminomethylenebisphosphonic acid, phytic acid, or N,N-bis(methylphosphono)-4-amino-benzoic acid.
  • a method of treating a PI3K-related cancer in a patient in need thereof comprising administering an effective amount of the compound.
  • the cancer may be selected from the group consisting of: brain cancer, lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, ovary cancer, prostate cancer, testes cancer, gastric cancer, genitourinary tract cancer, lymphatic cancer, rectum cancer, larynx cancer, pancreas cancer, esophagus cancer, stomach cancer, gall bladder cancer, cervix cancer, thyroid cancer, skin cancer, hematopoietic cancer, mesenchymal cancer, thyroid cancer, follicular cancer, multiple myeloma, and nervous system cancer.
  • Also provided herein is a method of inhibiting PD kinase in a cancer cell comprising administering to a patient in need thereof an effective amount of the compound. Also provided herein is a method of inhibiting tumor growth comprising administering to a patient in need thereof an effective amount of the compound.
  • a method for treating a PD-K related non-cancer disease comprising administering to a patient in need thereof an effective amount of the compound.
  • the disease may be selected from the group consisting of: inflammatory disease, pancreatitis, ulcers, age-related macular degeneration, hypertension, autoimmune disease, graft versus host disease, rheumatoid arthritis, atherosclerosis, thrombosis, PTEN-related disease, and diabetes.
  • the PTEN-related disease may be Cowden's disease.
  • Figure 1 shows the chemical structure of EDTMP, DOTMP, ABDTMP, BAD, MTX-BP and CF-BP.
  • Figure 2 shows the chemical structures of potential bone targeting agents.
  • Figure 3 shows the chemical reaction for modifying a phosphonate in a bone targeting agent.
  • Figure 4 shows the alkylation reaction to modify a phosphonate in a bone targeting agent.
  • Figure 5 shows a concept for chemically modifying EDTMP and DOTMP.
  • Figure 6 shows the inhibition of phagocytosis by LY294002 in J774 cells.
  • the columns indicate phagocytic index or percentage of cells positive for phagocytic response.
  • the phagocytic index is the number of sRBC's (sheep red blood cells) found per 100 J774 cells and the % of phagocytic cells is the % of J774 cells that have phagocytized at least 1 sRBC.
  • the error bars represent standard deviation of mean.
  • Figure 7A shows the UV and ELS Chromatograms of Compound 1126 (AO36-33).
  • Figure 7B shows the UV and ELS and mass spec Chromatograms of Compound SFl 126
  • Figure 8 shows the Positive Mass Spectrum of Compound 1126 (A036-33).
  • Figure 9 shows that Av ⁇ 3 targeted PI 3 kinase inhibitors abrogated the tube formation of
  • Figure 10 shows the crystal structure determined for SFl 110.
  • Figure 11 shows the carbon NMR spectrum of SFl 126.
  • Figure 12 shows the proton NMR spectrum of SFl 126.
  • Figure 13 shows LCMS chromatogram of SFl 103 (lot A102-62BS).
  • Figure 14 shows proton NMR spectrum of SFl 101 (lot A102-62BS).
  • Figure 15 shows carbon NMR spectrum of SFI lOl (lot A102-62BS).
  • Figure 16 shows the crystal structure of SFl 103.
  • Figure 17 shows that SFl 126 and rapamycin combination exhibit synergistic growth inhibition at 1.6 nM Rapamycin and 1.6 mM SFl 126 in RCC cells.
  • Figure 18 shows the drug response curves of RCC cells treated with combined SFl 126 and rapamycin.
  • Figure 19 shows the effect of SFl 126 (8 mM) and rapamycin (8 nM) in combination on
  • Figure 20 shows dose response curves of HBEC treated with SFl 126 and rapamycin.
  • Figure 21 shows the SFl 126 dose effect on cellular pAkt inhibition as stimulated by
  • Figure 22 shows the effects of SFl 101 and SFl 126 on dose-related apoptosis induction in 786-0 cells.
  • Figure 23 shows the combined effects of SFl 126 and rapamycin in 786-0 renal cancer cells.
  • Figure 24A shows the combined effect of SFl 126 and rapamycin on 786-0 renal tumor growth in nude mice.
  • Figure 24B shows the combined effect of SFl 126 and rapamycin on tumor weight in a
  • Figure 24C shows the combined effect of SFl 126 and rapamycin on mouse weight.
  • Figure 26 shows the combined effects of SFl 126 and docetaxel on PC3 tumor growth.
  • Figure 27 shows the combined effects of SFl 126 and docetaxel on PC3 tumor growth.
  • Figure 28 shows the effect of SFl 126 and docetaxel on PC3 xenograft tumors.
  • Figure 29 shows the effect of SFl 126 combined with various anti-cancer agents on PC3,
  • Figure 30 shows the effects of SFl 126 and docetaxel on growth inhibition of DU145 prostate cancer cells in nude mice.
  • Figure 31 shows the effect of docetaxel alone or in combination with SFl 126 on the inhibition of DU145 prostate cancer in nude mice.
  • branched refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group contains one or more subordinate branches from the main chain.
  • a branched group may contain from 1 to 12 backbone atoms, and may include isobutyl, t-butyl, isopropyl, -CH 2 CH 2 CH(CHS)CH 2 CH 3 , -CH 2 CH(CH 2 CH 3 )CH 2 CH 3 ,
  • unbranched refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group extends in a direct line.
  • An unbranched group may contain from 1 to 12 backbone atoms.
  • cyclic or "cyclo” as used herein alone or in combination refers to a group having one or more closed rings, whether unsaturated or saturated, possessing rings of from 3 to
  • lower refers to a group with 1 to 6 backbone atoms.
  • saturated refers to a group where all available valence bonds of the backbone atoms are attached to other atoms.
  • a saturated group may be butyl, cyclohexyl, piperidine or the like.
  • unsaturated refers to a group where at least one available valence bond of two adjacent backbone atoms is not attached to other atoms.
  • aliphatic refers to an unbranched, branched or cyclic hydrocarbon group, which may be substituted or unsubstituted, and which may be saturated or unsaturated, but which is not aromatic.
  • aliphatic further includes aliphatic groups, which comprise oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
  • aromatic refers to an unsaturated cyclic hydrocarbon group having 4n+2 delocalized ⁇ (pi) electrons, which may be substituted or unsubstituted.
  • aromatic further includes aromatic groups, which comprise a nitrogen atom replacing one or more carbons of the hydrocarbon backbone.
  • An aromatic group may be phenyl, naphthyl, thienyl, furanyl, pyridinyl, (is)oxazoyl or the like.
  • substituted refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group.
  • a substituted group may be substituted with one to five, or one to three substituents.
  • a substituent group may be an aliphatic group, aromatic group, alkyl, alkenyl, alkynyl, aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro, phosphate- containing group, sulfur-containing group, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio, thiocarboxylate, alkylsulfinyl, trifluoromethyl,
  • alkyl refers to a branched or unbranched, saturated aliphatic group.
  • An alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl or the like.
  • alkenyl as used herein alone or in combination refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon double bond which may occur at any stable point along the chain.
  • An alkenyl group may be ethenyl, E- or
  • alkynyl refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon triple bond which may occur at any stable point along the chain.
  • An alkynyl group may be ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl or the like.
  • aryl refers to a substituted or unsubstituted aromatic group, which may be optionally fused to other aromatic or non-aromatic cyclic groups.
  • An aryl group may be phenyl, benzyl, naphthyl, benzylidine, xylyl, styrene, styryl, phenethyl, phenylene, benzenetriyl or the like.
  • alkoxy refers to an alkyl, alkenyl or alkynyl group bound through a single terminal ether linkage.
  • An alkoxy may be methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, or trichloromethoxy.
  • aryloxy refers to an aryl group bound through a single terminal ether linkage.
  • halogen refers to fluorine ("F"), chlorine (“Cl”), bromine (“Br”), iodine ("I”), and astatine (“At”).
  • a halo group may be chloroacetamido, bromoacetamido, idoacetamido or the like.
  • hetero refers to a group that includes one or more atoms of any element other than carbon or hydrogen.
  • a hetero group may contain a heteroatom, which may be nitrogen, oxygen, sulfur or phosphorus.
  • heterocycle refers to a cyclic group containing a heteroatom.
  • a heterocycle may be pyridine, piperadine, pyrimidine, pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine, thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole, furan, benzofuran, dibenzofuran, thiophene, thiazole, benzothiazole, benzoxazole, benzothiophene, quinoline, isoquinoline, azapine, naphthopyran, furanobenzopyranone or the like.
  • carbonyl or “carboxy” as used herein alone or in combination refers to a group that contains a carbon-oxygen double bond.
  • Groups which contain a carbonyl may be aldehydes (i.e., formyls), ketones (i.e., acyls), carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e., imidos), esters, anhydrides or the like.
  • CH 2 C(Q)C(O)O- where Q is an aliphatic or aromatic group.
  • cyano refers to a carbon-nitrogren double bond.
  • a cyano group may be isocyanate, isothiocyanate or the like.
  • amino refers to a group containing a backbone nitrogen atom.
  • An amino group may be alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido or the like.
  • phosphate-containing group refers to a group containing at least one phosphorous atom in an oxidized state.
  • a phosphate-containing group may be phosphonic acid, phosphinic acid, phosphate ester, phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl, phosphoranylidene, phosphoroso or the like.
  • sulfur-containing group refers to a group containing a sulfur atom.
  • a sulfur-containing group may be sulfhydryl, sulfeno, sulfino, sulfinyl, sulfo, sulfonyl, thio, thioxo or the like.
  • “optionally substituted alkyl” means that the alkyl group may or may not be substituted and that the description includes both unsubstituted alkyl and alkyl where there is a substitution.
  • the term "effective amount,” when used in reference to a compound, product, or composition as provided herein, means a sufficient amount of the compound, product or composition to provide the desired result. The exact amount required will vary depending on the particular compound, product or composition used, its mode of administration and the like. Thus, it is not always possible to specify an exact "effective amount.” However, an appropriate effective amount may be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation.
  • suitable refers to a group that is compatible with the compounds, products, or compositions as provided herein for the stated purpose. Suitability for the stated purpose may be determined by one of ordinary skill in the art using only routine experimentation .
  • hydrolyzable refers to whether the group is capable of or prone to hydrolysis (i.e., splitting of the molecule or group into two or more new molecules or group).
  • the present invention provides a compound, which upon cleavage of one bond yields a compound of the formula:
  • Ring A is benzo
  • Z 1 and Z 2 independently are S or O;
  • R 1 and R 2 independently are H, optionally substituted aliphatic, optionally substituted aryl, hydroxyl, halogen, alkoxy, heterocycle, cyano, amino, or, are taken together to form an optionally substituted cycloaliphatic or optionally substituted aryl;
  • R 3 represents H, optionally substituted aliphatic, and optionally substituted aryl; and R 4 and R 5 independently are H, optionally substituted aliphatic, optionally substituted aryl, heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted heterocycle or optionally substituted heteroaryl.
  • the present invention also provides a compound of the formula:
  • Ring A is benzo
  • Z 1 and Z 2 independently are S or O;
  • R 1 and R 2 independently are H, optionally substituted aliphatic, optionally substituted aryl, hydroxyl, halogen, alkoxy, heterocycle, cyano, amino, or, are taken together to form an optionally substituted cycloaliphatic or optionally substituted aryl;
  • R 3 represents H, optionally substituted aliphatic, and optionally substituted aryl
  • R 4 and R 5 independently are H, optionally substituted aliphatic, optionally substituted aryl, heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted heterocycle or optionally substituted heteroaryl;
  • R 6 represents H, optionally substituted aliphatic, optionally substituted aryl, alkoxy, carboxy, amino, heterocycle, aryloxy, and optionally substituted therewith a targeting agent;
  • L represents a linker group
  • the positive charge is likely resonance delocalized between the oxygen (pyrylium ion) and on the nitrogen (quaternized Nitrogen form) as shown below to give the resonance form:
  • Compound 2 or 3 may be a compound wherein, R 1 -RnIg A-R 2 is selected from the group consisting of the following:
  • the compound may be a para-amino such as:
  • Compound 2 or 3 may also be a compound wherein, R 4 -N-R 5 is selected from the group consisting of the following:
  • Compound 2 or 3 may be a compound wherein, R 6 is selected from the group consisting of the following: a. Linker
  • Compound 3 may be a compound wherein the linker group is hydrolyzable.
  • the linker group of the prodrug may be cleaved by enzymatic cleavage or by hydrolysis to yield Compound 2.
  • the physiological conditions of this cleavage may include aqueous conditions in living animals.
  • the rate of hydrolysis of the linker group under physiological conditions may be from about 1 minute half-life to about 48 hour half-life.
  • hydrolyzable refers to whether the group is capable of or prone to hydrolysis (i.e., splitting of the molecule or group into two or more new molecules or groups due to the net insertion of a water molecule) at a rate of about 1 minute half- life to 48 hour half- life.
  • the linker group may be any group that may be hydrolyzed or enzymatically cleaved to yield Compound 2.
  • the linker group may be of the formula:
  • Z 3 and Z 4 independently are S or O;
  • R 7 represents -CH 2 -, -CH(CH 3 )-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-.
  • a compound disclosed herein may be a compound wherein R 6 further comprises one or more targeting agents (T) covalently attached thereto.
  • T targeting agents
  • Targeting agents allow the prodrugs of the present invention to be delivered selectively to specific types of cells, tissues, organs or extracellular structures.
  • Treatment with Compound 1 suffers from poor bioavailability, rapid metabolism and side effects because the compound is not tissue specific. Therefore, it is highly desirable to limit the location of the drug to that of the area of treatment or at least prevent it from reaching the tissues where if can cause side effects, and to ensure that at any particular time effective, but not excessive, amounts of the drug are used.
  • targeting agents may allow the prodrugs of the present invention to be concentrated at the site of treatment rather than evenly distributed throughout the entire body or to be metabolized prematurely or excreted too quickly.
  • the linker Once being delivered to the site of treatment, the linker may be enzymatically cleaved or hydrolyzed as described above to yield Compound 2.
  • the use of targeting agents may limit the dosage required to be administered in order to achieve an effective concentration of the drug at the site of treatment.
  • the use of targeting agents may also allow for more infrequent dosage or even alternative methods of administration in order to achieve an effective concentration of the drug at the site of treatment.
  • the targeting agent may be attached to the compounds of the present invention via a covalent bond, which may be formed by methods such as a nucleophilic or electrophilic group of the targeting agent that is covalently reacted with an electrophilic or nucleophilic group (respectively) on the linker.
  • Compound 2 or 3 may be a compound wherein, R 6 -T is selected from the group consisting of the following:
  • the targeting agent may be a carbohydrate, vitamin, peptide, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent or cartilage-seeking agent.
  • the targeting agent may also be a molecule that is bound by a receptor in a desired tissue and optionally transported into a cell by a receptor-mediated process.
  • the molecule may be a diazepine that binds to peripheral benzodiazepine receptors (PBRs) present in glial cells in the brain.
  • PBRs peripheral benzodiazepine receptors
  • the diazepine may be one discussed in G. Trapani, et al. Bioconjugate Chem.
  • the vitamin may be folate, vitamin B 12 or vitamin C.
  • the term "folate” encompasses folic acid derivatives with capacity to bind with folate-receptors.
  • the folate may be folic acid, folinic acid, pteropolyglutamic acid, or a folate receptor-binding pteridine such as a tetrahydropterin, dihydrofolate, or tetrahydrofolate or its deaza or dideaza analogs.
  • the folate may be a folate analog such as aminopterin, amethopterin (methotrexate), Nio-methylfolate, 2- deamino-hydroxyfolate, a deaza analog such as 1-deazamethopterin or 3-deazamethopterin, or 3'5'-dichloro4-amino-4-deoxy-N 1 o-methylpteroyl-glutamic acid (dichloromethotrexate).
  • a folate analog such as aminopterin, amethopterin (methotrexate), Nio-methylfolate, 2- deamino-hydroxyfolate, a deaza analog such as 1-deazamethopterin or 3-deazamethopterin, or 3'5'-dichloro4-amino-4-deoxy-N 1 o-methylpteroyl-glutamic acid (dichloromethotrexate).
  • the targeting agent may be a peptide or peptidomimetic, which may be an RGD- containing peptide selected from the group consisting of RGD, c(RGDfK), vitronectin, fibronectin, somatostatin-receptor agonist or somato statin-receptor antagonist.
  • the targeting agent may also be a molecule that binds to the avb3 integrin receptor and acts as an antagonist, as described in U.S. Patent Nos. 6,552,079, 6,426,353B, WO 2002/40505A2, and U.S. Patent Publications 2002/0055499, 2002/0061885, 2002/0065291, 2002/0072500, U.S.
  • the targeting agent may be an antibody or fragment thereof, such as a tumor-specific monoclonal antibody or fragment thereof.
  • the bone-seeking agent may be phosphonate, phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate, or hydroxyapatite -binding polypeptide.
  • the targeting agent may also be chlorotoxin (US 6,429,187Bl) or tissue factor (G. M. Lanza, et al. "Targeted Antiproliferative Drug Delivery to Vascular Smooth Muscle Cells with a Magnetic Resonance Imaging Nanoparticle Contrast Agent"; Circulation, 2002 volume 106 pp2842-2847).
  • the targeting agent may also be an antibody of the class IgG, IgM, IgA, IgD or IgE, or a fragment or derivative thereof, including Fab, F(ab') 2 , Fd, or a single chain antibody, diabody, bispecific antibody, bifunctional antibody or derivative thereof.
  • the antibody may be a monoclonal antibody, polyclonal antibody, affinity-purified antibody, or a mixture thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.
  • the antibody may also be a chimeric antibody.
  • the antibody may be directed against a variety of antigenic determinants including those associated with tumors, histocompatibility and other cell surface antigens, bacteria, fungi, viruses, enzymes, toxins, drugs and other biologically active molecules.
  • the antibody may specifically react to a tumor-associated antigen such as carcinoembryonic antigen (CEA), a mucin such as TAG-72, a human milk fat globule antigen, prostate serum antigen (PSA), prostate specific membrane antigen (PSMA), PS (phosphatidyl serine), or a receptor, which may be the IL-2, EGF, VEGF or transferrin receptor.
  • CEA carcinoembryonic antigen
  • PSMA prostate specific membrane antigen
  • PS phosphatidyl serine
  • the antigen may also be as described in Zalcberg and McKenzie, J. Clin. Oncology, Vol. 3; pp. 876-82 (1985),WO 01/68709A1, and U.S. Patent Publication US2004/
  • the targeting agent may also be glucose, galactose, mannose, mannose 6-phosphate, hormone (e.g., insulin, growth hormone, or the like), growth factor or cytokine (e.g., TGF ⁇ , EGF, insulin-like growth factor, or the like), YEE(GaINAc AH). sub.3 or a derivative, cobalamin, OC-2 macroglobulin, asialoglycoprotein, albumin, texaphyrin, metallotexaphyrin, antibody, antibody fragment (e.g., Fab), single-chain antibody variable region (scFv), transferrin, any vitamin or any coenzyme
  • the targeting agent may also be an agent that delivers the prodrug to bones.
  • the bone targeting agent may be EDTMP, DOTMP, or ABEDTMP, which are disclosed in U.S. Patent Nos. 4,937,333, 4,882,142, 5,064,633 and WO-94/00143, the contents of which are incorporated herein by reference.
  • DOTMP or EDTMP may be attached to the linker moiety by any method including the coupling chemistry shown in Figure 3 and the alkylation chemistry shown in Figure 4 where the R group can have an appropriate electrophilic or nucleophilic group that reacts with the nucleophilic or electrophilic (respectively) group of the linker moiety.
  • the targeting agent may be used to deliver the prodrug to bones as a slow release reservoir site for the compounds of the present invention.
  • the targeting agent may be a bone seeking (osteotropic) moiety attached to a compound disclosed herein via an acid cleavable linker attached to the quaternary amine.
  • the acid cleavable linker may be an ortho acid-amide linkage.
  • the protein-ACL-3 amide linkage is readily cleaved freeing the native amino group of the amide functionality as described in WO-94/00143 the contents of which are incorporated by reference.
  • the attachment tethering the prodrug to bone may be cleaved releasing the compounds of the present invention.
  • the targeting agent used to deliver the prodrugs to bones may be a molecule that binds with notch receptors. Notch signaling plays a key role in the development and differentiation of various hematopoietic lineages. As discussed in Jundt et al., Blood, 102(11): 928a (2003), ligand-induced notch signaling is a novel growth factor for multiple myeloma cells and suggests that these interactions contribute to lymphomagenesis of multiple myeloma in vivo. [0102]
  • the bone targeting agent may have a high affinity for calcium ions in hydroxyapatite, the major constituent of bone.
  • the compound of the invention can be targeted to calcium deposits in regions of the body other than bone, such as calcium deposits in the arteries, heart, kidney, or gall bladder.
  • the bone targeting agent ideally selectively binds to bone tissue.
  • the bone targeting agent may be attracted to the bone tissue of the subject, and may bind to the bone with a higher affinity than non-bone tissues, and may remain bound for a certain length of time, thereby delivering the composition to a bone environment.
  • the bone targeting agent may bind to bone tissue with at least 2-fold greater affinity (e.g., at least 3-fold, at least 5- fold, at least 10-fold, or at least 25-fold greater affinity) than the bone targeting agent binds to non-bone tissue.
  • the bone targeting agent may reversibly bind to bone tissue, meaning that the bone targeting agent may eventually be released from bone and expelled from the body.
  • the bone targeting agent may remain bound to bone tissue for a sufficient period of time to allow the quaternary prodrug to be hydrolyzed, thereby delivering the active drug to the target cells (e.g., bone marrow cells).
  • the bone targeting agent may remain bound to bone for about 1 day (e.g., about 2 days, about 3 days, or about 7 days) to about 1 year (e.g., about 330 days, about 365 days, or about 400 days), after which the bone targeting agent is expelled from the body.
  • the bone targeting agent may remain bound to bone for about 7 days (e.g., about 7 days, about 14 days, or about 21 days) to about 6 months (e.g., about 90 days, about 120 days, or about 150 days).
  • a bone targeted prodrug may remain bound to the bone for 30 days, during which time the drug may be released. After about 45 days the bone targeting agent may be released from the bone and eventually excreted.
  • the bone targeting agent may be selected based on binding kinetics to bone tissue.
  • a candidate bone targeting agent may be screened in vitro by determining affinity to bone tissue (e.g., hydroxyapatite) in, for example, a multi-well format.
  • a candidate bone targeting agent may also be screened in vivo by assessing the rate and timing of excretion of candidate bone targeting agents from the body.
  • the bone targeting agent may be expelled from the body via the kidneys.
  • the bone targeting agent may be selected from the group consisting of a phosphate, a phosphonate, a bisphosphonate, a hydroxybisphosphonate, an aminomethylenephosphonic acid, and an acidic peptide.
  • the bone targeting agent may carry one, more than one, or a mixture of these groups.
  • the bone targeting agent may be a phosphonate, meaning that the bone targeting agent may comprise one phosphonate, two phosphonates, or three or more phosphonates.
  • EDTMP ethylene diamine-N,N,N',N'-tetrakis(methylenephophonic acid), the chemical structure of which is set forth in Figure 1, currently FDA approved (QuadrametTM) as the radioactive 153 Sm complex for delivering a selective radiation dose to bone metastases for pain palliation.
  • EDTMP is a phosphonate that contains four phosphonic acid groups, and is therefore a tetraphosphonate.
  • Compounds such as 153 Sm-EDTMP are selectively localized in bone where tumors are present versus normal bone in a ratio of more than 10:1, probably because metabolic turnover of calcium is very high in the metastatic region.
  • the 153 Sm-EDTMP reportedly is rapidly taken up by the skeleton in osteoblastic bone metastases and cleared from the plasma. That portion of the compound that does not accumulate in the skeleton reportedly is rapidly excreted, and excretion is almost complete within 6 hours after administration (Jimonet et al., Heterocycles, 36, 2745 (1993)). The pain palliation is thought to be due to the radiation originating from the isotope bound to the osteoblastic bone metastases having some effect on the nearby metastatic tumor cells.
  • DOTMP the chemical structure of which is set forth in Figure 1, now in Phase III clinical trials (termed STR, skeletal targeted radiation) as the radioactive 166 Ho complex designed to deliver large doses of radiation selectively to the bone marrow for the treatment of multiple myeloma.
  • STR Phase III clinical trials
  • the radioactive 166 Ho-DOTMP complex localizes in the skeletal system and irradiates the nearby bone marrow which houses the malignant myeloma cells.
  • the phosphonate that does not localize in the bone is cleared through the urine and out the body.
  • the skeletal uptake is about 20 to about 50% of the injected dose, and the localization in areas of the skeleton with tumor infiltration is illustrated in Figure 7 of Bayouth et al., J. Nucl. Med., 36 b 730 (1995).
  • the bone targeting agent may be a polyphosphonic acid.
  • Polyphosphonic acid has been demonstrated to successfully target biologically-active molecules to bone tissue.
  • conjugation (via isothiocyanato chemistry) of polyaminophosphonic acids, such as ABDTMP (the chemical structure of which is set forth in Figure 1, to growth factors (to stimulate bone formation) successfully resulted in the targeting of the growth factors to the bones of rats (see, for example, International Patent Application WO 94/00145).
  • the bone targeting agent may be coupled to a protein.
  • bisphosphonates that were conjugated to human serum albumin successfully delivered the protein to bone in vitro (Biotechnol. Prog., 16, 258 (2000)) and in vivo (Biotechnol.
  • bone-seeking agents extends beyond delivery of proteins to bone and includes, for instance, small therapeutic molecules.
  • a conjugate comprising a bone-seeking bisphosphonate and an alkylating agent, such as BAD (the chemical structure of which is set forth in Figure 1, has been generated (see, for example, Wingen et al., J. Cancer Res. Clin. Oncol, 111, 209 (1986)).
  • BAD the chemical structure of which is set forth in Figure 1
  • the alkylating agent is not specific in its interaction with its target (DNA), and, thus, there is no requirement for cleavage between the bisphosphonate (i.e., bone-seeking agent) and the alkylating moiety.
  • the bisphosphonate-alkylating agent demonstrated efficacy in a rat osteosarcoma model using BAD.
  • Another series of studies have been performed using the antifolate antineoplastic agent methotrexate that has been covalently attached to bisphosphonates, designated MTX-BP and shown in Figure 1 (see, for example, Sturtz et al., Eur. J. Med. Chem., 27, 825 (1992); Sturtz et al, Eur. J. Med. Chem., 28, 899 (1993); and Hosain et al, J. Nucl. Med., 37, 105 (1996)).
  • the CF group is a fluorescent marker to quantitate pharmacokinetics and biodistribution, and is connected to the bone targeting agent through an ester bond which is susceptible to hydrolysis in vivo.
  • the bone-seeking agent may also be a peptide, such as (Asp) 6 and (GIu) 6 .
  • the acid-rich peptide sequence of the glycoprotein osteonectin which is found in abundance in bone and dentin, has a strong affinity to hydroxyapatite (Fujisawa et al., Biochimica et Biophysica Acta, 53, 1292 (1996)).
  • peptide ligands comprising acidic amino acids are ideal candidates for bone targeting agents.
  • estradiol- (Asp) 6 conjugates to bone has been demonstrated in ovariectomized animals with concomitant inhibition of osteoporectic-type bone loss (Kasugai et al., Journal of Bone and Mineral Research (Suppl 1), 14, S534 (1999)). It is believed that the (Asp) 6 tether to bone is metabolized during the bone resorption process mediated by osteoclasts. Therefore, the acidic peptide ligand provides not only a means of recruiting compounds to bone, but also provides a mechanism of slowly releasing compounds to bone cells and surrounding tissue.
  • the bone targeting agent may also be amino- and hydroxy-alkyl phosphonic or diphosphonic acid; a hydroxybisphosphonic acid such as alendronate, pamidronate, 4- aminobutylphosphonic acid, 1-hydroxyethane- 1,1 -diphosphonic acid, or aminomethylenebisphosphonic acid; a phosphate such as phytic acid; or a aminomethylenephosphonic acid such as N,N-bis(methylphosphono)-4-amino-benzoic acid or nitrilotri(methylphosphonic acid).
  • a hydroxybisphosphonic acid such as alendronate, pamidronate, 4- aminobutylphosphonic acid, 1-hydroxyethane- 1,1 -diphosphonic acid, or aminomethylenebisphosphonic acid
  • a phosphate such as phytic acid
  • aminomethylenephosphonic acid such as N,N-bis(methylphosphono)-4-amino-benzoic acid or nitrilotri(methylphosphonic acid.
  • the bone targeting agent may be an aminomethylenephosphonic acid.
  • aminomethylenephosphonic acid is meant a compound that contains an -NCH 2 PO 3 H moiety, where the amino group has one, two, or three methylenephosphonic acid groups attached, and may be further substituted with other chemical moieties.
  • An aminomethylenephosphonic acid may include one or more phosphonic acid groups and one or more amino groups. Examples of these aminomethylenephosphonic acids include the compounds F through N set forth in Figure 2.
  • the bone targeting agent may be attached through one of the heteroatoms or by chemical modification that installs an additional attachment point.
  • EDTMP can be connected to a linker by one of the phosphorous oxygens to create a phosphonate linkage, as illustrated in Figure 3 (see for example Vieira de Almedia et al, Tetrahedron, 55, 12997-13010 (1999).)
  • the phosphorous oxygen can also be alkylated as shown in Figure 4, where the R group can have, for example, a pendant amino group, to provide a secondary attachment point for ligation to, for example, an activated PEG.
  • alkylation examples similar to that involving DOTMP, as has been further described in Chavez et al., Biomedical Imaging: Reporters, Dyes, & Instumentation, Contag & Sevick-Muracia, Eds., Proc. SPIE, Vol. 3600, 99-106 (July, 1999), or as shown for other phosphonic acids further described in, for example, U.S. Patent 5,177,064, U.S. Patent 5,955,453, de Lombaert et al., J Med. Chem., 37, 498-511 (1994), and Iyer et al., Tetrahedron Letters, 30(51), 7141-7144 (1989).
  • EDTMP can be, for example , modified to generate ABDTMP by installation of an aniline group (as further described in, for example, Figure 1 of International Patent Application WO 94/00145).
  • the aniline amine is then available to form, for example, an amide bond.
  • DOMTP could be similarly modified, as outlined in Figure 5.
  • the terms "phosphonate, phosphate, and aminomethylenephosphonate” are meant to encompass the phosphonic acids, the phosphoric acids, and aminomethylenephosphonic acids, respectively, as well as any salts, hydrolyzable esters, and prodrugs of the phosphorous-based acids thereof.
  • a certain portion of the phosphate or phosphonate of the bone targeting agent may be deprotonated and replaced with a counterion.
  • the exchange of proton for calcium is an inherent event for the binding of the bone targeting agent to the hydroxyapatite in the invention.
  • preparation and administration of the composition containing the bone targeting agent may or may not require complete protonation of the phosphorous acids therein. Therefore, the phosphonic acid, phosphoric acid, and aminomethylenephosphonic acid are drawn and utilized interchangeably with phosphate, phosphonate, and aminomethylenephosphonate.
  • Biologically hydrolyzable esters of the phosphorus-based acids may also be utilized in the in vivo use of the bone targeting prodrugs.
  • prodrugs of the phosphorous-based acids may also be utilized in vivo to mask the acidity of the composition during, for example, formulation and administration.
  • the targeting agent may also be an agent that targets based upon properties of the particular tissue.
  • the bone targeting agent may be a polymer that is selectively localized in tumor tissues due to the EPR effect (enhanced permeability and retention) as described in H. Maeda et al "Tumor vascular permeability and the EPR effect in macromolecular therapeutics: A Review”; Journal of Controlled Release, 2000 vol 63, pp 271-284, the contents of which are incorporated by reference.
  • the polymer may also be N-(2-hydroxypropyl)methacrylamide (HPMA) or (poly)L-glutamic acid.
  • the targeting agent may also comprise an RGD moiety.
  • RGD moieties target RGD fusion proteins to vasculature by interacting with interacts with cell adhesion receptors, including ⁇ v ⁇ 3 integrin. 3. Synthesis a. Main Ring System
  • the compounds of the present invention may be synthesized using LY294002 (Compound 1) as a starting product.
  • LY294002 (Compound 1) may be obtained commercially or synthesized as described in Example 1 or as described in U.S. Patent No. 5, 703, 075, the contents of which are incorporated herein by reference.
  • One of ordinary skill in the art may also synthesize the compounds of the present invention using Compound 2 as a starting product.
  • the main ring system of Compounds 2 and 3 may be derivatives of the main ring system of LY294002 (Compound 1).
  • Derivatives of the main ring system of Compound 3 may be prepared as disclosed in U.S. Patent No. 5,703,075, the contents of which are incorporated herein by reference, for the preparation of main ring derivatives of LY294002 (Compound 1).
  • Derivatives of the main ring system of Compound 3 may also be prepared by using a commercially available compound such as substituted 2-hydroxy-acetophenone.
  • the amine derivatives of Compound 3 may be prepared by the displacement of the thioalkyl group in Example 1 under conditions ranging from room temperature to forcing conditions (excess nucleophile and heating to 110 0 C). Any primary or secondary nitrogen- containing nucleophile may react to give alternative amine substitutions to the morpholine ring structure (including different morpholine analogs).
  • the synthesis of representative examples of such amine derivatives of Compound 3 has been described previously in US 6,949,537, the contents of which are incorporated herein by reference. d. Preparation of Esters
  • esters may be used to form the compounds of the present invention.
  • a compound disclosed herein may be formed using halo esters.
  • the compound may be formed using chloromethyl esters.
  • Numerous chlorlomethyl esters useful in the preparation of the compounds of the present invention are available from commercial sources.
  • chloromethyl esters may be synthesized as described in WO 02/42265, WO 94/23724, and U.S. Patent Nos. 4,444,686, 4,264,765, and 4,342,768, the contents of which are incorporated herein. e. Alkylation
  • the prodrugs of the present invention may be prepared by alkylating the keto oxygen of Compound 1 or Compound 2 with a halomethyl ester.
  • the alkylated compounds are generally not reversible under mild conditions. However, the positively charged alkylated compounds of the present invention are readily hydrolyzable as discussed above.
  • Halomethyl esters that may be used to alkylate the keto oxygen of Compound 1 or Compound 2 are commercially available or may be prepared as described in the Examples below. f.
  • the prodrugs of the present invention may also be prepared by alkylating a keto oxygen of Compound 1 or Compound 2 with a linker comprising at least two functional groups.
  • the linker may be any natural or synthetic linker that is capable of alkylating a keto oxygen of the chromanone ring and is also capable of being covalently attached to a targeting molecule or may already be attached to a targeting molecule.
  • the prodrugs of the present inventions may also be prepared by acylation or sulfonylation and the like.
  • the linker may be an atom such as oxygen or sulfur, a unit such as -NH-, -CH 2 -, -C(O)-, - C(O)NH-, or a chain of atoms.
  • the molecular mass of the linker may be in the range of about 14 to 200, or may be in the range of 14 to 96 with a length of up to about six atoms.
  • the linker may also be a saturated or unsaturated aliphatic group which is optionally substituted, and wherein one or two saturated carbons of the chain are optionally replaced by -C(O)-, -C(O)C(O)-, -CONH-, -CONHNH-, -C(O)O-, -OC(O)-, -NHCO 2 -, -O-, -NHCONH-, -OC(O)NH-, -NHNH-, -NHCO-, -S-, -SO-, -SO 2 -, -NH-, -SO 2 NH-, or -NHSO 2 -.
  • the first functional group of the linker is used to alkylate the keto oxygen as discussed above.
  • a first functional group may be a halomethyl ester such as chloromethylester or iodomethyl ester.
  • the second functional group of the linker may be used to covalently attach a targeting agent.
  • the second functional group may be an electrophilic group or a nucleophilic group.
  • Second functional groups for covalently attaching targeting groups may be isothiocyanate, haloacetamide maleimide, imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyl disulfide, phenyl azide, carboxyl (and acid chlorides thereof), amino, acyl hydrozide, semicarbazide, thiosemicarbazide, diazonium, hydrazine, azide, aminoalkylurea, aminoalkylthiourea, halotriazine, or meta (dihydroxyboryl)phenylthiourea.
  • Suitable reactive moieties which may be suitable for covalently attaching the prodrugs of the present invention to targeting agents may be disulfides, nitrenes, sulfonamides, carbodiimides, sulfonyl chlorides, benzimidates, -COCH 3 or -SO 3 H.
  • the appropriate second functional group will depend on the functional group of the targeting agent with which the covalent bond will be formed and by its susceptibility to loss of biological activity as a consequence of forming a given type of linkage.
  • the targeting agent is a protein
  • the second functional group may be reactive with side chain groups of amino acids making up the polypeptide backbone.
  • side chain groups include the carboxyl groups of aspartic acid and glutamic acid residues, the amino groups of lysine residues, the aromatic groups of tyrosine and histidine, and the sulfhydryl groups of cysteine residues.
  • Carboxyl side groups presented by a targeting agent such as a polypeptide backbone may be reacted with amine second functional groups by means of a soluble carbodiimide reaction.
  • Amino side groups presented by a targeting agent may be reacted with isothiocyanate, isocyanate or halotriazine second functional groups to effect linkage to the prodrugs of the present invention.
  • amino side groups on the targeting agent may be linked to the prodrugs compounds of this invention bearing amine reactive groups by means of bifunctional agents such as dialdehydes and imidoesters.
  • Aromatic groups presented by a targeting agent may be coupled to the prodrugs of this invention via diazonium derivatives.
  • Sulfhydryl groups on targeting agent molecules may be reacted with maleimides or with haloalkyl targeting agent reactive groups such as iodoacetamide.
  • Free sulhydryl groups suitable for such reactions may be generated from the disulfide bonds of protein immunoglobulin or may be introduced by chemical derivatization. Linkage to free sulfhydryl groups generated in the intra-heavy chain region of immunoglobulins does not interfere with the antigen binding site of the immunoglobulin but may render the antibody incapable of activating complement.
  • the targeting agent is a glycosylated protein
  • an alternative to forming a linkage to the compounds of the present invention via the polypeptide backbone is to form a covalent linkage with the carbohydrate side chains of the glycoprotein according to the methods such as those of McKearn, et al., EPO 88,695.
  • the carbohydrate side chains of antibodies may be selectively oxidized to generate aldehydes which may then be reacted either with amine reactive groups to form a Schiff base or with hydrazine, semicarbazide or thiosemicarbazide reactive groups, to give the corresponding hydrazone, semicarbazone or thiosemicarbazone linkages.
  • Suitable targeting agent reactive moiety useful for linkage to carbohydrates and polysaccharides without the necessity for prior oxidation is the dihydroxyboryl groups, such as is present in meta (dihydroxyboryl)phenylthiourea derivatives. This group is reactive with targeting agents containing a 1,2-cis-diol, forming a 5-membered cyclic borate ester, and thus is of use with those carbohydrates, polysaccharides and glycoproteins which contain this group.
  • the dihydroxyboryl derivatives may also be used to link the prodrugs of this invention to ribonucleosides, ribonucleotides and ribonucleic acids, since ribose contains a 1,2-cis-diol group at the 2',3' position, as disclosed by Rosenberg, et al., Biochemistry, 11, 3623-28 (1972).
  • Deoxyribonucleotides and DNA targeting agents may not be linked to the present prodrugs in this fashion as the 3' hydroxyl group is absent.
  • the latter targeting agents may be conjugated to isothiocyanate derivatives of prodrugs by first forming an allylamine derivative of the deoxyribonucleotide as disclosed by Engelhardt, et al., EPO 97,373.
  • the targeting agent to be linked with the prodrugs of this invention is an intact cell, either polypeptide reactive or carbohydrate reactive moieties may be employed.
  • Hwang and Wase, Biochim. Biophys. Acta, 512, 54-71 (1978) disclose the use of the diazonium derivative of the bifunctional EDTA chelator of Sundberg, et al., J. Med. Chem., 17, 1304 (1974), to label erythrocytes and platelets with indium- 111.
  • the dihydroxyboryl group is reactive with a variety of bacteria, viruses and microorganisms, see Zittle, Advan. Enzym., 12 493 (1951) and Burnett, et al., Biochem. Biophys. Res. Comm., 96, 157-62 (1980).
  • the compound may have one of the following structures:
  • the compound may be produced by the following reaction:
  • the linker may be used to covalently alkylate Compound 1 or Compound 2 and may have the formula:
  • X represents a halo group
  • Compound 4 of the present invention are those compounds wherein, X represents Cl or I;
  • Y represents -CH 2 -, -CH(CH 3 )-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-;
  • Compound 4 of the present invention are those compounds wherein,
  • X represents Cl or I
  • Y represents -CH 2 -, -CH(CH 3 )-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-;
  • Compound 4 provides linkers with both an alkyl and aryl carboxylic backbone which provides flexibility in the cleavage rate of the final quaternary nitrogen.
  • the linkers of Compound 4 may be prepared using commercially available starting products as described in Example 5. g. Purification
  • the compounds of the present invention may be isolated using standard purification methods.
  • the hydrolyzable bond of the compounds of the present may be prone to hydrolysis during the purification of the compounds.
  • the present invention is also directed to methods of purifying the compounds of the present invention comprising adding the compounds to a solution comprising at least 0.1% acid (v/v) to solubilize the compound.
  • the compound is then purified by performing chromatography, such as HPLC. h. Testing
  • the prodrugs of the present invention may be tested to determine the rate of hydrolysis of the hydrolyzable bond and the products of hydrolysis by performing HPLC analysis of the prodrug exposed to cleavage conditions as a function of time.
  • the biological activity of the compounds of the present invention may be measured by methods including blocking phagocytosis in macrophage cell line J774 cells.
  • the biological activity of the compounds of the present invention may also be measured by PDK enzyme assays as described by U.S. Patent No. 5,480,906; K. Fuchikami et al J. Biomol Screen, 2002 Oct. pp441-450; VI Silveria et al J. Biomol. Screen, 2002, Dec. 7(6), 507-514; BE Drees Combinatorial Chemistry and Highthroughput Screening 2003, vol 6, 321-330, the contents of which are incorporated by reference. i. Salts
  • compositions may be prepared from the active ingredients or their pharmaceutically acceptable salts in combination with pharmaceutically acceptable carriers.
  • salts of the compounds of the present invention which are suitable for use in the methods and compositions of the present invention include salts formed with a variety of organic and inorganic acids such as hydrogen chloride, hydroxymethane sulfonic acid, hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, glycolic acid, stearic acid, lactic acid, malic acid, pamoic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethonic acid, and include various other pharmaceutically acceptable salts, such as, e.g., nitrates, phosphates, borates, tartrates,
  • a salt of a compound disclosed herein may be a hydrochloride salt, methanesulfonic acid salt, trifluoroacetic acid salt, or methanesulfonic acid salt.
  • pharmaceutically acceptable salts of the compounds of the present invention may be formed with alkali metals such as sodium, potassium and lithium; alkaline earth metals such as calcium and magnesium; organic bases such as dicyclohexylamine, tributylamine, and pyridine; and amino acids such as arginine, lysine and the like.
  • the pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base, in a suitable solvent or solvent combination. [0141] In general, the counterions of the salts of the compounds of the present invention are determined by the reactants used to synthesized the compounds. There may be a mixture of counterions of the salts, depending on the reactants. For example, where NaI is added to facilitate the reaction the counterion may be a mixture of Cl and I counter anions.
  • the present invention also encompasses a composition comprising one or more compounds of the present invention.
  • the composition may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
  • additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
  • a composition disclosed herein may be in the form of tablets or lozenges formulated in a conventional manner.
  • tablets and capsules for oral administration may contain conventional excipients including a binding agent, filler, lubricant, disintegrant or wetting agent.
  • the binding agent may be syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone.
  • the filler may be lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, or sorbitol.
  • the lubricant may be magnesium stearate, stearic acid, talc, polyethylene glycol, or silica.
  • the disintegrant may be potato starch or sodium starch glycollate.
  • the wetting agent may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
  • the composition may also be a liquid formulation such as an aqueous or oily suspension, solution, emulsion, syrup, or elixir.
  • the composition may also be formulated as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain an additive such as a suspending agent, emulsifying agent, nonaqueous vehicle or preservative.
  • the suspending agent may be sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, or hydrogenated edible fat.
  • the emulsifying agent may be lecithin, sorbitan monooleate, or acacia.
  • the nonaqueous vehicle may be an edible oil, almond oil, fractionated coconut oil, oily ester, propylene glycol, or ethyl alcohol.
  • the preservative may be methyl or propyl p-hydroxybenzoate or sorbic acid.
  • the composition may also be formulated as a suppository, which may contain a suppository base such as cocoa butter or glycerides.
  • the composition may also be formulated for inhalation, which may be in a form such as a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
  • the composition may also be formulated in a transdermal formulation comprising an aqueous or nonaqueous vehicle such as a cream, ointment, lotion, paste, medicated plaster, patch, or membrane.
  • the composition may also be formulated for parenteral administration such as by injection or continuous infusion.
  • Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain a formulation agent such as a suspending, stabilizing, or dispersing agent.
  • the composition may also be provided in a powder form for reconstitution with a suitable vehicle including sterile, pyrogen-free water.
  • the composition may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
  • the composition may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
  • the composition may also be formulated as a liposome preparation.
  • the liposome preparation may comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell.
  • liposomes such as those described in U.S. Patent No. 5,077,211 of Yarosh, U.S. Patent No. 4,621,023 of Redziniak et a or U.S. Patent No. 4,508,703 of Redziniak et ah can be used.
  • the composition may be intended to target skin conditions, and may be administered before, during, or after exposure of the skin of the mammal to UV or agents causing oxidative damage.
  • the composition may comprise niosomes.
  • Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
  • the present invention also encompasses a method of treating a patient suffering from a condition associated with PDK activity.
  • the PDK activity may be abnormal, excessive, or constitutively active.
  • the present invention also encompasses a method for treating inflammatory disease comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • diseases and adverse health effects attributable to inappropriate PDK signaling activity have been disclosed in the art, for example U.S.
  • the present invention also encompasses a method for enhancing p53 mediated programmed cell death comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for enhancing the chemosensitivity of tumor cells comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for enhancing the radiosensitivity of tumor cells comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for inhibiting tumor induced angiogenesis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for inhibiting angiogenic processes associated with non-cancer diseases comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for treatment of cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for treating age-related macular degeneration (AMD) comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • AMD age-related macular degeneration
  • VEGF inhibits blood vessel overgrowth associated with AMD.
  • the compounds of the present invention may treat AMD by inhibiting angiogenesis.
  • the present invention also encompasses a method for treating hypertension comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • inhibition of PI3K may prevent the low extracellular concentrations of Mg 2+ that are associated with hypertension.
  • the present invention also encompasses a method for suppressing differentiation of progenitor cells, such as myeloid progenitor cells, comprising adding an effective amount of a compound of the present invention to progenitor cells.
  • progenitor cells such as myeloid progenitor cells
  • inhibition of the PI3K pathway suppresses myeloid progenitor cell.
  • the present invention also encompasses a method for treating liver cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • LY294002 inhibits phosphorylation of Akt (serine/threonine protein kinase B), which is an indicator in human liver tissues.
  • the present invention also encompasses a method for treating conditions associated with a mutant PTEN comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • PTEN is a tumor suppressor gene located on chromosome 10q23 that has been identified in patients with Cowden disease. As discussed in Vega et al, Journal of Investigative Dermatology, 121(6): 1356-1359 (2003), mutant PTEN has reduced ability to inhibit the activation of the proto-oncogene Akt. Inhibitors of PI3K may inhibit phosphorylation of Akt, thereby reducing the effect of the mutant PTEN.
  • the compound may be administered simultaneously or metronomic ally with other anticancer treatments such as chemotherapy and radiation therapy.
  • the term “simultaneous” or “simultaneously” as used herein, means that the other anti-cancer treatment and the compound of the present invention may be administered within 48 hours, 24 hours, 12 hours, 6 hours, or 3 hours or less, of each other.
  • the anti-cancer treatment may be administered 1-7 days prior to administering the compound.
  • the term “metronomically” as used herein means the administration of the compounds at times different from the chemotherapy and at certain frequency relative to repeat administration and/or the chemotherapy regimen.
  • the chemotherapy treatment may comprise administration of an anti-cancer agent, which may be a cytotoxic agent or cytostatic agent, or combination thereof.
  • Cytotoxic agents prevent cancer cells from multiplying by: (1) interfering with the cell's ability to replicate DNA and (2) inducing cell death and/or apoptosis in the cancer cells. Cytostatic agents act via modulating, interfering or inhibiting the processes of cellular signal transduction which regulate cell proliferation and sometimes at low continuous levels.
  • Classes of compounds that may be used as cytotoxic agents include the following: alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphat
  • proliferative cytotoxic agents are navelbene, CPT-I l, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
  • a microtubule affecting agent may include allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC 608832 or docetaxel), thiocolchicine NSC 361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones such as epothilone A
  • cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cis-platin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors.
  • Cytostatic agents that may be used include hormones and steroids (including synthetic analogs): 17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl- testosterone, prednisolone, triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, zoladex.
  • cytostatic agents are antiangiogenics such as matrix metalloproteinase inhibitors, and other VEGF inhibitors, such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included.
  • VEGF inhibitors such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included.
  • Anti-Her2 antibodies from Genetech may also be utilized.
  • a suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are Imclone antibody C225 immuno specific for the EGFR, and src inhibitors.
  • cytostatic agent also suitable for use as an cytostatic agent is Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative.
  • Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative.
  • antiestrogen Tamoxifen® which inhibits the proliferation or growth of estrogen dependent breast cancer.
  • Inhibitors of the transduction of cellular proliferative signals are cytostatic agents. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-I kinase inhibitors, MAPK kinase inhibitors, PI-3 inhibitors, Src kinase inhibitors, and PDGF inhibitors.
  • the PI-3 inhibitor may be rapamycin or rapamycin analogs, which may be an inhibitor of mTOR, which may be component downstream of PI3K in the PI-3 signaling pathway.
  • proliferative signal inhibitors include: CCI-779, temsirolimus, trastuzumab, cetuximab, sunitib, lapatinib, imatinib mesylate, bortezumib, and sorafanib.
  • carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non- Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma;
  • the present invention also encompasses a method for treating pancreatitis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • a method for treating pancreatitis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • inhibition of PDK may prevent pancreatitis.
  • the present invention also encompasses a method for treating ulcers comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • the present invention also encompasses a method for treating gastric cancer, such as stomach cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • gastric cancer such as stomach cancer
  • PI3K is involved in the adhesion of Helicobacter pylori to gastric cells.
  • Osaki et al. Journal of Cancer Research and Clinical Oncology, 130(1): 8-14 (2004) indicates that a PI3K inhibitor, such as LY294002, may be useful as an anti-tumor agent for gastric carcinoma.
  • the present invention also encompasses a method of improving the performance of a stent comprising administering a therapeutically effective amount of a compound of the present invention to a patient with a stent, such as a cardiovascular stent.
  • a stent such as a cardiovascular stent.
  • inhibition of PI3K may prevent the "stretch" damage that accompanies stent placement in vessels.
  • the compounds of the present invention in the stent or polymer matrix thereof may improve solubility in the stent coating matrix, improve aqueous/serum solubility, or improve perfusion into the cells immediately adjacent to the stent placement.
  • the present invention also encompasses a method for treating age-related macular degeneration (AMD) comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • AMD age-related macular degeneration
  • inhibition of VEGF inhibits blood vessel overgrowth associated with AMD.
  • the compounds of the present invention may treat AMD by inhibiting angiogenesis.
  • the present invention also encompasses a method for treating hypertension comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • inhibition of PI3K may prevent the low extracellular concentrations of Mg 2+ that are associated with hypertension.
  • the present invention also encompasses a method for suppressing differentiation of progenitor cells, such as myeloid progenitor cells, comprising adding an effective amount of a compound of the present invention to progenitor cells.
  • progenitor cells such as myeloid progenitor cells
  • inhibition of the PI3K pathway suppresses myeloid progenitor cell.
  • the present invention also encompasses a method for treating liver cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • LY294002 inhibits phosphorylation of Akt (serine/threonine protein kinase B), which is an indicator in human liver tissues.
  • the present invention also encompasses a method for treating conditions associated with a mutant PTEN comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
  • PTEN is a tumor suppressor gene located on chromosome 10q23 that has been identified in patients with Cowden disease. As discussed in Vega et al, Journal of Investigative Dermatology, 121(6): 1356-1359 (2003), mutant PTEN has reduced ability to inhibit the activation of the proto-oncogene Akt. Inhibitors of PI3K may inhibit phosphorylation of Akt, thereby reducing the effect of the mutant PTEN. a. Administration
  • compositions of the present invention may be administered in any manner including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof.
  • Parenteral administration includes intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular.
  • the compositions of the present invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion. b. Dosage
  • a therapeutically effective amount of the compound required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and is ultimately determined by the attendant physician.
  • doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day.
  • the dose may be about 1 ⁇ g/kg to about 100 ⁇ g/kg per day.
  • the desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. Multiple doses often are desired, or required.
  • the compounds of the present invention may be administered at a wide range of dosages. When given in combination with other therapeutics, the dosage of the compounds of the present invention may be given at relatively lower dosages. In addition, the use of targeting agents may allow the necessary dosage to be relatively low. Certain compounds of the present invention may be administered at relatively high dosages due to factors including low toxicity, high clearance, low rates of cleavage of the tertiary amine. As a result, the dosage of a compound of the present invention may be from about 1 ng/kg to about 100 mg/kg.
  • the dosage of a compound of the present invention may be at any dosage including about 1 ⁇ g/kg, 25 ⁇ g/kg, 50 ⁇ g/kg, 75 ⁇ g/kg, 100 ⁇ g/kg, 125 ⁇ g/kg, 150 ⁇ g/kg, 175 ⁇ g/kg, 200 ⁇ g/kg, 225 ⁇ g/kg, 250 ⁇ g/kg, 275 ⁇ g/kg, 300 ⁇ g/kg, 325 ⁇ g/kg, 350 ⁇ g/kg, 375 ⁇ g/kg, 400 ⁇ g/kg, 425 ⁇ g/kg, 450 ⁇ g/kg, 475 ⁇ g/kg, 500 ⁇ g/kg, 525 ⁇ g/kg, 550 ⁇ g/kg, 575 ⁇ g/kg, 600 ⁇ g/kg, 625 ⁇ g/kg, 650 ⁇ g/kg, 675 ⁇ g/kg, 700 ⁇ g/kg, 725 ⁇ g/kg, 750 ⁇ g/kg, 775
  • a lO g sample of LY294002 was prepared according to Scheme 1 based on the procedure described in Vlahos et al, J. Biol. Chem. 269(7): 5241 (1994), the contents of which are incorporated by reference.
  • the displacement of the thiomethyl group of thoichromones such as 12 by amines has been described previously (Bantick et al, J. Heterocyclic Chem, 18:679 (1981), the contents of which are incorporated by reference) as has the cyclization of methyl phenyl ketones such as jj_ with carbon disulfide with concomitant alkylation of the thiol anion (Vlahos et al. and Bantick et al).
  • keto oxygen of LY294002 is alkylated using iodomethane or benzyl chlorides under forcing conditions, which can be methyl alkylated, phthalimido alkylated, paracarboxy benzyl alkylated, and a para- sen-benzyl alkylated prodrugs.
  • Chloromethyl intermediates were prepared following the procedure described in Tsujihara, Synth Commun, 24, 767, 1994. Briefly, the appropriate carboxylic acid was diluted in a 50/50 mixture of dichloromethane/water. The mixture was cooled in an ice-water bath and sodium bicarbonate (4 equiv) and n-tetrabutyl ammonium hydrogen sulfate (0.05 equiv) was added. After stirring for 5 min, chloromethyl chlorosulfate (1.1 equiv) was added. The solution was stirred vigorously overnight. The mixture was transferred to a separatory funnel with more dichloromethane and washed with saturated sodium chloride solution.
  • LY294002 (Compound 1) is dissolved in acetonitrile and then each of the chloromethyl esters (1-1.5 equiv) from Example 3 is added along with 1-2 equivalents of sodium iodide. At room temperature, the reaction proceeds only slowly with the chloromethylesters to give very small amounts of the alkylated product along with the precipitation of sodium chloride. At 65 0 C, the reaction proceeds to completion usually in 4 hours. The reaction when complete (as judged by analysis by LC-MS) is filtered; concentrated and then purified on reverse phase HPLC. The fractions are collected and lyophilized to give the desired products as fluffy powders.
  • Halomethyl ester linkers were prepared (Scheme 2 and chart).
  • Compound B was prepared from Compound A (commercially available) as described in Example 3. This compound was converted into the more reactive iodomethyl ester (Compound C) by a Finklestein reaction by dissolving in acetone or 2-butanone and then dissolving 2-5 equivalents of sodium iodide whereupon the sodium chloride precipitated and the iodomethyl ester (Compound C) was produced in solution.
  • Compound C was isolated by stripping off the solvent and dissolving in a water immiscible solvent such as methylene chloride and extracting with water to remove the residual sodium iodide.
  • Compound E was prepared from Compound D (commercially available) Compounds F and G were prepared in a manner similar to the production of Compounds B and C, respectively.
  • Halomethyl esters including those of Example 5, are used to alkylate LY294002 using conditions similar to the methodology in Example 4.
  • Compound 1111 was produced by the method as shown below. Compound 1110 was treated with neat trifluoroacetic acid for 1-3 hours and the TFA was blown off with argon and dried under vacuum to give a glassy solid comprised of Compound 1113 (t-butyl ester group of Compound 1110 cleaved to give the corresponding carboxylic acid). Compound 1113 was then dissolved in 1-3 ml of thionyl chloride and heated at 65 0 C for 3-8 hours. The thionyl chloride was blown off with argon and then dried under high vacuum to give Compound 1111 (acid chloride of Compound 1113) in good yields as a glassy yellow solid. Compound 1111 can be reacted as a typical acid chloride with various nitrogen-containing and hydroxyl-containing nucleophiles for example by simply dissolving in methanol to give the corresponding methyl ester Compound 1112.
  • Proteins are conjugated in largely aqueous solution (pH 7-9) (phosphate buffer to carbonate buffer) using a 2-10 fold excess of Compound 1111 relative to the keto oxygen to be modified.
  • the acid chloride Compound 1111 can be introduced in a mixed organic-aqueous solution (such at 50/50 water/acetonitrile or 50/50 water/THF) or stirred in methylene chloride in a two-phase reaction system at room temperature for 1-24 hours.
  • Protein-conjugates can be purified by dialysis or ultrafiltration and used directly.
  • peptide arg-gly-asp-ser (RGDS) was prepared on wang resin using standard FMOC/HOBT coupling peptide chemistry using all natural amino acids.
  • the resin-bound peptide was reacted with Compound 1111 in DMF from 1-24 hours, filtered and the resin washed with DMF and then methylene chloride and then treated with trifluoroacetic acid to cleave the conjugate Compound 1126 from the resin (Scheme 4).
  • Scheme 4 SCHEME 4
  • the third trace down from the top of Figure 7 A is the evaporative light scattering detector signal.
  • the bottom trace is the single ion monitoring trace for the SFl 126 at 853 m/z+.
  • Figure 7 is shown the chromatogram of a similarly purified batch (lot A036-33) of SFl 126 the x-axis is time in minutes and the y-axis for the top chromatogram is milli-absorbance units for the UV detector at 254 nm and for the bottom chromatogram is millivolts detected by the evaporative light scattering detector.
  • the x-axis is the mass- to-charge ratio (m/z) and the y-axis is the intensity of the mass ion count.
  • mice of 4-6 weeks in age weighing around 30 grams were inoculated subcutaneously in the right flank with 5 million tumor cells (human non-small cell lung cancer cells: H1299) on day 0. After 14 days of allowing the tumors to grow the animals were divided into 3 groups of 5 animals each.
  • One group received vehicle control alone.
  • One group received twice-per-day tail vein injections (i.v) of 50 uL volume of 24.4 millimolar solution of Compound 1126 in phosphate buffered saline corresponding to 25 mg/kg/day dosing level of the active component of the prodrug (i.e. compound 1).
  • the last group received twice-per-day tail vein injections (i.v) of 50 uL volume of 4.9 millimolar solution of Compound 1126 in phosphate buffered saline corresponding to 5 mg/kg/day dosing level of the active component of the prodrug (compound 1).
  • the tumors were measured every three days using calipers to determine the tumor volume and the animals weights were recorded when the animals were sacrificed on day 27.
  • the results are shown in Table 2 and indicate strong tumor volume reduction versus control for both dose levels at the first datapoint only 3 days after treatment (Day 17) and continuing through to the end of the study:
  • Alpha v targeted PI 3 kinase inhibitors abrogated the tube formation of EDC-CBFl endothelial cells on Matrigel.
  • Tube formation represents to some extent the formation of angiogenesis in vivo.
  • PI 3 kinase inhibitors including targeted PI3 kinase inhibitor prodrugs
  • Matrigel was plated into 12-well plate wells and solidified in 37°C for 2 hours. 1 x 10 EDC-CBFl endothelial cells were then put on the top of the Matrigel layer in the presence of PBS, RADfV (cyclic negative control peptide), RGDfV (cyclic positive control peptide), RADS (linear negative control peptide), compound 1, or Compound 1126 at 20 0 M concentration overnight. Pictures were then taken using a microscope.
  • Well formed tubes can be visualized in the PBS control wells (top left panel of Figure). There was not much difference in the RGDfV-, RADfV-, or RGDS- containing wells compared with PBS control. Tube formation was significantly less in Compound 1101- and Compound 1126- containing wells.
  • This experiment tested the effect of PI 3 kinase inhibitors (Compound 1 and the targeted prodrug version of Compound l;Compound 1126) on the induction of p53 luciferase activity.
  • the transfection procedure was similar to that described in the literature to monitor p53 transcription.
  • Compound 1 (6 hour exposure) induced more than two fold higher luciferase activity than the control and the targeted version of compound 1 (Compound 1126) had even better ability of inducing the p53 luciferase activity (to almost 3 fold).
  • This induction of p53 function was demonstrated to be abrogated by the p53 inhibitor, pifithrin alpha at 20 uM concentration.
  • Reaction mixture A044-84 (2.33 g) was weighed out into separate 0.33 g samples and dissolved immediately before preparative chromatography in 800 ⁇ l of a solution containing 1 part by volume acetonitrile, 1 part by volume water, and 1% by volume acetic acid. 400 ⁇ l of this solution was injected for each preparative chromatography run.
  • the pump A eluant was B&J water (365-4) with 0.1% acetic acid added, and the pump B eluant was B&J acetonitrile (015-4) with 0.1% acetic acid added.
  • the eluant was 10% B, then linearly ramped to 34% B over a 4 minute period, then linearly ramped to 95% B at 4.25 minutes and held there until 5.25 minutes, then linearly ramped back to the starting concentration of 10% B at 5.50 minutes.
  • mice were injected with a million non-small cell lung cancer cells (H1299) subcutaneously and allowed to grow about 7 days until the tumor mass was approximately 10 to 15 mm by 7 to 9 mm in dimensions.
  • Animals were injected with the targeted prodrug, Compound 1126, either i.v. (50 uL) or i.p. (50 uL) with 32.6 mMolar solutions of Compound 1126 in phosphate buffered saline. After 60 minutes the mice were sacrificed and the tumors removed. Three small pieces of the tumors were retrieved and minced. After aging for 24 hours to allow all of the prodrug to convert to compound 1 the tumor samples were extracted with acetonitrile.
  • Example 20 Synthesis of Tumor Localizing Conjugate
  • the electrophilic group-bearing compounds (such as compound 1111 and 1113) can be reacted with polymers bearing nucleohilic groups such as alcohols, amino, and thiol groups.
  • N- (2-hyroxypropyl)methylacrylamide (HPMA) having molecular weight of 2000 to 100,000 is reacted with excess compound 1111 in a nonprotic organic solvent such as methylene chloride or tetrahydrofuran in the presensce of triethyl amine or diisopropylethyl amine and then separated by size exclusion chromatography, ultracentrifugation, or precipitation in another solvent such as methanol or ether.
  • a nonprotic organic solvent such as methylene chloride or tetrahydrofuran
  • polyglutamic acids can be converted to poly-nucleophilic bearing groups by reaction of the carboxylic acids with excess diamines using carbodiimide coupling followed by size exclusion chromatography or reverse phase HPLC purification to obtain poly-nucleophilic versions of polyglutamic acids.
  • polymers can then be reacted directly with excess portions of compound 1111 in an aprotic organic solvent such as methylene chloride or tetrahydrofuran in the presensce of triethyl amine or diisopropylethyl amine and then separated by size exclusion chromatography, ultracentrifugation, or precipitation in another solvent such as methanol or ether.
  • an aprotic organic solvent such as methylene chloride or tetrahydrofuran in the presensce of triethyl amine or diisopropylethyl amine
  • another solvent such as methanol or ether.
  • the poly- conjugated polymer thus precipitated or separated is substantially free of low molecular weight residual prodrug and is used as a tumor localizing conjugate that releases active compound 1 overtime in the vicinity of the tumor resulting in antitumor and anti-angiogenic effects.
  • SFl 126 (lot number A 102-65; having retention time 1.903 minutes with reference SFI lOl having retention time 3.236 minutes) was dissolved in D2O containing 0.1% TFA-d at about 55 mg/mL and studied using the following NMR experiments:
  • COSY Correlation Spectroscopy
  • COSY Correlation Spectroscopy
  • DEPT Distortionless Enhancement by Polarization Transfer
  • the experiment as acquired includes three sets of spectra, which from bottom to top correspond to the 45°, 90°, and 135° proton decoupler pulses; or: all protonated carbons, methines only, and methines and methyls up and methylenes down, respectively.
  • HMQC Heteronuclear Multiple Quantum Coherence
  • HMBC Heteronuclear Multiple Bond Correlation
  • HMBC Heteronuclear Multiple Bond Correlation
  • the data support the O-linked structure versus an alternative N-alkylated structure in that there is no ketone carbon downfield between 210 to 178 ppm.
  • the furthest downfield carbon found was at 174.4 ppm assigned to an amide carbonyl carbon.
  • the possibility of the ketone also existing as a hydrate since SFl 126 in the solid form typically has 4-6% by weight of water was dismissed due to the mapping of this particular carbon to be an olefinic carbon with double bond character thus supporting the enol structure in the O-alkylated arrangement.
  • the HMBC supports the O-alkylated structure by the correlations to the olefin labeled as Carbon 1 at 166 ppm: i.e., Protons 22, 2, and 6 all correlated to Carbon 1.
  • the structure with atoms numbered is shown below: NMR Assignments for SFl 126 (as O-alkylated form) in D 2 O Solution (w/0.1% TFA-d).
  • a 53 mg portion of A102-62BS was weighed out and dissolved in 150 uL of dimethylformamide (DMF) at 73°C and then allowed to cool. These crystals were then analyzed by X-ray and the crystal structure solved to indicate alkylation had occurred on the oxygen and not on the nitrogen to give the O-alkylated version of SFl 103 shown in Figure 16.
  • DMF dimethylformamide
  • SFl 126 acts synergistically in combination with rapamycin in inhibiting tumor cell growth and promoting tumor cell death
  • SFl 126 acts synergistically in reducing tumor cell growth and promoting tumor cell death when used in combination with rapamycin, which inhibits the PDK pathway component mTOR.
  • Rapamycin was chosen as the mTOR inhibitor because it is readily available for research purposes and is a marketed drug.
  • Several rapamycin analogs are in development, and temsirolimus has been recently approved for patients with advance renal cell carcinoma (RCC).
  • RCC renal cell carcinoma
  • RCC cell lines are available including 786-0 which is widely used in PDK research and has been investigated with LY294002 in an in vivo model.
  • HBEC Human brain endothelial cell
  • RCC renal carcinoma cells
  • CI Combination Index
  • mice were randomly divided up into six groups of six mice each, and treated with rapamycin alone (1.5 mg/kg, i.p.) three times weekly (M, W, F), SFl 126 alone (20 mg/kg, i.v.) three times weekly (M, W, F), or a combination of the two agents in three different sequences: (1) simultaneous treatment combination (simultaneous rapamycin and SFl 126 administration); (2) rapamycin administration followed by SFl 126 one week later; and (3) SFl 126 administration followed by rapamycin one week later.
  • the dose level of rapamycin and SFl 126 in the combination treatment groups was the same as those in the single agent treatment ones.
  • VEGF is a well known proangiogenic factor and Bv8 (prokineticin-2) is a related factor that has recently been characterized. These two angiogenic stimulants along with IGF were evaluated in 786-0 cells ( Figure 21A) and HBECs ( Figure 21B) for their effect on PDK pathway signaling. A significant increase in pAkt was noted in HBEC exposed to VEGF (0.1 ⁇ g/mL), Bv8 (2.5 ⁇ g/mL), and IGF (0.1 ⁇ g/mL). A clear dose dependant reduction in pAkt levels in both 786-0 and HBECs was observed with SFl 126 in the presence or absence of these PDK pathway activators.
  • PDK signaling Another important aspect in PDK signaling is apoptosis versus cell cycle arrest. Some PDK inhibitors are widely believed to be only cell cycle arrest agents and not capable of inducing apoptosis.
  • the effect of SFl 126 on the level of apoptosis in 786-0 cells was studied using annexin V staining as a marker for early apoptosis detection. Exposure of 786-0 cells to SFl 126 at 5, 10, and 20 mM for 18 hours resulted in a 37%, 64%, and 92% apoptosis increase (respectively) in cells compared to untreated controls ( Figure 22).
  • rapamycin shows a flat dose response as a single agent and in combinations with SFl 126.
  • In vitro cell culture work such as this should ultimately be helpful in guiding clinical decisions such as scheduling of combination agents.
  • the above in vitro results were confirmed in tumor treatment by usingw a 786-0 xenograft tumor model ( Figure 24A). Over a four-week period the no-treatment control group showed a 480% increase in tumor volume, SFl 126 alone showed a 328% increase in tumor volume (41% growth inhibition versus control; p ⁇ 0.05), and rapamycin alone (1.5 mg/kg Lp., 3X per week) showed complete growth inhibition (0% increase in tumor growth).
  • mice were either not treated as control (Group 1), treated only with SFl 126 at 50 mg/kg dose s.c administration 3 times weekly for 3 weeks (Group 2), or treated only with docetaxel at 12 mg/kg dose iv administration on MWF of the first week (Group 3).
  • Groups 4 and 5 of mice were treated with SFl 126 s.c administration 3 times weekly for the whole period of 3 weeks and docetaxel was administered during the second week to group 4 mice (MWF) and during the first week (MWF) to group 5 mice.
  • Mice in group 6 received docetaxel during the first week (MWF) and SFl 126 was given in the following 2 weeks thereafter.
  • Example 26 SFl 126 inhibits PC3 tumor growth and sensitizes tumor response to docetaxel
  • This example shows that SFl 126 inhibits PC3 tumor growth and sensitizes tumor response to paclitaxel in a nuce mouse xenograft model.
  • Athymic nude mice bearing subcutaneous PC-3 tuors were randomly divided in groups of six when tumor volume reached 300mm 3 . Mice were treated with either docetaxel or SFl 126 alone or in combination.
  • Docetaxel was intravenously administered at 6 mg/kg dose every other day for a total of three doses.
  • SFl 126 was subcutaneously administered at a 50 mg/kg dose three times weekly for six weeks.
  • PC3 cells were plated in 50 ⁇ L RPMI 1640 media with 10% FBS in each well of 96 well plates in hexaplicated settings. After adding 50 ul of SFl 126 and docetaxel as individual or in combination in complete media to various doses, cells were incubated for 0, 1, 2 or 3 days followed by measuring viability using WST staining (adding 10 ⁇ L WST solution [Roche Diagnostics] for 4 hours). Treatment of cells with either SFl 126 or docetaxel alone resulted in significant growth inhibition reflected by a decrease in the optical density reading after WST staining of viable cells. Combining the two agents at the same concentration showed further statistically significant inhibition of cell growth (Figure 27).
  • mice were randomly divided up into 3 groups and treated with either SFl 126 alone as a positive control at 20 mg/kg dose intravenously administered three times weekly (M, W, F) or SFl 126 in combination with a single 10 mg/kg dose of docetaxel given at different schedules.
  • Docetaxel was given either at the same day when SFl 126 treatment was started or administered one week after the starting of SFl 126 treatment. There was a 3 hour interval between SFl 126 injection and docetaxel administration when administered on the same day. Treatment with SFl 126 lasted for 6 weeks and tumor size was monitored three times weekly.
  • Example 27 Combined effect of SFl 126 and docetaxel on various prostate cancer cell lines
  • SFl 126 acts synergistically with docetaxel and rapamycin on tumor cells.
  • docetaxel TXT
  • rapamycin 2 x 10 3 prostate cancer PC3, LNCaP or DU145 cells were plated in 50 ul RPMI 1640 media with 10% FBS in each well of 96 well plates in hexaplicated settings.
  • mice were implanted with 2 x 106 of DU- 145 cells and randomly divided into 4 groups of 5 when average tumor volume reached 400 mm3.
  • One group was untreated control, two other groups were treated with SFl 126 (20 mg/kg iv 3x weekly for 4 weeks) or docetaxel (6 mg/kg iv QOD on first week) as single agent.
  • the last group was SFl 126 and docetaxel combination with the same dose and frequency.
  • the solution was diluted with dichloromethane (50 mL) and washed with 0.1% aqueous citric acid solution (3 x 50 mL), 5% aqueous sodium bicarbonate solution (3 x 50 mL), and brine solution (2 x 50 mL).
  • the organic layer was dried over magnesium sulfate, filtered, and concentrated in-vacuo to give a yellow residue (798 mg).
  • the yellow residue (317 mg) was dissolved in dichloromethane (5 niL), anisole (0.1 niL), and trifluoroacetic acid (5 niL), and stirred at room temperature for 3 hours.
  • the solution was concentrated in-vacuo and immediately purified by preparative high-pressure liquid chromatography.

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Abstract

The invention provides compounds, compositions, and therapeutic methods including methods for treating cancer by administering prodrugs of the PI-3 kinase inhibitor LY294002 alone or in combination with another treatment or anti-cancer agent. The inhibitor comprises a reversibly O-alkylated substituent. Also provided is a composition comprising a combination of the prodrug PI-3 kinase inhibitor and an anti-cancer agent.

Description

O-ALKYLATED PI-3 KINASE INHIBITOR PRODRUG COMBINATION
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Prov. Appl. No. 61/031,308 filed February 25, 2008 and U.S. Prov. Appl. No. 61/044,780 filed April 14, 2008, the contents of which are incorported herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates to prodrugs of O-alkylated PI-3 kinase inhibitors, combinations thereof, and methods of using these prodrugs and combinations.
2. Description of Related Art
[0003] PI-3 kinases are a large family of lipid kinases that phosphorylate phosphatidylinositol in the D3 position to generate an important second messenger, phosphatidylinositol 3 '-phosphate. Members of the PI-3 kinase family are divided into 3 classes based on sequence homology and the product formed by enzyme catalysis. The class I PI-3 kinases are composed of 2 subunits: a 110 kd catalytic subunit and an 85 kd regulatory subunit. Class I PI-3 kinases are involved in important signal transduction events downstream of cytokines, integrins, growth factors and immunoreceptors, which suggests that control of this pathway may lead to important therapeutic effects.
[0004] Inhibition of class I PI-3 kinase induces apoptosis, blocks tumor induced angiogenesis in vivo, and increases the radiosensitivity of certain tumors. LY294002 (2-(4-morpholinyl)-8- phenyl-4H-l-benzopyran-4-one) (Compound 1) is a well known specific inhibitor of class I PI-3 kinases and has been demonstrated to possess anti-cancer properties.
Figure imgf000004_0001
(Compound 1)
However, the anti-cancer applications of LY294002 are severely limited by its lack of aqueous solubility and its poor pharmacokinetics and its toxicity. Moreover, LY294002 has no tissue specific properties and has been demonstrated to be rapidly metabolized in animals. Because of these factors, LY294002 would need to be administered at frequent intervals and thus has the potential to also inhibit PI-3 kinases in normal cells thereby leading to undesirable side effects. [0005] There continues to be a need for class I PI-3 kinase (PI3K) inhibitors with improved pharmacokinetic and pharmacodynamic properties. The present invention fulfills these needs and provides other related advantages.
SUMMARY OF THE INVENTION
[0006] Provided herein are O-alkylated PI-3 kinase inhibitors that are useful alone or in combination with other agent(s) or treatment(s) for therapeutic use including for treating cancer. [0007] Also provided herein is a compound of the formula:
Figure imgf000004_0002
wherein
Ring A is benzo; Z1 is S or O;
Z2 is S or O;
R1 and R2 independently are H, optionally substituted Ci_24aliphatic, optionally substituted aryl, hydroxyl, halogen, Ci_24alkoxy, C3_12heterocycle, cyano, amino, or, are taken together to form an optionally substituted C3_12cycloaliphatic or optionally substituted aryl;
R3 represents H, optionally substituted Ci-^aliphatic, and optionally substituted aryl;
R4 and R5 independently are H, optionally substituted Ci_12aliphatic, optionally substituted aryl, C3_12heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted C3_12heterocycle or optionally substituted heteroaryl;
Re represents H, optionally substituted Ci_24aliphatic, optionally substituted aryl, alkoxy, carboxy, amino, C3_12heterocycle, aryloxy, any of which may be optionally substituted with a targeting agent, selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate; and
L represents a linker group selected from oxygen, sulphur, -NH-, -CH2-, -C(O)-, - C(O)NH- or saturated or unsaturated aliphatic group of up to 6 carbon atoms wherein one or two saturated carbons of the chain are optionally replaced by -C(O) , C(O)C(O) , -CONH-, - CONHNH-, -C(O)O-, -OC(O)-, -NHCO2-, -0-, -NHCONH-, -OC(O)NH-, -NHNH-, -NHCO-, - S-, -SO-, -SO2-, -NH-, -SO2NH- or NHS02-; wherein the bond between Z1 and L of the compound is hydrolyzable. [0008] The compound may have the formula:
Figure imgf000005_0001
wherein, Z3 and Z4 independently are S or O; and
R7 represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2). [0009] The R1-RnIgA-R2 of the compound may have a structure may have a formula selected from the group consisting of:
Figure imgf000006_0001
Figure imgf000006_0002
Figure imgf000006_0003
wherein R4-N-R5 are selected from the group consisting of:
Figure imgf000007_0001
and wherein R6 is selected from the group consisting of:
Figure imgf000008_0001
[0010] The compound may have the formula:
Figure imgf000009_0001
[0011] The compound may also have the formula:
Figure imgf000009_0002
wherein T is selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate. The R6-T of the compound may have a structure selected from the group consisting of:
Figure imgf000010_0001
[0012] The compound may also have the formula:
Figure imgf000011_0001
and salts thereof.
[0013] T of the compound may be a vitamin, which may be folate or vitamin C. T may also be a peptide, which may be an RGD-containing peptide selected from the group consisting of RGDs, c(RGDfK), vitronectin, fibronectin, somato statin-receptor agonists and somatostatin-receptor antagonists. T may also be a bone-seeking agent, which may be selected from the group consisting of a phosphonate, phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate, and hydroxyapatite-binding polypeptides. The bone-seeking agent may also be EDTMP, DOTMP, ABDTMP, BAD, MTX-BP, CF-BP, (Asp)6, (Glu)6, alendronate, pamidronate, 4 aminobutylphosphonic acid, l-hydroxyethane-l,l-diphosphonic acid, aminomethylenebisphosphonic acid, phytic acid, or N,N-bis(methylphosphono)-4-amino-benzoic acid.
[0014] Also provided herein is a method of treating a PI3K-related cancer in a patient in need thereof, comprising administering an effective amount of the compound. The cancer may be selected from the group consisting of: brain cancer, lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, ovary cancer, prostate cancer, testes cancer, gastric cancer, genitourinary tract cancer, lymphatic cancer, rectum cancer, larynx cancer, pancreas cancer, esophagus cancer, stomach cancer, gall bladder cancer, cervix cancer, thyroid cancer, skin cancer, hematopoietic cancer, mesenchymal cancer, thyroid cancer, follicular cancer, multiple myeloma, and nervous system cancer.
[0015] Also provided herein is a method of inhibiting PD kinase in a cancer cell comprising administering to a patient in need thereof an effective amount of the compound. Also provided herein is a method of inhibiting tumor growth comprising administering to a patient in need thereof an effective amount of the compound.
[0016] Also provided herein is a method for treating a PD-K related non-cancer disease comprising administering to a patient in need thereof an effective amount of the compound. The disease may be selected from the group consisting of: inflammatory disease, pancreatitis, ulcers, age-related macular degeneration, hypertension, autoimmune disease, graft versus host disease, rheumatoid arthritis, atherosclerosis, thrombosis, PTEN-related disease, and diabetes. The PTEN-related disease may be Cowden's disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 shows the chemical structure of EDTMP, DOTMP, ABDTMP, BAD, MTX-BP and CF-BP.
[0018] Figure 2 shows the chemical structures of potential bone targeting agents.
[0019] Figure 3 shows the chemical reaction for modifying a phosphonate in a bone targeting agent.
[0020] Figure 4 shows the alkylation reaction to modify a phosphonate in a bone targeting agent.
[0021] Figure 5 shows a concept for chemically modifying EDTMP and DOTMP.
[0022] Figure 6 shows the inhibition of phagocytosis by LY294002 in J774 cells. The columns indicate phagocytic index or percentage of cells positive for phagocytic response. The phagocytic index is the number of sRBC's (sheep red blood cells) found per 100 J774 cells and the % of phagocytic cells is the % of J774 cells that have phagocytized at least 1 sRBC. The error bars represent standard deviation of mean.
[0023] Figure 7A shows the UV and ELS Chromatograms of Compound 1126 (AO36-33).
[0024] Figure 7B shows the UV and ELS and mass spec Chromatograms of Compound SFl 126
(lot A051-19).
[0025] Figure 8 shows the Positive Mass Spectrum of Compound 1126 (A036-33).
[0026] Figure 9 shows that Avβ3 targeted PI 3 kinase inhibitors abrogated the tube formation of
EDC-CBFl endothelial cells on Matrigel.
[0027] Figure 10 shows the crystal structure determined for SFl 110.
[0028] Figure 11 shows the carbon NMR spectrum of SFl 126. [0029] Figure 12 shows the proton NMR spectrum of SFl 126.
[0030] Figure 13 shows LCMS chromatogram of SFl 103 (lot A102-62BS).
[0031] Figure 14 shows proton NMR spectrum of SFl 101 (lot A102-62BS).
[0032] Figure 15 shows carbon NMR spectrum of SFI lOl (lot A102-62BS).
[0033] Figure 16 shows the crystal structure of SFl 103.
[0034] Figure 17 shows that SFl 126 and rapamycin combination exhibit synergistic growth inhibition at 1.6 nM Rapamycin and 1.6 mM SFl 126 in RCC cells.
[0035] Figure 18 shows the drug response curves of RCC cells treated with combined SFl 126 and rapamycin.
[0036] Figure 19 shows the effect of SFl 126 (8 mM) and rapamycin (8 nM) in combination on
HBEC.
[0037] Figure 20 shows dose response curves of HBEC treated with SFl 126 and rapamycin.
[0038] Figure 21 shows the SFl 126 dose effect on cellular pAkt inhibition as stimulated by
Bv8, VEGF, and IGF in 786-0 renal carcinoma cells.
[0039] Figure 22 shows the effects of SFl 101 and SFl 126 on dose-related apoptosis induction in 786-0 cells.
[0040] Figure 23 shows the combined effects of SFl 126 and rapamycin in 786-0 renal cancer cells.
[0041] Figure 24A shows the combined effect of SFl 126 and rapamycin on 786-0 renal tumor growth in nude mice.
[0042] Figure 24B shows the combined effect of SFl 126 and rapamycin on tumor weight in a
786-0 renal cell carcinoma xenograft model.
[0043] Figure 24C shows the combined effect of SFl 126 and rapamycin on mouse weight.
[0044] Figure 25 shows the effects of different scheduling of SFl 126 and docetaxel on treatment efficacy in PC-3 xenograft tumor models (n=5).
[0045] Figure 26 shows the combined effects of SFl 126 and docetaxel on PC3 tumor growth.
[0046] Figure 27 shows the combined effects of SFl 126 and docetaxel on PC3 tumor growth.
[0047] Figure 28 shows the effect of SFl 126 and docetaxel on PC3 xenograft tumors.
[0048] Figure 29 shows the effect of SFl 126 combined with various anti-cancer agents on PC3,
LNCaP, and DU145 growth. [0049] Figure 30 shows the effects of SFl 126 and docetaxel on growth inhibition of DU145 prostate cancer cells in nude mice.
[0050] Figure 31 shows the effect of docetaxel alone or in combination with SFl 126 on the inhibition of DU145 prostate cancer in nude mice.
DETAILED DESCRIPTION
[0051] Before the compounds, products, compositions and methods that relate to the present invention are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly dictates otherwise.
[0052] Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
1. Definitions
[0053] The term "branched" as used herein refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group contains one or more subordinate branches from the main chain. A branched group may contain from 1 to 12 backbone atoms, and may include isobutyl, t-butyl, isopropyl, -CH2CH2CH(CHS)CH2CH3, -CH2CH(CH2CH3)CH2CH3,
-CH2CH2C(CH3)2CH3, -CH2CH2C(CH3)3 or the like.
[0054] The term "unbranched" as used herein refers to a group containing from 1 to 24 backbone atoms wherein the backbone chain of the group extends in a direct line. An unbranched group may contain from 1 to 12 backbone atoms.
[0055] The term "cyclic" or "cyclo" as used herein alone or in combination refers to a group having one or more closed rings, whether unsaturated or saturated, possessing rings of from 3 to
12 backbone atoms, or 3 to 7 backbone atoms.
[0056] The term "lower" as used herein refers to a group with 1 to 6 backbone atoms.
[0057] The term "saturated" as used herein refers to a group where all available valence bonds of the backbone atoms are attached to other atoms. A saturated group may be butyl, cyclohexyl, piperidine or the like. [0058] The term "unsaturated" as used herein refers to a group where at least one available valence bond of two adjacent backbone atoms is not attached to other atoms. An unsaturated group may include -CH2CH2CH=CH2, phenyl, pyrrole or the like. [0059] The term "aliphatic" as used herein refers to an unbranched, branched or cyclic hydrocarbon group, which may be substituted or unsubstituted, and which may be saturated or unsaturated, but which is not aromatic. The term aliphatic further includes aliphatic groups, which comprise oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone.
[0060] The term "aromatic" as used herein refers to an unsaturated cyclic hydrocarbon group having 4n+2 delocalized π(pi) electrons, which may be substituted or unsubstituted. The term aromatic further includes aromatic groups, which comprise a nitrogen atom replacing one or more carbons of the hydrocarbon backbone. An aromatic group may be phenyl, naphthyl, thienyl, furanyl, pyridinyl, (is)oxazoyl or the like.
[0061] The term "substituted" as used herein refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group. A substituted group may be substituted with one to five, or one to three substituents. An atom with two substituents is denoted with "di," whereas an atom with more than two substituents is denoted by "poly." A substituent group may be an aliphatic group, aromatic group, alkyl, alkenyl, alkynyl, aryl, alkoxy, halo, aryloxy, carbonyl, acryl, cyano, amino, nitro, phosphate- containing group, sulfur-containing group, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio, thiocarboxylate, alkylsulfinyl, trifluoromethyl, azido, heterocyclyl, alkylaryl, heteroaryl, semicarbazido, thiosemicarbazido, maleimido, oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino, arylcycloalkyl, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl, arylphosphinyl, arylalkylphosphinyl, arylcycloalkylphosphinyl, arylphosphonyl, arylalkylphosphonyl, arylcycloalkylphosphonyl, arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, a combination thereof, or substitution thereto.
[0062] The term "unsubstituted" as used herein refers to a group that does not have any further groups attached thereto or substituted therefor. [0063] The term "alkyl" as used herein alone or in combination refers to a branched or unbranched, saturated aliphatic group. An alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl or the like.
[0064] The term "alkenyl" as used herein alone or in combination refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon double bond which may occur at any stable point along the chain. An alkenyl group may be ethenyl, E- or
Z-pentenyl, decenyl or the like.
[0065] The term "alkynyl" as used herein alone or in combination refers to a branched or unbranched, unsaturated aliphatic group containing at least one carbon-carbon triple bond which may occur at any stable point along the chain. An alkynyl group may be ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyl or the like.
[0066] The term "aryl" as used herein alone or in combination refers to a substituted or unsubstituted aromatic group, which may be optionally fused to other aromatic or non-aromatic cyclic groups. An aryl group may be phenyl, benzyl, naphthyl, benzylidine, xylyl, styrene, styryl, phenethyl, phenylene, benzenetriyl or the like.
[0067] The term "alkoxy" as used herein alone or in combination refers to an alkyl, alkenyl or alkynyl group bound through a single terminal ether linkage. An alkoxy may be methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, 2-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3- pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, or trichloromethoxy.
[0068] The term "aryloxy" as used herein alone or in combination refers to an aryl group bound through a single terminal ether linkage.
[0069] The term "halogen," "halide" or "halo" as used herein alone or in combination refers to fluorine ("F"), chlorine ("Cl"), bromine ("Br"), iodine ("I"), and astatine ("At"). A halo group may be chloroacetamido, bromoacetamido, idoacetamido or the like.
[0070] The term "hetero" as used herein combination refers to a group that includes one or more atoms of any element other than carbon or hydrogen. A hetero group may contain a heteroatom, which may be nitrogen, oxygen, sulfur or phosphorus. [0071] The term "heterocycle" as used herein refers to a cyclic group containing a heteroatom. A heterocycle may be pyridine, piperadine, pyrimidine, pyridazine, piperazine, pyrrole, pyrrolidinone, pyrrolidine, morpholine, thiomorpholine, indole, isoindole, imidazole, triazole, tetrazole, furan, benzofuran, dibenzofuran, thiophene, thiazole, benzothiazole, benzoxazole, benzothiophene, quinoline, isoquinoline, azapine, naphthopyran, furanobenzopyranone or the like.
[0072] The term "carbonyl" or "carboxy" as used herein alone or in combination refers to a group that contains a carbon-oxygen double bond. Groups which contain a carbonyl may be aldehydes (i.e., formyls), ketones (i.e., acyls), carboxylic acids (i.e., carboxyls), amides (i.e., amidos), imides (i.e., imidos), esters, anhydrides or the like.
[0073] The term "acryl" as used herein alone or in combination refers to a group represented by
CH2=C(Q)C(O)O- where Q is an aliphatic or aromatic group.
[0074] The term "cyano," "cyanate," or "cyanide" as used herein alone or in combination refers to a carbon-nitrogren double bond. A cyano group may be isocyanate, isothiocyanate or the like.
[0075] The term "amino" as used herein alone or in combination refers to a group containing a backbone nitrogen atom. An amino group may be alkylamino, dialkylamino, arylamino, diarylamino, alkylarylamino, alkylcarbonylamino, arylcarbonylamino, carbamoyl, ureido or the like.
[0076] The term "phosphate-containing group" as used herein refers to a group containing at least one phosphorous atom in an oxidized state. A phosphate-containing group may be phosphonic acid, phosphinic acid, phosphate ester, phosphinidene, phosphino, phosphinyl, phosphinylidene, phospho, phosphono, phosphoranyl, phosphoranylidene, phosphoroso or the like.
[0077] The term "sulfur-containing group" as used herein refers to a group containing a sulfur atom. A sulfur-containing group may be sulfhydryl, sulfeno, sulfino, sulfinyl, sulfo, sulfonyl, thio, thioxo or the like.
[0078] The term "optional" or "optionally" as used herein means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, the phrase
"optionally substituted alkyl" means that the alkyl group may or may not be substituted and that the description includes both unsubstituted alkyl and alkyl where there is a substitution. [0079] The term "effective amount," when used in reference to a compound, product, or composition as provided herein, means a sufficient amount of the compound, product or composition to provide the desired result. The exact amount required will vary depending on the particular compound, product or composition used, its mode of administration and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate effective amount may be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation.
[0080] The term "suitable" as used herein refers to a group that is compatible with the compounds, products, or compositions as provided herein for the stated purpose. Suitability for the stated purpose may be determined by one of ordinary skill in the art using only routine experimentation .
[0081] The term "hydrolyzable" as used herein refers to whether the group is capable of or prone to hydrolysis (i.e., splitting of the molecule or group into two or more new molecules or group). 2. Compounds
[0082] The present invention provides a compound, which upon cleavage of one bond yields a compound of the formula:
Figure imgf000018_0001
(Compound 2)
wherein,
Ring A is benzo;
Z1 and Z2 independently are S or O;
R1 and R2 independently are H, optionally substituted aliphatic, optionally substituted aryl, hydroxyl, halogen, alkoxy, heterocycle, cyano, amino, or, are taken together to form an optionally substituted cycloaliphatic or optionally substituted aryl;
R3 represents H, optionally substituted aliphatic, and optionally substituted aryl; and R4 and R5 independently are H, optionally substituted aliphatic, optionally substituted aryl, heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted heterocycle or optionally substituted heteroaryl.
Cleavage of one bond of the compound may yield LY294002 (Compound 1).
[0083] The present invention also provides a compound of the formula:
Figure imgf000019_0001
(Compound 3)
wherein,
Ring A is benzo;
Z1 and Z2 independently are S or O;
R1 and R2 independently are H, optionally substituted aliphatic, optionally substituted aryl, hydroxyl, halogen, alkoxy, heterocycle, cyano, amino, or, are taken together to form an optionally substituted cycloaliphatic or optionally substituted aryl;
R3 represents H, optionally substituted aliphatic, and optionally substituted aryl;
R4 and R5 independently are H, optionally substituted aliphatic, optionally substituted aryl, heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted heterocycle or optionally substituted heteroaryl; and
R6 represents H, optionally substituted aliphatic, optionally substituted aryl, alkoxy, carboxy, amino, heterocycle, aryloxy, and optionally substituted therewith a targeting agent; and
L represents a linker group.
The positive charge is likely resonance delocalized between the oxygen (pyrylium ion) and on the nitrogen (quaternized Nitrogen form) as shown below to give the resonance form:
Figure imgf000020_0001
Quaternized N Form Pyrylium Form Resonance Form
Since all forms differ only by moving electrons around they can be used interchangeably to describe the same compound.
[0084] Compound 2 or 3 may be a compound wherein, R1-RnIg A-R2 is selected from the group consisting of the following:
Figure imgf000021_0001
Figure imgf000021_0002
[0085] The compound may be a para-amino such as:
Figure imgf000022_0001
[0086] Compound 2 or 3 may also be a compound wherein, R4-N-R5 is selected from the group consisting of the following:
Figure imgf000022_0002
[0087] Compound 2 or 3 may be a compound wherein, R6 is selected from the group consisting of the following:
Figure imgf000023_0001
a. Linker
[0088] In another embodiment, Compound 3 may be a compound wherein the linker group is hydrolyzable. The linker group of the prodrug may be cleaved by enzymatic cleavage or by hydrolysis to yield Compound 2. The physiological conditions of this cleavage may include aqueous conditions in living animals. The rate of hydrolysis of the linker group under physiological conditions may be from about 1 minute half-life to about 48 hour half-life. b. Hydrolysis
[0089] The term "hydrolyzable" as used herein refers to whether the group is capable of or prone to hydrolysis (i.e., splitting of the molecule or group into two or more new molecules or groups due to the net insertion of a water molecule) at a rate of about 1 minute half- life to 48 hour half- life.
[0090] The linker group may be any group that may be hydrolyzed or enzymatically cleaved to yield Compound 2. The linker group may be of the formula:
Figure imgf000024_0001
wherein,
Z3 and Z4 independently are S or O; and
R7 represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-. c. Targeting Agent
[0091] In another embodiment, a compound disclosed herein may be a compound wherein R6 further comprises one or more targeting agents (T) covalently attached thereto. Targeting agents allow the prodrugs of the present invention to be delivered selectively to specific types of cells, tissues, organs or extracellular structures. As discussed above, treatment with Compound 1 (LY294002) suffers from poor bioavailability, rapid metabolism and side effects because the compound is not tissue specific. Therefore, it is highly desirable to limit the location of the drug to that of the area of treatment or at least prevent it from reaching the tissues where if can cause side effects, and to ensure that at any particular time effective, but not excessive, amounts of the drug are used. The use of targeting agents may allow the prodrugs of the present invention to be concentrated at the site of treatment rather than evenly distributed throughout the entire body or to be metabolized prematurely or excreted too quickly. Once being delivered to the site of treatment, the linker may be enzymatically cleaved or hydrolyzed as described above to yield Compound 2. Moreover, the use of targeting agents may limit the dosage required to be administered in order to achieve an effective concentration of the drug at the site of treatment. The use of targeting agents may also allow for more infrequent dosage or even alternative methods of administration in order to achieve an effective concentration of the drug at the site of treatment.
[0092] The targeting agent may be attached to the compounds of the present invention via a covalent bond, which may be formed by methods such as a nucleophilic or electrophilic group of the targeting agent that is covalently reacted with an electrophilic or nucleophilic group (respectively) on the linker.
[0093] Compound 2 or 3 may be a compound wherein, R6-T is selected from the group consisting of the following:
Figure imgf000026_0001
[0094] The targeting agent may be a carbohydrate, vitamin, peptide, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent or cartilage-seeking agent. The targeting agent may also be a molecule that is bound by a receptor in a desired tissue and optionally transported into a cell by a receptor-mediated process. The molecule may be a diazepine that binds to peripheral benzodiazepine receptors (PBRs) present in glial cells in the brain. The diazepine may be one discussed in G. Trapani, et al. Bioconjugate Chem. 2003, vol 14, pp830-839 "Peripheral Benzodiazepine Receptor Ligand-Melphalan Conjugates for Potential Selective Drug Delivery to Brain Tumors," the contents of which are incorporated by reference. [0095] The vitamin may be folate, vitamin B12 or vitamin C. The term "folate" encompasses folic acid derivatives with capacity to bind with folate-receptors. The folate may be folic acid, folinic acid, pteropolyglutamic acid, or a folate receptor-binding pteridine such as a tetrahydropterin, dihydrofolate, or tetrahydrofolate or its deaza or dideaza analogs. The folate may be a folate analog such as aminopterin, amethopterin (methotrexate), Nio-methylfolate, 2- deamino-hydroxyfolate, a deaza analog such as 1-deazamethopterin or 3-deazamethopterin, or 3'5'-dichloro4-amino-4-deoxy-N1o-methylpteroyl-glutamic acid (dichloromethotrexate). Methods of conjugating molecules to folates that are suitable for covalent attachment to compounds of the present invention are disclosed in U.S. Patent Nos. 6,576,239, 5,820,847, 5,688,488, 5,108,921, 5,635,382, and 5,416,016 the contents of which are incorporated herein by reference. Methods of conjugating molecules to vitamin C that are suitable for covalent attachment of the compound of the present invention are dislosed in S. Manfrdini J. Med. Chem. VoI 45, pp559-562, 2002 the contents of which are incorporated herein by reference.
[0096] The targeting agent may be a peptide or peptidomimetic, which may be an RGD- containing peptide selected from the group consisting of RGD, c(RGDfK), vitronectin, fibronectin, somatostatin-receptor agonist or somato statin-receptor antagonist. The targeting agent may also be a molecule that binds to the avb3 integrin receptor and acts as an antagonist, as described in U.S. Patent Nos. 6,552,079, 6,426,353B, WO 2002/40505A2, and U.S. Patent Publications 2002/0055499, 2002/0061885, 2002/0065291, 2002/0072500, U.S. 2002/0072518; W. Arap et al. Science vol 279, number 16, 1998, pp 377-380;RJ Kok et al. Biojonjugate Chem. 2002, vol 13, ppl28-135; DA Sipkins et al. Nature Medicine vol 4, number 5, 1998 pp623-626; PM Winter et al. Cancer Research 2003, vol 63, pp5838-5843; and JD Hood et al. Science vol 296, pp2404-2407; the contents of which are incorporated herein by reference. The targeting agent may be an antibody or fragment thereof, such as a tumor- specific monoclonal antibody or fragment thereof. The bone-seeking agent may be phosphonate, phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate, or hydroxyapatite -binding polypeptide. The targeting agent may also be chlorotoxin (US 6,429,187Bl) or tissue factor (G. M. Lanza, et al. "Targeted Antiproliferative Drug Delivery to Vascular Smooth Muscle Cells with a Magnetic Resonance Imaging Nanoparticle Contrast Agent"; Circulation, 2002 volume 106 pp2842-2847). [0097] The targeting agent may also be an antibody of the class IgG, IgM, IgA, IgD or IgE, or a fragment or derivative thereof, including Fab, F(ab')2, Fd, or a single chain antibody, diabody, bispecific antibody, bifunctional antibody or derivative thereof. The antibody may be a monoclonal antibody, polyclonal antibody, affinity-purified antibody, or a mixture thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be directed against a variety of antigenic determinants including those associated with tumors, histocompatibility and other cell surface antigens, bacteria, fungi, viruses, enzymes, toxins, drugs and other biologically active molecules. The antibody may specifically react to a tumor-associated antigen such as carcinoembryonic antigen (CEA), a mucin such as TAG-72, a human milk fat globule antigen, prostate serum antigen (PSA), prostate specific membrane antigen (PSMA), PS (phosphatidyl serine), or a receptor, which may be the IL-2, EGF, VEGF or transferrin receptor. The antigen may also be as described in Zalcberg and McKenzie, J. Clin. Oncology, Vol. 3; pp. 876-82 (1985),WO 01/68709A1, and U.S. Patent Publication US2004/0009122A1, the contents of which are incorporated herein by reference.
[0098] The targeting agent may also be glucose, galactose, mannose, mannose 6-phosphate, hormone (e.g., insulin, growth hormone, or the like), growth factor or cytokine (e.g., TGFβ, EGF, insulin-like growth factor, or the like), YEE(GaINAc AH). sub.3 or a derivative, cobalamin, OC-2 macroglobulin, asialoglycoprotein, albumin, texaphyrin, metallotexaphyrin, antibody, antibody fragment (e.g., Fab), single-chain antibody variable region (scFv), transferrin, any vitamin or any coenzyme
[0099] The targeting agent may also be an agent that delivers the prodrug to bones. The bone targeting agent may be EDTMP, DOTMP, or ABEDTMP, which are disclosed in U.S. Patent Nos. 4,937,333, 4,882,142, 5,064,633 and WO-94/00143, the contents of which are incorporated herein by reference. DOTMP or EDTMP may be attached to the linker moiety by any method including the coupling chemistry shown in Figure 3 and the alkylation chemistry shown in Figure 4 where the R group can have an appropriate electrophilic or nucleophilic group that reacts with the nucleophilic or electrophilic (respectively) group of the linker moiety. Further details of the coupling chemistry are provided in Tetrahedron 1999, 55, ppl2997-13010, the contents of which are incorporated by reference. Further details of the alkylation chemistry are provided in Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3600 (Biomedical Imagn. Reporters Dyes & Instrumental, pp99-106; U.S. Patent No. 5,177,054; J Med. Chem. 1994, 37, 498-511; Tetrahedron Letters, 1989, 30 #51 pp7141-7144; and U.S. Patent No. 5,955,453, the contents of which are incorporated by reference.
[0100] The targeting agent may be used to deliver the prodrug to bones as a slow release reservoir site for the compounds of the present invention. The targeting agent may be a bone seeking (osteotropic) moiety attached to a compound disclosed herein via an acid cleavable linker attached to the quaternary amine. The acid cleavable linker may be an ortho acid-amide linkage. Under acidic conditions, the protein-ACL-3 amide linkage is readily cleaved freeing the native amino group of the amide functionality as described in WO-94/00143 the contents of which are incorporated by reference. During osteoclastic bone resorption, which involves an acidic mediated mechanism, the attachment tethering the prodrug to bone may be cleaved releasing the compounds of the present invention.
[0101] The targeting agent used to deliver the prodrugs to bones may be a molecule that binds with notch receptors. Notch signaling plays a key role in the development and differentiation of various hematopoietic lineages. As discussed in Jundt et al., Blood, 102(11): 928a (2003), ligand-induced notch signaling is a novel growth factor for multiple myeloma cells and suggests that these interactions contribute to lymphomagenesis of multiple myeloma in vivo. [0102] The bone targeting agent may have a high affinity for calcium ions in hydroxyapatite, the major constituent of bone. The compound of the invention can be targeted to calcium deposits in regions of the body other than bone, such as calcium deposits in the arteries, heart, kidney, or gall bladder. However, the bone targeting agent ideally selectively binds to bone tissue. The bone targeting agent may be attracted to the bone tissue of the subject, and may bind to the bone with a higher affinity than non-bone tissues, and may remain bound for a certain length of time, thereby delivering the composition to a bone environment. In other words, the bone targeting agent may bind to bone tissue with at least 2-fold greater affinity (e.g., at least 3-fold, at least 5- fold, at least 10-fold, or at least 25-fold greater affinity) than the bone targeting agent binds to non-bone tissue. The bone targeting agent may reversibly bind to bone tissue, meaning that the bone targeting agent may eventually be released from bone and expelled from the body. [0103] The bone targeting agent may remain bound to bone tissue for a sufficient period of time to allow the quaternary prodrug to be hydrolyzed, thereby delivering the active drug to the target cells (e.g., bone marrow cells). The bone targeting agent may remain bound to bone for about 1 day (e.g., about 2 days, about 3 days, or about 7 days) to about 1 year (e.g., about 330 days, about 365 days, or about 400 days), after which the bone targeting agent is expelled from the body. The bone targeting agent may remain bound to bone for about 7 days (e.g., about 7 days, about 14 days, or about 21 days) to about 6 months (e.g., about 90 days, about 120 days, or about 150 days). For example, a bone targeted prodrug may remain bound to the bone for 30 days, during which time the drug may be released. After about 45 days the bone targeting agent may be released from the bone and eventually excreted. Thus, the bone targeting agent may be selected based on binding kinetics to bone tissue. A candidate bone targeting agent may be screened in vitro by determining affinity to bone tissue (e.g., hydroxyapatite) in, for example, a multi-well format. A candidate bone targeting agent may also be screened in vivo by assessing the rate and timing of excretion of candidate bone targeting agents from the body. In this respect, the bone targeting agent may be expelled from the body via the kidneys. [0104] The bone targeting agent may be selected from the group consisting of a phosphate, a phosphonate, a bisphosphonate, a hydroxybisphosphonate, an aminomethylenephosphonic acid, and an acidic peptide. The bone targeting agent may carry one, more than one, or a mixture of these groups. For example, the bone targeting agent may be a phosphonate, meaning that the bone targeting agent may comprise one phosphonate, two phosphonates, or three or more phosphonates. One suitable bone targeting agent for use in the invention is EDTMP (ethylene diamine-N,N,N',N'-tetrakis(methylenephophonic acid), the chemical structure of which is set forth in Figure 1, currently FDA approved (Quadramet™) as the radioactive 153Sm complex for delivering a selective radiation dose to bone metastases for pain palliation. EDTMP is a phosphonate that contains four phosphonic acid groups, and is therefore a tetraphosphonate. Compounds such as 153Sm-EDTMP are selectively localized in bone where tumors are present versus normal bone in a ratio of more than 10:1, probably because metabolic turnover of calcium is very high in the metastatic region. The 153Sm-EDTMP reportedly is rapidly taken up by the skeleton in osteoblastic bone metastases and cleared from the plasma. That portion of the compound that does not accumulate in the skeleton reportedly is rapidly excreted, and excretion is almost complete within 6 hours after administration (Jimonet et al., Heterocycles, 36, 2745 (1993)). The pain palliation is thought to be due to the radiation originating from the isotope bound to the osteoblastic bone metastases having some effect on the nearby metastatic tumor cells. Another clinically useful bone-targeting system is DOTMP (the chemical structure of which is set forth in Figure 1, now in Phase III clinical trials (termed STR, skeletal targeted radiation) as the radioactive 166Ho complex designed to deliver large doses of radiation selectively to the bone marrow for the treatment of multiple myeloma. It should be noted that the radioactive 166Ho-DOTMP complex localizes in the skeletal system and irradiates the nearby bone marrow which houses the malignant myeloma cells. Like the 153Sm-EDTMP system, the phosphonate that does not localize in the bone is cleared through the urine and out the body. In general, the skeletal uptake is about 20 to about 50% of the injected dose, and the localization in areas of the skeleton with tumor infiltration is illustrated in Figure 7 of Bayouth et al., J. Nucl. Med., 36b 730 (1995).
[0105] The bone targeting agent may be a polyphosphonic acid. Polyphosphonic acid has been demonstrated to successfully target biologically-active molecules to bone tissue. For example, conjugation (via isothiocyanato chemistry) of polyaminophosphonic acids, such as ABDTMP (the chemical structure of which is set forth in Figure 1, to growth factors (to stimulate bone formation) successfully resulted in the targeting of the growth factors to the bones of rats (see, for example, International Patent Application WO 94/00145). Similarly, the bone targeting agent may be coupled to a protein. For example bisphosphonates that were conjugated to human serum albumin successfully delivered the protein to bone in vitro (Biotechnol. Prog., 16, 258 (2000)) and in vivo (Biotechnol. Prog., 16, 1116 (2000)). The utility of bone-seeking agents extends beyond delivery of proteins to bone and includes, for instance, small therapeutic molecules. A conjugate comprising a bone-seeking bisphosphonate and an alkylating agent, such as BAD (the chemical structure of which is set forth in Figure 1, has been generated (see, for example, Wingen et al., J. Cancer Res. Clin. Oncol, 111, 209 (1986)). In this molecule, the alkylating agent is not specific in its interaction with its target (DNA), and, thus, there is no requirement for cleavage between the bisphosphonate (i.e., bone-seeking agent) and the alkylating moiety. The bisphosphonate-alkylating agent demonstrated efficacy in a rat osteosarcoma model using BAD. Another series of studies have been performed using the antifolate antineoplastic agent methotrexate that has been covalently attached to bisphosphonates, designated MTX-BP and shown in Figure 1 (see, for example, Sturtz et al., Eur. J. Med. Chem., 27, 825 (1992); Sturtz et al, Eur. J. Med. Chem., 28, 899 (1993); and Hosain et al, J. Nucl. Med., 37, 105 (1996)). Using Tc-99m labeled MTX-BP, it was determined that around 15% of the injected dose was localized in the skeleton after 4 hours with about 61% of the dose being excreted (Hosain, supra). MTX- BP further demonstrated five times greater anticancer activity compared with methotrexate alone in animal models of transplanted osteosarcoma (Sturtz 1992, supra). Similar work has been described using the conjugate CF-BP, a carboxyfluorescein group with an appended bisphosphonate whose chemical structure is set forth in Figure 1 (Fujisaki et al., Journal of Drug Targeting, 4, 117 (1994)). In this molecule, the CF group is a fluorescent marker to quantitate pharmacokinetics and biodistribution, and is connected to the bone targeting agent through an ester bond which is susceptible to hydrolysis in vivo. Studies in rats injected intravenously indicated that CF-BP localized in the bone and served as a slow release mechanism for CF generated via general hydrolysis of the ester linkage (Fujisaki, supra).
[0106] The bone-seeking agent may also be a peptide, such as (Asp)6 and (GIu)6. The acid-rich peptide sequence of the glycoprotein osteonectin, which is found in abundance in bone and dentin, has a strong affinity to hydroxyapatite (Fujisawa et al., Biochimica et Biophysica Acta, 53, 1292 (1996)). Thus, peptide ligands comprising acidic amino acids are ideal candidates for bone targeting agents. Indeed, (GIu)1O, when attached to biotin, successfully recruited labeled strepavidin to hydroxyapatite (described further in Chu and Orgel, Bioconjugate Chem., 8, 103 (1997), and International Patent Application WO 98/35703). In addition, the biological half-life of the fluorescein isothiocyanate conjugated to (Asp)6 was 14 days in the femur (Kasugai et al., Journal of Bone and Mineral Research, 15(5), 936 (2000)), which is an acceptable half-life for the bone targeting agent of the invention. Likewise, delivery of estradiol- (Asp)6 conjugates to bone has been demonstrated in ovariectomized animals with concomitant inhibition of osteoporectic-type bone loss (Kasugai et al., Journal of Bone and Mineral Research (Suppl 1), 14, S534 (1999)). It is believed that the (Asp)6 tether to bone is metabolized during the bone resorption process mediated by osteoclasts. Therefore, the acidic peptide ligand provides not only a means of recruiting compounds to bone, but also provides a mechanism of slowly releasing compounds to bone cells and surrounding tissue.
[0107] The bone targeting agent may also be amino- and hydroxy-alkyl phosphonic or diphosphonic acid; a hydroxybisphosphonic acid such as alendronate, pamidronate, 4- aminobutylphosphonic acid, 1-hydroxyethane- 1,1 -diphosphonic acid, or aminomethylenebisphosphonic acid; a phosphate such as phytic acid; or a aminomethylenephosphonic acid such as N,N-bis(methylphosphono)-4-amino-benzoic acid or nitrilotri(methylphosphonic acid). Nonlimiting examples of some bone targeting agents are shown in Figure 2.
[0108] The bone targeting agent may be an aminomethylenephosphonic acid. By "aminomethylenephosphonic acid" is meant a compound that contains an -NCH2PO3H moiety, where the amino group has one, two, or three methylenephosphonic acid groups attached, and may be further substituted with other chemical moieties. An aminomethylenephosphonic acid may include one or more phosphonic acid groups and one or more amino groups. Examples of these aminomethylenephosphonic acids include the compounds F through N set forth in Figure 2.
[0109] The bone targeting agent may be attached through one of the heteroatoms or by chemical modification that installs an additional attachment point. For example, EDTMP can be connected to a linker by one of the phosphorous oxygens to create a phosphonate linkage, as illustrated in Figure 3 (see for example Vieira de Almedia et al, Tetrahedron, 55, 12997-13010 (1999).) The phosphorous oxygen can also be alkylated as shown in Figure 4, where the R group can have, for example, a pendant amino group, to provide a secondary attachment point for ligation to, for example, an activated PEG. Other types of alkylation that could be utilized in the invention include examples similar to that involving DOTMP, as has been further described in Chavez et al., Biomedical Imaging: Reporters, Dyes, & Instumentation, Contag & Sevick-Muracia, Eds., Proc. SPIE, Vol. 3600, 99-106 (July, 1999), or as shown for other phosphonic acids further described in, for example, U.S. Patent 5,177,064, U.S. Patent 5,955,453, de Lombaert et al., J Med. Chem., 37, 498-511 (1994), and Iyer et al., Tetrahedron Letters, 30(51), 7141-7144 (1989). Alternatively, for chemical modification, EDTMP can be, for example , modified to generate ABDTMP by installation of an aniline group (as further described in, for example, Figure 1 of International Patent Application WO 94/00145). The aniline amine is then available to form, for example, an amide bond. DOMTP could be similarly modified, as outlined in Figure 5. [0110] The terms "phosphonate, phosphate, and aminomethylenephosphonate" are meant to encompass the phosphonic acids, the phosphoric acids, and aminomethylenephosphonic acids, respectively, as well as any salts, hydrolyzable esters, and prodrugs of the phosphorous-based acids thereof. At the biological pH of 7.4 in the blood, or the more acidic pH around the bone, a certain portion of the phosphate or phosphonate of the bone targeting agent may be deprotonated and replaced with a counterion. Furthermore, the exchange of proton for calcium is an inherent event for the binding of the bone targeting agent to the hydroxyapatite in the invention. However, preparation and administration of the composition containing the bone targeting agent may or may not require complete protonation of the phosphorous acids therein. Therefore, the phosphonic acid, phosphoric acid, and aminomethylenephosphonic acid are drawn and utilized interchangeably with phosphate, phosphonate, and aminomethylenephosphonate. Biologically hydrolyzable esters of the phosphorus-based acids may also be utilized in the in vivo use of the bone targeting prodrugs. Similarly, prodrugs of the phosphorous-based acids may also be utilized in vivo to mask the acidity of the composition during, for example, formulation and administration.
[0111] The targeting agent may also be an agent that targets based upon properties of the particular tissue. The bone targeting agent may be a polymer that is selectively localized in tumor tissues due to the EPR effect (enhanced permeability and retention) as described in H. Maeda et al "Tumor vascular permeability and the EPR effect in macromolecular therapeutics: A Review"; Journal of Controlled Release, 2000 vol 63, pp 271-284, the contents of which are incorporated by reference. The polymer may also be N-(2-hydroxypropyl)methacrylamide (HPMA) or (poly)L-glutamic acid.
[0112] The targeting agent may also comprise an RGD moiety. As discussed in Curnis et al, Cancer Research, 64(2): 565-571 (2004), RGD moieties target RGD fusion proteins to vasculature by interacting with interacts with cell adhesion receptors, including αvβ3 integrin. 3. Synthesis a. Main Ring System
[0113] The compounds of the present invention may be synthesized using LY294002 (Compound 1) as a starting product. LY294002 (Compound 1) may be obtained commercially or synthesized as described in Example 1 or as described in U.S. Patent No. 5, 703, 075, the contents of which are incorporated herein by reference. One of ordinary skill in the art may also synthesize the compounds of the present invention using Compound 2 as a starting product. b. Preparation of Derivatives of Main Ring System
[0114] The main ring system of Compounds 2 and 3 may be derivatives of the main ring system of LY294002 (Compound 1). Derivatives of the main ring system of Compound 3 may be prepared as disclosed in U.S. Patent No. 5,703,075, the contents of which are incorporated herein by reference, for the preparation of main ring derivatives of LY294002 (Compound 1). Derivatives of the main ring system of Compound 3 may also be prepared by using a commercially available compound such as substituted 2-hydroxy-acetophenone. c. Preparation of Derivatives of Morpholine Ring
[0115] The amine derivatives of Compound 3 may be prepared by the displacement of the thioalkyl group in Example 1 under conditions ranging from room temperature to forcing conditions (excess nucleophile and heating to 1100C). Any primary or secondary nitrogen- containing nucleophile may react to give alternative amine substitutions to the morpholine ring structure (including different morpholine analogs). The synthesis of representative examples of such amine derivatives of Compound 3 has been described previously in US 6,949,537, the contents of which are incorporated herein by reference. d. Preparation of Esters
[0116] As described above, esters may be used to form the compounds of the present invention. A compound disclosed herein may be formed using halo esters. The compound may be formed using chloromethyl esters. Numerous chlorlomethyl esters useful in the preparation of the compounds of the present invention are available from commercial sources. In addition, chloromethyl esters may be synthesized as described in WO 02/42265, WO 94/23724, and U.S. Patent Nos. 4,444,686, 4,264,765, and 4,342,768, the contents of which are incorporated herein. e. Alkylation
[0117] The prodrugs of the present invention may be prepared by alkylating the keto oxygen of Compound 1 or Compound 2 with a halomethyl ester. The alkylated compounds are generally not reversible under mild conditions. However, the positively charged alkylated compounds of the present invention are readily hydrolyzable as discussed above. Halomethyl esters that may be used to alkylate the keto oxygen of Compound 1 or Compound 2 are commercially available or may be prepared as described in the Examples below. f. Linkers
[0118] The prodrugs of the present invention may also be prepared by alkylating a keto oxygen of Compound 1 or Compound 2 with a linker comprising at least two functional groups. The linker may be any natural or synthetic linker that is capable of alkylating a keto oxygen of the chromanone ring and is also capable of being covalently attached to a targeting molecule or may already be attached to a targeting molecule. The prodrugs of the present inventions may also be prepared by acylation or sulfonylation and the like.
[0119] The linker may be an atom such as oxygen or sulfur, a unit such as -NH-, -CH2-, -C(O)-, - C(O)NH-, or a chain of atoms. The molecular mass of the linker may be in the range of about 14 to 200, or may be in the range of 14 to 96 with a length of up to about six atoms. The linker may also be a saturated or unsaturated aliphatic group which is optionally substituted, and wherein one or two saturated carbons of the chain are optionally replaced by -C(O)-, -C(O)C(O)-, -CONH-, -CONHNH-, -C(O)O-, -OC(O)-, -NHCO2-, -O-, -NHCONH-, -OC(O)NH-, -NHNH-, -NHCO-, -S-, -SO-, -SO2-, -NH-, -SO2NH-, or -NHSO2-.
[0120] The first functional group of the linker is used to alkylate the keto oxygen as discussed above. A first functional group may be a halomethyl ester such as chloromethylester or iodomethyl ester. The second functional group of the linker may be used to covalently attach a targeting agent.
[0121] The second functional group may be an electrophilic group or a nucleophilic group. Second functional groups for covalently attaching targeting groups may be isothiocyanate, haloacetamide maleimide, imidoester, thiophthalimide, N-hydroxysuccinimyl ester, pyridyl disulfide, phenyl azide, carboxyl (and acid chlorides thereof), amino, acyl hydrozide, semicarbazide, thiosemicarbazide, diazonium, hydrazine, azide, aminoalkylurea, aminoalkylthiourea, halotriazine, or meta (dihydroxyboryl)phenylthiourea. Other suitable reactive moieties which may be suitable for covalently attaching the prodrugs of the present invention to targeting agents may be disulfides, nitrenes, sulfonamides, carbodiimides, sulfonyl chlorides, benzimidates, -COCH3 or -SO3H.
[0122] The appropriate second functional group will depend on the functional group of the targeting agent with which the covalent bond will be formed and by its susceptibility to loss of biological activity as a consequence of forming a given type of linkage. If the targeting agent is a protein, the second functional group may be reactive with side chain groups of amino acids making up the polypeptide backbone. Such side chain groups include the carboxyl groups of aspartic acid and glutamic acid residues, the amino groups of lysine residues, the aromatic groups of tyrosine and histidine, and the sulfhydryl groups of cysteine residues. [0123] Carboxyl side groups presented by a targeting agent such as a polypeptide backbone may be reacted with amine second functional groups by means of a soluble carbodiimide reaction. Amino side groups presented by a targeting agent may be reacted with isothiocyanate, isocyanate or halotriazine second functional groups to effect linkage to the prodrugs of the present invention. Alternatively, amino side groups on the targeting agent may be linked to the prodrugs compounds of this invention bearing amine reactive groups by means of bifunctional agents such as dialdehydes and imidoesters. Aromatic groups presented by a targeting agent may be coupled to the prodrugs of this invention via diazonium derivatives. Sulfhydryl groups on targeting agent molecules may be reacted with maleimides or with haloalkyl targeting agent reactive groups such as iodoacetamide. Free sulhydryl groups suitable for such reactions may be generated from the disulfide bonds of protein immunoglobulin or may be introduced by chemical derivatization. Linkage to free sulfhydryl groups generated in the intra-heavy chain region of immunoglobulins does not interfere with the antigen binding site of the immunoglobulin but may render the antibody incapable of activating complement.
[0124] When the targeting agent is a glycosylated protein, an alternative to forming a linkage to the compounds of the present invention via the polypeptide backbone is to form a covalent linkage with the carbohydrate side chains of the glycoprotein according to the methods such as those of McKearn, et al., EPO 88,695. Thus, the carbohydrate side chains of antibodies may be selectively oxidized to generate aldehydes which may then be reacted either with amine reactive groups to form a Schiff base or with hydrazine, semicarbazide or thiosemicarbazide reactive groups, to give the corresponding hydrazone, semicarbazone or thiosemicarbazone linkages. These same methods may also be employed to link the prodrugs of this invention to non- proteinaceous targeting agents such as carbohydrates and polysaccharides. [0125] An alternative targeting agent reactive moiety useful for linkage to carbohydrates and polysaccharides without the necessity for prior oxidation is the dihydroxyboryl groups, such as is present in meta (dihydroxyboryl)phenylthiourea derivatives. This group is reactive with targeting agents containing a 1,2-cis-diol, forming a 5-membered cyclic borate ester, and thus is of use with those carbohydrates, polysaccharides and glycoproteins which contain this group. The dihydroxyboryl derivatives may also be used to link the prodrugs of this invention to ribonucleosides, ribonucleotides and ribonucleic acids, since ribose contains a 1,2-cis-diol group at the 2',3' position, as disclosed by Rosenberg, et al., Biochemistry, 11, 3623-28 (1972). Deoxyribonucleotides and DNA targeting agents may not be linked to the present prodrugs in this fashion as the 3' hydroxyl group is absent. The latter targeting agents may be conjugated to isothiocyanate derivatives of prodrugs by first forming an allylamine derivative of the deoxyribonucleotide as disclosed by Engelhardt, et al., EPO 97,373.
[0126] When the targeting agent to be linked with the prodrugs of this invention is an intact cell, either polypeptide reactive or carbohydrate reactive moieties may be employed. Hwang and Wase, Biochim. Biophys. Acta, 512, 54-71 (1978), disclose the use of the diazonium derivative of the bifunctional EDTA chelator of Sundberg, et al., J. Med. Chem., 17, 1304 (1974), to label erythrocytes and platelets with indium- 111. The dihydroxyboryl group is reactive with a variety of bacteria, viruses and microorganisms, see Zittle, Advan. Enzym., 12 493 (1951) and Burnett, et al., Biochem. Biophys. Res. Comm., 96, 157-62 (1980). [0127] The compound may have one of the following structures:
Figure imgf000038_0001
[0128] The compound may be produced by the following reaction:
Figure imgf000039_0001
[0129] The linker may be used to covalently alkylate Compound 1 or Compound 2 and may have the formula:
Figure imgf000039_0002
(Compound 4)
[0130] wherein,
X represents a halo group;
Y represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)- Z1 and Z2 independently are S or O; and n = 0 to 4.
[0131] In one embodiment, Compound 4 of the present invention are those compounds wherein, X represents Cl or I;
Y represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-; Z1 and Z2 independently are O; and n = 0.
[0132] In another embodiment, Compound 4 of the present invention are those compounds wherein,
X represents Cl or I;
Y represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2)-;
Z1 and Z2 independently are O; and n = 1.
[0133] Compound 4 provides linkers with both an alkyl and aryl carboxylic backbone which provides flexibility in the cleavage rate of the final quaternary nitrogen. The linkers of Compound 4 may be prepared using commercially available starting products as described in Example 5. g. Purification
[0134] The compounds of the present invention may be isolated using standard purification methods. The hydrolyzable bond of the compounds of the present may be prone to hydrolysis during the purification of the compounds.
[0135] The present invention is also directed to methods of purifying the compounds of the present invention comprising adding the compounds to a solution comprising at least 0.1% acid (v/v) to solubilize the compound. The compound is then purified by performing chromatography, such as HPLC. h. Testing
[0136] The prodrugs of the present invention may be tested to determine the rate of hydrolysis of the hydrolyzable bond and the products of hydrolysis by performing HPLC analysis of the prodrug exposed to cleavage conditions as a function of time. The biological activity of the compounds of the present invention may be measured by methods including blocking phagocytosis in macrophage cell line J774 cells. The biological activity of the compounds of the present invention may also be measured by PDK enzyme assays as described by U.S. Patent No. 5,480,906; K. Fuchikami et al J. Biomol Screen, 2002 Oct. pp441-450; VI Silveria et al J. Biomol. Screen, 2002, Dec. 7(6), 507-514; BE Drees Combinatorial Chemistry and Highthroughput Screening 2003, vol 6, 321-330, the contents of which are incorporated by reference. i. Salts
[0137] The compounds of the present invention are useful in various pharmaceutically acceptable salt forms. The term "pharmaceutically acceptable salt" refers to those salt forms which would be apparent to the pharmaceutical chemist, i.e., those which are substantially nontoxic and which provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity and flowability of the resulting bulk drug. Conveniently, pharmaceutical compositions may be prepared from the active ingredients or their pharmaceutically acceptable salts in combination with pharmaceutically acceptable carriers.
[0138] Pharmaceutically acceptable salts of the compounds of the present invention which are suitable for use in the methods and compositions of the present invention include salts formed with a variety of organic and inorganic acids such as hydrogen chloride, hydroxymethane sulfonic acid, hydrogen bromide, methanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid, glycolic acid, stearic acid, lactic acid, malic acid, pamoic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethonic acid, and include various other pharmaceutically acceptable salts, such as, e.g., nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates, salicylates, and the like. Cations such as quaternary ammonium ions are contemplated as pharmaceutically acceptable counterions for anionic moieties.
[0139] A salt of a compound disclosed herein may be a hydrochloride salt, methanesulfonic acid salt, trifluoroacetic acid salt, or methanesulfonic acid salt. In addition, pharmaceutically acceptable salts of the compounds of the present invention may be formed with alkali metals such as sodium, potassium and lithium; alkaline earth metals such as calcium and magnesium; organic bases such as dicyclohexylamine, tributylamine, and pyridine; and amino acids such as arginine, lysine and the like.
[0140] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base, in a suitable solvent or solvent combination. [0141] In general, the counterions of the salts of the compounds of the present invention are determined by the reactants used to synthesized the compounds. There may be a mixture of counterions of the salts, depending on the reactants. For example, where NaI is added to facilitate the reaction the counterion may be a mixture of Cl and I counter anions. Furthermore preparatory HPLC may cause the original counterion to be exchanged by acetate anions when acetic acid is present in the eluent. The counterions of the salts may be exchanged to a different counterion. The counterions are preferably exchanged for a pharmaceutically acceptable counterion to form the salts described above. Procedures for exchanging counterions are described in WO 2002/042265, WO 2002/042276 and S. D. Clas, "Quaternized Colestipol, an improved bile salt adsorbent: In Vitro studies." Journal of Pharmaceutical Sciences, 80(2): 128-131 (1991), the contents of which are incorporated herein by reference. For clarity reasons the counterions are not explicitly shown in the chemical structures herein and the characterization of the compounds is based on the identified quarternary cation. 4. Composition
[0142] The present invention also encompasses a composition comprising one or more compounds of the present invention. The composition may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like. a. Formulation
[0143] A composition disclosed herein may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including a binding agent, filler, lubricant, disintegrant or wetting agent. The binding agent may be syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone. The filler may be lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, or sorbitol. The lubricant may be magnesium stearate, stearic acid, talc, polyethylene glycol, or silica. The disintegrant may be potato starch or sodium starch glycollate. The wetting agent may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
[0144] The composition may also be a liquid formulation such as an aqueous or oily suspension, solution, emulsion, syrup, or elixir. The composition may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain an additive such as a suspending agent, emulsifying agent, nonaqueous vehicle or preservative. The suspending agent may be sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, or hydrogenated edible fat. The emulsifying agent may be lecithin, sorbitan monooleate, or acacia. The nonaqueous vehicle may be an edible oil, almond oil, fractionated coconut oil, oily ester, propylene glycol, or ethyl alcohol. The preservative may be methyl or propyl p-hydroxybenzoate or sorbic acid. [0145] The composition may also be formulated as a suppository, which may contain a suppository base such as cocoa butter or glycerides. The composition may also be formulated for inhalation, which may be in a form such as a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane. The composition may also be formulated in a transdermal formulation comprising an aqueous or nonaqueous vehicle such as a cream, ointment, lotion, paste, medicated plaster, patch, or membrane.
[0146] The composition may also be formulated for parenteral administration such as by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain a formulation agent such as a suspending, stabilizing, or dispersing agent. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including sterile, pyrogen-free water. [0147] The composition may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The composition may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
[0148] The composition may also be formulated as a liposome preparation. The liposome preparation may comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell. For example, liposomes such as those described in U.S. Patent No. 5,077,211 of Yarosh, U.S. Patent No. 4,621,023 of Redziniak et a or U.S. Patent No. 4,508,703 of Redziniak et ah can be used. The composition may be intended to target skin conditions, and may be administered before, during, or after exposure of the skin of the mammal to UV or agents causing oxidative damage. The composition may comprise niosomes. Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
5. Treatment
[0149] The present invention also encompasses a method of treating a patient suffering from a condition associated with PDK activity. The PDK activity may be abnormal, excessive, or constitutively active. The present invention also encompasses a method for treating inflammatory disease comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. Such diseases and adverse health effects attributable to inappropriate PDK signaling activity have been disclosed in the art, for example U.S.
2002/0150954A1; US 5,504,103; US 6,518,277Bl; U.S. 6,403,588; U.S. 6,482,623; U.S.
6,518,277; U.S.6,667,300; U.S.20030216389; U.S.20030195211; U.S.20020037276 and U.S.
5,703,075 the contents of which are incorporated by reference.
[0150] The present invention also encompasses a method for enhancing p53 mediated programmed cell death comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
[0151] The present invention also encompasses a method for enhancing the chemosensitivity of tumor cells comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
[0152] The present invention also encompasses a method for enhancing the radiosensitivity of tumor cells comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
[0153] The present invention also encompasses a method for inhibiting tumor induced angiogenesis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
[0154] The present invention also encompasses a method for inhibiting angiogenic processes associated with non-cancer diseases comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention.
[0155] The present invention also encompasses a method for treatment of cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. [0156] The present invention also encompasses a method for treating age-related macular degeneration (AMD) comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Retina, February 18, 2004, inhibition of VEGF inhibits blood vessel overgrowth associated with AMD. The compounds of the present invention may treat AMD by inhibiting angiogenesis. [0157] The present invention also encompasses a method for treating hypertension comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Northcott and Watts, Hypertension, 43(1): 125-130 (2004), inhibition of PI3K may prevent the low extracellular concentrations of Mg2+ that are associated with hypertension.
[0158] The present invention also encompasses a method for suppressing differentiation of progenitor cells, such as myeloid progenitor cells, comprising adding an effective amount of a compound of the present invention to progenitor cells. As discussed in Lewis et al, Experimental Hematology, 32(1): 36-44 (2004), inhibition of the PI3K pathway suppresses myeloid progenitor cell.
[0159] The present invention also encompasses a method for treating liver cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Leng et al, Hepatology 38(4) Suppl 1: 40 IA (2003), LY294002 inhibits phosphorylation of Akt (serine/threonine protein kinase B), which is an indicator in human liver tissues.
[0160] The present invention also encompasses a method for treating conditions associated with a mutant PTEN comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. PTEN is a tumor suppressor gene located on chromosome 10q23 that has been identified in patients with Cowden disease. As discussed in Vega et al, Journal of Investigative Dermatology, 121(6): 1356-1359 (2003), mutant PTEN has reduced ability to inhibit the activation of the proto-oncogene Akt. Inhibitors of PI3K may inhibit phosphorylation of Akt, thereby reducing the effect of the mutant PTEN. [0161] The compound may be administered simultaneously or metronomic ally with other anticancer treatments such as chemotherapy and radiation therapy. The term "simultaneous" or "simultaneously" as used herein, means that the other anti-cancer treatment and the compound of the present invention may be administered within 48 hours, 24 hours, 12 hours, 6 hours, or 3 hours or less, of each other. The anti-cancer treatment may be administered 1-7 days prior to administering the compound. The term "metronomically" as used herein means the administration of the compounds at times different from the chemotherapy and at certain frequency relative to repeat administration and/or the chemotherapy regimen. [0162] The chemotherapy treatment may comprise administration of an anti-cancer agent, which may be a cytotoxic agent or cytostatic agent, or combination thereof. Cytotoxic agents prevent cancer cells from multiplying by: (1) interfering with the cell's ability to replicate DNA and (2) inducing cell death and/or apoptosis in the cancer cells. Cytostatic agents act via modulating, interfering or inhibiting the processes of cellular signal transduction which regulate cell proliferation and sometimes at low continuous levels.
[0163] Classes of compounds that may be used as cytotoxic agents include the following: alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide; antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine; natural products and their derivatives (for example, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins): vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel (paclitaxel is commercially available as Taxol®), mithramycin, deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (e.g., IFN-α), etoposide, and teniposide. [0164] Other proliferative cytotoxic agents are navelbene, CPT-I l, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.
[0165] Microtubule affecting agents interfere with cellular mitosis and are well known in the art for their cytotoxic activity. A microtubule affecting agent may include allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC 608832 or docetaxel), thiocolchicine NSC 361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574), natural and synthetic epothilones such as epothilone A, epothilone B, and discodermolide (see Service, (1996) Science, 274:2009) estramustine, nocodazole, MAP4, and the like. Examples of such agents are also described in Bulinski (1997) J. Cell Sci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou (1997) Nature 387:268-272; Vasquez (1997) MoI. Biol. Cell. 8:973-985; and Panda (1996) J. Biol. Chem 271:29807-29812. [0166] Also suitable are cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cis-platin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors. [0167] Cytostatic agents that may be used include hormones and steroids (including synthetic analogs): 17.alpha.-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl- testosterone, prednisolone, triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, zoladex.
[0168] Other cytostatic agents are antiangiogenics such as matrix metalloproteinase inhibitors, and other VEGF inhibitors, such as anti-VEGF antibodies and small molecules such as ZD6474 and SU6668 are also included. Anti-Her2 antibodies from Genetech may also be utilized. A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Also included are Imclone antibody C225 immuno specific for the EGFR, and src inhibitors.
[0169] Also suitable for use as an cytostatic agent is Casodex® (bicalutamide, Astra Zeneca) which renders androgen-dependent carcinomas non-proliferative. Yet another example of a cytostatic agent is the antiestrogen Tamoxifen® which inhibits the proliferation or growth of estrogen dependent breast cancer. Inhibitors of the transduction of cellular proliferative signals are cytostatic agents. Representative examples include epidermal growth factor inhibitors, Her-2 inhibitors, MEK-I kinase inhibitors, MAPK kinase inhibitors, PI-3 inhibitors, Src kinase inhibitors, and PDGF inhibitors. For example the PI-3 inhibitor may be rapamycin or rapamycin analogs, which may be an inhibitor of mTOR, which may be component downstream of PI3K in the PI-3 signaling pathway. Examples of other proliferative signal inhibitors that may be used include: CCI-779, temsirolimus, trastuzumab, cetuximab, sunitib, lapatinib, imatinib mesylate, bortezumib, and sorafanib.
[0170] A variety of cancers may be treated according to the present invention including: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non- Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma. [0171] The compound may be used to treat accelerated or metastatic cancers of the bladder, pancreatic cancer, prostate cancer, non-small cell lung cancer, colorectal cancer, and breast cancer.
[0172] The present invention also encompasses a method for treating pancreatitis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Gukovsky et al., Gastroenterology, 126(2):554-66 (2004), inhibition of PDK may prevent pancreatitis.
[0173] The present invention also encompasses a method for treating ulcers comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. The present invention also encompasses a method for treating gastric cancer, such as stomach cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Bacon et al., Digestive Disease Week Abstracts and Itinerary Planner, Vol. 2003, Abstract No. M921 (2003) and Rokutan et al., Digestive Disease Week Abstracts and Itinerary Planner, Vol. 2003, Abstract No. 354 (2003), PI3K is involved in the adhesion of Helicobacter pylori to gastric cells. Furthermore, Osaki et al., Journal of Cancer Research and Clinical Oncology, 130(1): 8-14 (2004) indicates that a PI3K inhibitor, such as LY294002, may be useful as an anti-tumor agent for gastric carcinoma.
[0174] The present invention also encompasses a method of improving the performance of a stent comprising administering a therapeutically effective amount of a compound of the present invention to a patient with a stent, such as a cardiovascular stent. As discussed in Zhou et al., Arteriosclerosis Thrombosis and Vascular Biology, 23(11): 2015-2020 (2003), inhibition of PI3K may prevent the "stretch" damage that accompanies stent placement in vessels. The compounds of the present invention in the stent or polymer matrix thereof may improve solubility in the stent coating matrix, improve aqueous/serum solubility, or improve perfusion into the cells immediately adjacent to the stent placement.
[0175] The present invention also encompasses a method for treating age-related macular degeneration (AMD) comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Retina, February 18, 2004, inhibition of VEGF inhibits blood vessel overgrowth associated with AMD. The compounds of the present invention may treat AMD by inhibiting angiogenesis. [0176] The present invention also encompasses a method for treating hypertension comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Northcott and Watts, Hypertension, 43(1): 125-130 (2004), inhibition of PI3K may prevent the low extracellular concentrations of Mg2+ that are associated with hypertension.
[0177] The present invention also encompasses a method for suppressing differentiation of progenitor cells, such as myeloid progenitor cells, comprising adding an effective amount of a compound of the present invention to progenitor cells. As discussed in Lewis et al, Experimental Hematology, 32(1): 36-44 (2004), inhibition of the PI3K pathway suppresses myeloid progenitor cell.
[0178] The present invention also encompasses a method for treating liver cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. As discussed in Leng et al., Hepatology 38(4) Suppl 1: 40 IA (2003), LY294002 inhibits phosphorylation of Akt (serine/threonine protein kinase B), which is an indicator in human liver tissues.
[0179] The present invention also encompasses a method for treating conditions associated with a mutant PTEN comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the present invention. PTEN is a tumor suppressor gene located on chromosome 10q23 that has been identified in patients with Cowden disease. As discussed in Vega et al, Journal of Investigative Dermatology, 121(6): 1356-1359 (2003), mutant PTEN has reduced ability to inhibit the activation of the proto-oncogene Akt. Inhibitors of PI3K may inhibit phosphorylation of Akt, thereby reducing the effect of the mutant PTEN. a. Administration
[0180] Compositions of the present invention may be administered in any manner including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof. Parenteral administration includes intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular. The compositions of the present invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion. b. Dosage
[0181] A therapeutically effective amount of the compound required for use in therapy varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the patient, and is ultimately determined by the attendant physician. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 1 μg/kg to about 100 μg/kg per day. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. Multiple doses often are desired, or required.
[0182] A number of factors may lead to the compounds of the present invention being administered at a wide range of dosages. When given in combination with other therapeutics, the dosage of the compounds of the present invention may be given at relatively lower dosages. In addition, the use of targeting agents may allow the necessary dosage to be relatively low. Certain compounds of the present invention may be administered at relatively high dosages due to factors including low toxicity, high clearance, low rates of cleavage of the tertiary amine. As a result, the dosage of a compound of the present invention may be from about 1 ng/kg to about 100 mg/kg. The dosage of a compound of the present invention may be at any dosage including about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg, 125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, 450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600 μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg, 775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925 μg/kg, 950 μg/kg, 975 μg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg. [0183] The present invention has multiple aspects, illustrated by the following non-limiting examples.
Example 1 Preparation of LY294002
[0184] A lO g sample of LY294002 was prepared according to Scheme 1 based on the procedure described in Vlahos et al, J. Biol. Chem. 269(7): 5241 (1994), the contents of which are incorporated by reference. The displacement of the thiomethyl group of thoichromones such as 12 by amines has been described previously (Bantick et al, J. Heterocyclic Chem, 18:679 (1981), the contents of which are incorporated by reference) as has the cyclization of methyl phenyl ketones such as jj_ with carbon disulfide with concomitant alkylation of the thiol anion (Vlahos et al. and Bantick et al). Preparation of methyl ketones (e.g., H) in a one-step reaction from the carboxylic acid (K)) was performed using the procedure described in Rubottom et al., J. Org. Chem., 48:1550 (1983), the contents of which are incorporated by reference.
SCHEME 1
Figure imgf000052_0001
Example 2 Preparation of Alkylated Analogs of LY294002
[0185] The keto oxygen of LY294002 is alkylated using iodomethane or benzyl chlorides under forcing conditions, which can be methyl alkylated, phthalimido alkylated, paracarboxy benzyl alkylated, and a para- sen-benzyl alkylated prodrugs.
Example 3 Preparation of Chloromethyl Esters
[0186] Chloromethyl intermediates were prepared following the procedure described in Tsujihara, Synth Commun, 24, 767, 1994. Briefly, the appropriate carboxylic acid was diluted in a 50/50 mixture of dichloromethane/water. The mixture was cooled in an ice-water bath and sodium bicarbonate (4 equiv) and n-tetrabutyl ammonium hydrogen sulfate (0.05 equiv) was added. After stirring for 5 min, chloromethyl chlorosulfate (1.1 equiv) was added. The solution was stirred vigorously overnight. The mixture was transferred to a separatory funnel with more dichloromethane and washed with saturated sodium chloride solution. The organics were dried over sodium sulfate and the solvent removed to provide the product. The material was characterized by LC-MS and in some cases by IH NMR spectroscopy. By this general procedure the following representative chloromethyl esters were prepared from the corresponding carboxylic acids:
Table 1
Figure imgf000052_0002
Figure imgf000053_0001
51
Figure imgf000054_0001
* HPLC-MS retention time using UV detection;
** HPLC-MS retention time of the starting carboxylic acid using UV detection;
UD= undetectable due to lack of UV absorbance and no ionization by MS
Example 4 Conversion of LY294002 to Alkylated Prodrug
[0187] LY294002 (Compound 1) is dissolved in acetonitrile and then each of the chloromethyl esters (1-1.5 equiv) from Example 3 is added along with 1-2 equivalents of sodium iodide. At room temperature, the reaction proceeds only slowly with the chloromethylesters to give very small amounts of the alkylated product along with the precipitation of sodium chloride. At 650C, the reaction proceeds to completion usually in 4 hours. The reaction when complete (as judged by analysis by LC-MS) is filtered; concentrated and then purified on reverse phase HPLC. The fractions are collected and lyophilized to give the desired products as fluffy powders.
Example 5 Halomethyl Ester Linkers
[0188] Halomethyl ester linkers were prepared (Scheme 2 and chart). Compound B was prepared from Compound A (commercially available) as described in Example 3. This compound was converted into the more reactive iodomethyl ester (Compound C) by a Finklestein reaction by dissolving in acetone or 2-butanone and then dissolving 2-5 equivalents of sodium iodide whereupon the sodium chloride precipitated and the iodomethyl ester (Compound C) was produced in solution. Compound C was isolated by stripping off the solvent and dissolving in a water immiscible solvent such as methylene chloride and extracting with water to remove the residual sodium iodide. [0189] Compound E was prepared from Compound D (commercially available) Compounds F and G were prepared in a manner similar to the production of Compounds B and C, respectively.
SCHEME 2
Figure imgf000055_0001
Example 6 Alkylation of LY294402 with Halomethyl Linkers
[0190] Halomethyl esters, including those of Example 5, are used to alkylate LY294002 using conditions similar to the methodology in Example 4.
Example 7 Preparation of Prodrugs Using Compound 1111
[0191] Compound 1111 was produced by the method as shown below. Compound 1110 was treated with neat trifluoroacetic acid for 1-3 hours and the TFA was blown off with argon and dried under vacuum to give a glassy solid comprised of Compound 1113 (t-butyl ester group of Compound 1110 cleaved to give the corresponding carboxylic acid). Compound 1113 was then dissolved in 1-3 ml of thionyl chloride and heated at 650C for 3-8 hours. The thionyl chloride was blown off with argon and then dried under high vacuum to give Compound 1111 (acid chloride of Compound 1113) in good yields as a glassy yellow solid. Compound 1111 can be reacted as a typical acid chloride with various nitrogen-containing and hydroxyl-containing nucleophiles for example by simply dissolving in methanol to give the corresponding methyl ester Compound 1112.
SCHEME 3
Figure imgf000056_0001
[0192] A sample of Compound 1111 was dissolved in acetonitrile and treated with at least 5 equivalents of different alcohols in separate vials. After 1 hour the samples were analyzed by HPLC-MS and showed good conversion >90% of Compound 1111 to corresponding esters.
54
INCORPORATED BY REFERENCE (RULE 20.6) Example 8 Preparation of Protein Conjugate Prodrugs Using Compound 1111
[0193] Proteins are conjugated in largely aqueous solution (pH 7-9) (phosphate buffer to carbonate buffer) using a 2-10 fold excess of Compound 1111 relative to the keto oxygen to be modified. The acid chloride Compound 1111 can be introduced in a mixed organic-aqueous solution (such at 50/50 water/acetonitrile or 50/50 water/THF) or stirred in methylene chloride in a two-phase reaction system at room temperature for 1-24 hours. Protein-conjugates can be purified by dialysis or ultrafiltration and used directly.
[0194] A 500 μl aliquot of 5 mg/ml transferrin protein (Sigma) in 50 mM sodium bicarbonate buffer was mixed with 100 up of 30 mM A024-79 (100 molar equivalents) in DMSO. After 1 hour and 20 minutes of reacting at room temperature a 50 up sample was removed and passed through a Sephadex G- 10 (700 molecular weight cutoff) column to separate protein from small molecules. An aliquot of the purified conjugated protein eluent was then extracted with acetonitrile and no detectable Compound 1 was observed by LC-MS. The purified conjugated protein eluent was allowed to stand at room temperature 39 hours at which time the protein mix was again extracted with acetonitrile and this time 15% of the maximum theoretical amount of Compound 1 was detected. These results indicate a molar ratio of 15 moles of prodrug were attached per mole of transferrin. These results demonstrated the attachment of an electrophilic linker-bearing prodrug to a representative protein and demonstrated that over time a substantial amount of a PI3 kinase inhibitor (compound 1) was released from the protein under aqueous conditions.
Example 9 Preparation of Resin-Bound Prodrugs Using Compound 1111
[0195] The peptide arg-gly-asp-ser (RGDS) was prepared on wang resin using standard FMOC/HOBT coupling peptide chemistry using all natural amino acids. The resin-bound peptide was reacted with Compound 1111 in DMF from 1-24 hours, filtered and the resin washed with DMF and then methylene chloride and then treated with trifluoroacetic acid to cleave the conjugate Compound 1126 from the resin (Scheme 4). SCHEME 4
Figure imgf000058_0001
Example 10 Synthesis of Compound 1126
[0196] In order to prepare chloromethyl-t-butylsuccinate, mono t-butyl succinate (Aldrich), 2.0 g, that was dissolved in 8.0 ml methylene chloride was added to a glass vial containing 4.0 g potassium carbonate and 0.24 g tetra n-butyl ammonium hydrogen sulfate in 8.0 ml water with stirring in an ice bath. After 15 minutes, 1.3 mL chloromethyl chlorosulfate (Acros) was added to the methylene chloride layer and the reaction mixture was stirred with the temperature slowly coming to room temperature. The organic layer was separated and washed with Ix 10 ml water and 1 x 10 ml saturated brine solution. The solution was dried over anhydrous sodium sulfate and the solvent removed under reduced pressure. 3 g of a pale yellow oil was obtained and assigned lot number A047-71.
[0197] The 3 g of A047-71 from above were dissolved in 36 mL of acetonitrile and treated with 1.8 g of Compound 1101 and 1.8 g of NaI and put on a heater with stirring for 16 hours. The reaction mix (including precipitate) was partitioned between water and methylene chloride and the methylene chloride layer was separated, washed with brine, dried over sodium sulfate and evaporated to give a dark oil. This oil was dissolved in 5 mL of acetonitrile and stored in the freezer for two days. The yellow precipitate that formed was then filtered and washed with 2X5 mL of acetonitrile and dried to give 2.4946g of SFl 110 as lot A046-67SM with retention time of about 2.7 minutes and m/z + of 494. A 2 gm portion of SFl 110 (A046-67SM) was teated with 8 mL of neat trifluoroacetic acid (TFA) for about one hour to give SFl 113 (carboxylic acid) with a retention time of about 2.2 minues and m/z+ of 438. The TFA was removed under a stream of argon and then the reaction mix was dried under vacuum to give 2.8435g of SFl 113 (lot A046- 67A). All of this SFl 113 carboxylic acid product was dissolved in 10 mL of thionyl chloride and heated to 65°C for 4 hours. The excess thionyl chloride was removed under vacuum and the yellow oil dried under high vacuum to yield 1.9906g of the acid chloride (Compound 1111, lot A046-67B) as a yellow crunchy solid. This solid was used directly in subsequent reactions. [0198] In order to couple the Compound 1111 acid chloride and de-FMOCed RGDS peptide, 1.26 g acid chloride of Compound 1111 and 5.6 g de-FMOC removed RGDS peptide (both dried in vacuum dessicator over Phosphorous pentoxide) were mixed in a 50 ml round bottom flask under Argon gas. To the solids was added 270 uL dry pyridine in 28 ml methylene chloride and the mixture shaken to dissolve the acid chloride. The mixture was placed on an orbital shaker for one hour. The solution was drained through a fritted plastic syringe and washed with 2 x 10 ml methylene chloride and the solvent drained. To the resin was added 500 uL anisole followed by 20 ml of a 50/50 TFA/ methylene chloride solution. The resin was allowed to stand in the TFA solution for three hours with occasional shaking. The TFA solution was then drained away from the resin. The resin was washed with 10 ml methylene chloride which was combined with the TFA solution. The TFA-methylene chloride solution was placed into four vials and then blown dry with Argon gas in a well-ventilated fume hood. Each vial was treated with multiple ether washes which precipitated the crude product. The ether washes were decanted and the crude solids blown dry with Argon gas to give a total of 2.3363g of crude product (lot A044-84). The product was identified in the crude product by LCMS (retention time about 1.8 minutes) and was then purified in seventeen runs using preparative reverse phase LCMS. The combined runs yielded 163.7 mg of 95% pure product (assigned lot A051-19) with a retention time of 1.768 minutes, M+ = 853 and [M+H]/2 at 427 m/z. In Figure 7A is the chromatogram of this purifed lot of SFl 126. The top two traces are the 254nm and 214nm UV signals respectively. In the 254 nm trace the SFl 126 retention time is about 1.8 minutes and a small amount of SFl 101 can be noted at retention time of 3.15 minutes. The third trace down from the top of Figure 7 A is the evaporative light scattering detector signal. The bottom trace is the single ion monitoring trace for the SFl 126 at 853 m/z+. In Figure 7 is shown the chromatogram of a similarly purified batch (lot A036-33) of SFl 126 the x-axis is time in minutes and the y-axis for the top chromatogram is milli-absorbance units for the UV detector at 254 nm and for the bottom chromatogram is millivolts detected by the evaporative light scattering detector. In Figure 8 the x-axis is the mass- to-charge ratio (m/z) and the y-axis is the intensity of the mass ion count.
Figure imgf000060_0001
(Compound 1101)
SCHEME 5
Figure imgf000060_0002
A047-71
58 INCORPORATED BY REFERENCE (RULE 20.6) Compound 1101 NaI, Acetonitπle, heat
Figure imgf000061_0001
Compound 1113
Figure imgf000061_0002
59 INCORPORATED BY REFERENCE (RULE 20.6) Compound 1126
Figure imgf000062_0001
Example 11 Synthesis of Compound A042-70
Figure imgf000062_0002
[0199] A solution of 111.1 mg of the phosphonated diamine (prepared via A042-32) in 1 niL dichloromethane was treated with 194 mg of bromotrimethylsilane. After 5 hr, methanol (1 mL) was added, the mixture was stirred for 1 hr, and the solvent was removed to provide 113.9 mg of a tan solid. The presence of the title compound was confirmed by electrospray LC-MS using method B; tR = 1.0 min. MS [M=C18H37N5O13P4] m/z 328 [(M+2H/2)2+)], 656 (MH+). The compound was also analyzed by proton NMR spectroscopy: H (CDCl3) δ: 7.77 (d, 2H, / 8.1 Hz), 7.43 (d, 2H, / 8.2 Hz), 4.1-3.3 (m, 33H).
Example 12 Preparation of a bone targeted prodrug of Compound 1.
[0200] A 20 uL portion of 75 mMolar A042-70 (1.5 uMoles) in water was added to a vial containing 500 mMolar phosphate buffer (11 vials at 11 different pHs ranging from 3.0 to 8.0 in 0.5 unit increments). After mixing each vial was then treated with 50 uL of 60 uMolar Compound 1111 (acid chloride) in acetonitrile (3.0 uMoles= 2 equivalents relative to amino
60 INCORPORATED BY REFERENCE (RULE 20.6) group of bone targeting agent) and mixed by shaking. After one hour a 3 uL aliquot of each vial was injected on HPLC and the UV peak area determined for starting material and desired product and by product of aqueous hydrolysis, Compound 1101. Only the samples at pH of 6, 6.5, 7, 7.5 and 8 were found to have significant amounts of desired bone targeted prodrug A046- 89P (retention time 2.50 minutes; [M+] found for 1075 m/z C42H59N6O19P4) [M+2]/2=538 m/z also found). This example demonstrates that the optimum pH for synthesis of bone targeted prodrug under these condition is pH=7.0 which gave about 42% of theoretical yield of the desired bone targeted prodrug of compound 1 possessing 4 phosphonic acid groups. After 24 hours analysis of this same solution standing for that time at pH=7.0 indicated that the targeted prodrug had converted completely back to compound 1 demonstratrating reversibility under physiologically relevant conditions.
Figure imgf000063_0001
Example 13 In Vivo Efficacy of Compound 1126 Against Non-Small Cell Lung Cancer
[0201] Male nude mice of 4-6 weeks in age weighing around 30 grams were inoculated subcutaneously in the right flank with 5 million tumor cells (human non-small cell lung cancer cells: H1299) on day 0. After 14 days of allowing the tumors to grow the animals were divided into 3 groups of 5 animals each. One group received vehicle control alone. One group received twice-per-day tail vein injections (i.v) of 50 uL volume of 24.4 millimolar solution of Compound 1126 in phosphate buffered saline corresponding to 25 mg/kg/day dosing level of the active component of the prodrug (i.e. compound 1). The last group received twice-per-day tail vein injections (i.v) of 50 uL volume of 4.9 millimolar solution of Compound 1126 in phosphate buffered saline corresponding to 5 mg/kg/day dosing level of the active component of the prodrug (compound 1). The tumors were measured every three days using calipers to determine the tumor volume and the animals weights were recorded when the animals were sacrificed on day 27. The results are shown in Table 2 and indicate strong tumor volume reduction versus control for both dose levels at the first datapoint only 3 days after treatment (Day 17) and continuing through to the end of the study:
Table 2
Figure imgf000064_0001
*Compared to the vehicle only control animals
[0202] The twice per day doses were well tolerated over the two week administration period. The efficacy results above were also accompanied by a lack of statistically significant difference in the animal body weights between the control group and the two treatment groups which as a general measure of gross toxicity indicated the targeted prodrug has a desirable lack of toxicity.
Example 14 In Vivo Efficacy of Compound 1126 Against Brain Cancer
[0203] An animal study was run as described in Example above except using a human brain cancer cell line (U87MG) and with treatment starting on day 7 such that the first tumor volume measurement occurred on day 10. The results of Compound 1126 against this cancer cell line are shown in Table 3 and indicated effectiveness and a desirable lack of toxicity: Table 3
Figure imgf000065_0001
*Compared to the vehicle only control animals
Example 15
Alpha v targeted PI 3 kinase inhibitors abrogated the tube formation of EDC-CBFl endothelial cells on Matrigel.
[0204] Tube formation represents to some extent the formation of angiogenesis in vivo. In this example it was determined to what degree PI 3 kinase inhibitors (including targeted PI3 kinase inhibitor prodrugs) could inhibit tube formation. Matrigel was plated into 12-well plate wells and solidified in 37°C for 2 hours. 1 x 10 EDC-CBFl endothelial cells were then put on the top of the Matrigel layer in the presence of PBS, RADfV (cyclic negative control peptide), RGDfV (cyclic positive control peptide), RADS (linear negative control peptide), compound 1, or Compound 1126 at 20 0M concentration overnight. Pictures were then taken using a microscope. Well formed tubes can be visualized in the PBS control wells (top left panel of Figure). There was not much difference in the RGDfV-, RADfV-, or RGDS- containing wells compared with PBS control. Tube formation was significantly less in Compound 1101- and Compound 1126- containing wells.
Example 16 Targeted PI 3 kinase inhibitors induced p53 transcriptional activity in HBECs
[0205] This experiment tested the effect of PI 3 kinase inhibitors (Compound 1 and the targeted prodrug version of Compound l;Compound 1126) on the induction of p53 luciferase activity. The transfection procedure was similar to that described in the literature to monitor p53 transcription. Compound 1 (6 hour exposure) induced more than two fold higher luciferase activity than the control and the targeted version of compound 1 (Compound 1126) had even better ability of inducing the p53 luciferase activity (to almost 3 fold). This induction of p53 function was demonstrated to be abrogated by the p53 inhibitor, pifithrin alpha at 20 uM concentration. Co-transfection of catalytic active Akt also inhibited the p53 function induced by these compounds. This result shows that the p53 transcription induced by PI3 kinase inhibitors is downstream of Akt in the whole signaling cascade and the targeted prodrug Compound 1126 gave an enhanced induction of p53 versus the untargeted drug, Compound 1.
Example 17 Purification of Compound 1126
[0206] Reaction mixture A044-84 (2.33 g) was weighed out into separate 0.33 g samples and dissolved immediately before preparative chromatography in 800 μl of a solution containing 1 part by volume acetonitrile, 1 part by volume water, and 1% by volume acetic acid. 400 μl of this solution was injected for each preparative chromatography run. The pump A eluant was B&J water (365-4) with 0.1% acetic acid added, and the pump B eluant was B&J acetonitrile (015-4) with 0.1% acetic acid added. Initially, the eluant was 10% B, then linearly ramped to 34% B over a 4 minute period, then linearly ramped to 95% B at 4.25 minutes and held there until 5.25 minutes, then linearly ramped back to the starting concentration of 10% B at 5.50 minutes. The total pump flow was 20 niL/minute. Re-equilibration of the system was accomplished while the autosampler was sampling for the next run. Using this gradient, the product with positive mass spectral peaks at 853 (m/z=l) and 427 (m/z=2) eluted at 3.37 minutes. Fractions were collected during the preparative chromatography runs when the signal detected at the ELSD exceeded 10 mv. Fractions containing product were diluted with a two-fold excess of water (by volume) and frozen in a lyophilization vessel using a dry ice-acetone bath immediately after collection. After lyophilization over a 24-48 hour period a total of 180 mg of Compound 1126 as a white fluffy solid with a purity of 95% was obtained. This example demonstrates that with careful pH control the labile prodrug Compound 1126 can be isolated in high purity using aqueous based reverse phase separation methods. Example 18 Use of Prodrug to Deliver Compound 1 in Mice
[0207] Mice were injected with a million non-small cell lung cancer cells (H1299) subcutaneously and allowed to grow about 7 days until the tumor mass was approximately 10 to 15 mm by 7 to 9 mm in dimensions. Animals were injected with the targeted prodrug, Compound 1126, either i.v. (50 uL) or i.p. (50 uL) with 32.6 mMolar solutions of Compound 1126 in phosphate buffered saline. After 60 minutes the mice were sacrificed and the tumors removed. Three small pieces of the tumors were retrieved and minced. After aging for 24 hours to allow all of the prodrug to convert to compound 1 the tumor samples were extracted with acetonitrile. Quantitation by LC-MS indicated that the concentration of extractable compound 1 (as the sum of free compound 1 and derived from Compound 1126) was 157+7 nanomolar in the tumor pieces for the LP. injection and 271+17 nanomolar in the tumor pieces for the LV. injection. This example demonstrates the delivery of Compound 1 to tumor tissue using a targeted prodrug.
Example 19 Reversibility of Prodrugs to form Compound 1
[0208] Prodrug Compounds were dissolved in water or in DMSO (if not freely soluble in water) and then diluted at least 10-fold into 50 mM phosphate buffer at pH= 7.4 or pH=4.8 and allowed to stand at room temperature. The final concentration of the compounds in aqueous environment ranged from 50 to 500 uMolar. Aliquots over time were taken and analyzed by LC-MS to determine both disappearance of prodrug and confirm appearance of drug (compound 1). Compound 1126 was found to have a half-life of about 1 hour at pH=7.5 and a half-life of about 64 hours at pH= 4.8. Compound 1110 was found to have a half-life of about 10 hours at pH=7.4 and greater than 120 hours at pH= 4.8. These examples demonstrate that chemically the prodrugs converted to drug (compound 1) and the disappearance of the prodrug is very pH dependant with conversion taking place much faster at physiological pH and substantially slower at acidic pH. Example 20 Synthesis of Tumor Localizing Conjugate
[0209] The electrophilic group-bearing compounds (such as compound 1111 and 1113) can be reacted with polymers bearing nucleohilic groups such as alcohols, amino, and thiol groups. N- (2-hyroxypropyl)methylacrylamide (HPMA) having molecular weight of 2000 to 100,000 is reacted with excess compound 1111 in a nonprotic organic solvent such as methylene chloride or tetrahydrofuran in the presensce of triethyl amine or diisopropylethyl amine and then separated by size exclusion chromatography, ultracentrifugation, or precipitation in another solvent such as methanol or ether. The polymer thus precipitated or separated is substantially free of 1111 and is used as a tumor localizing conjugate that releases active compound 1 overtime in the vicinity of the tumor resulting in antitumor and anti-angiogenic effects. Likewise polyglutamic acids can be converted to poly-nucleophilic bearing groups by reaction of the carboxylic acids with excess diamines using carbodiimide coupling followed by size exclusion chromatography or reverse phase HPLC purification to obtain poly-nucleophilic versions of polyglutamic acids. These polymers can then be reacted directly with excess portions of compound 1111 in an aprotic organic solvent such as methylene chloride or tetrahydrofuran in the presensce of triethyl amine or diisopropylethyl amine and then separated by size exclusion chromatography, ultracentrifugation, or precipitation in another solvent such as methanol or ether. The poly- conjugated polymer thus precipitated or separated is substantially free of low molecular weight residual prodrug and is used as a tumor localizing conjugate that releases active compound 1 overtime in the vicinity of the tumor resulting in antitumor and anti-angiogenic effects.
Example 21 Characterization of SF1126 as O-Alkylated Species
[0210] SFl 126 (lot number A 102-65; having retention time 1.903 minutes with reference SFI lOl having retention time 3.236 minutes) was dissolved in D2O containing 0.1% TFA-d at about 55 mg/mL and studied using the following NMR experiments:
[0211] COSY, or Correlation Spectroscopy, correlates J-coupled protons. This is employed mainly to identify adjacent proton couplings, but geminal proton, as well as some long-range coupling will also be present. [0212] DEPT, or Distortionless Enhancement by Polarization Transfer, identifies the number of attached protons to each protonated carbon. The experiment as acquired includes three sets of spectra, which from bottom to top correspond to the 45°, 90°, and 135° proton decoupler pulses; or: all protonated carbons, methines only, and methines and methyls up and methylenes down, respectively.
[0213] HMQC, or Heteronuclear Multiple Quantum Coherence, correlates the carbon(s) attached to each proton(s). As acquired it is an indirect detection experiment using gradient selection. [0214] HMBC, or Heteronuclear Multiple Bond Correlation, correlates multiple bond carbon couplings to each proton(s). As acquired it is an indirect detection experiment using gradient selection, yielding strong 3-bond couplings, with some 2, 4, and 5 bond couplings being evident. As the experiment is sensitive to coupling constants, not all expected correlations will be evident. [0215] Using the above experimental data a complete carbon hydrogen map was established assigning all the shifts to the atoms of the O-linked structure. The data support the O-linked structure versus an alternative N-alkylated structure in that there is no ketone carbon downfield between 210 to 178 ppm. The furthest downfield carbon found was at 174.4 ppm assigned to an amide carbonyl carbon. The possibility of the ketone also existing as a hydrate (since SFl 126 in the solid form typically has 4-6% by weight of water) was dismissed due to the mapping of this particular carbon to be an olefinic carbon with double bond character thus supporting the enol structure in the O-alkylated arrangement.
[0216] Besides the lack of a ketone peak in the carbon spectrum, which would be expected to be evident from 210 to 178 ppm, the HMBC supports the O-alkylated structure by the correlations to the olefin labeled as Carbon 1 at 166 ppm: i.e., Protons 22, 2, and 6 all correlated to Carbon 1. Proton 2 also exhibits an HMBC correlation to the C=N of Carbon 3 at 162 ppm. [0217] The structure with atoms numbered is shown below: NMR Assignments for SFl 126 (as O-alkylated form) in D2O Solution (w/0.1% TFA-d).
Figure imgf000070_0001
Assignment C λ (ppm) H λ (ppm)
1 166.0
2 86.7 6.59
3 162.4
4 148.5
5 114.8
6 123.0 7.86
7 126.8 7.46
8 135.9 7.65
9 129.9
10 133.9
11 - 13 128.9 & 128.8 7.38
14 173.0
15 174.4
16 156.5
17 174.3
18 170.9
19 171.8
20 173.6
21 172.6
22 84.7 6.03
23 & 24 29.2 & 28.5 2.7 & 2.6
25 53.6 4.03
26 27.6 1.6
27 24.1 1.5
28 40.3 3.00
29 42.3 3.74
30 49.6 4.59
31 35.1 2.7 & 2.6
32 54.7 4.30
33 60.8 3.8 & 3.7
34 65.3 & 65.1 3.8 & 3.7
35 46.8 & 45.8 3.9 & 3.6
Example 22
[0218] The alkylation of SFl 101 with chloromethly esters as in the production of SFl 110 locks in the site of alkylation- oxygen versus nitrogen. A sample of SFl 110 (lot number Al 13-08) was recrystallized from ethyl acetate and the crystal structure was determined and it was found to be the O-alkylated structure (see Figure 10).
[0219] Additionally, the C- 13 NMR spectrum of this sample in DMSO d8 of SFl 110 showed a lack of carbonyl carbon with the furthest downfield carbon being 171.0 ppm which is supportive for lack of ketone functionality found in the O-alkylated structure. The carbon and proton NMR spectra of this lot of SFl 110 are shown in Figure 11 and 12 respectively.
Example 23 Preparation and Characterization of SFl 103
[0220] An O-acylated form of SFl 103 was prepared as described below. A 730 mg sample of bromomethyl acetate was dissolved in 5 mL of dry acetonitrile. To this solution was added 585 mg of compound 1 (SFl 101). The reaction mix was heated in a water bath for a few minutes at 600C until all but a trace of the material was soluble. The small residual material was removed by filtration of about half of the solution through a 0.2 micron syringe filter. The filtrate was allowed to stand for three days. The supernatant was decanted and the crystals washed with two portions of 1 mL of acetonitrile. The crystalline solid was then dried under vacuum to give 232.3 mg of solid (SFl 103) and assigned lot number A102-62BS.
[0221] A lmg portion of this material was dissolved in 1 mL of 50/50 water/acetonitrile containing 0.1% TFA and analyzed by LC-MS. The chromatogram is shown in Figure 13. The top two traces are the 254nm and 214nm UV signals respectively. In the 254 nm trace the SFl 103 retention time is about 2.3 minutes (m/z=380+) and a small amount of SFI lOl (m+H/z= 308+ ) can be noted at retention time of 3.3 minutes. The third trace down from the top of Figure 13 is the evaporative light scattering detector signal. A sample of A102-62BS was placed in DMSO d6 and the proton and carbon- 13 NMR spectra were obtained and are shown in Figure 14 and 15 respectively.
[0222] A 53 mg portion of A102-62BS was weighed out and dissolved in 150 uL of dimethylformamide (DMF) at 73°C and then allowed to cool. These crystals were then analyzed by X-ray and the crystal structure solved to indicate alkylation had occurred on the oxygen and not on the nitrogen to give the O-alkylated version of SFl 103 shown in Figure 16.
Example 24
SFl 126 acts synergistically in combination with rapamycin in inhibiting tumor cell growth and promoting tumor cell death
[0223] This example demonstrates that SFl 126 acts synergistically in reducing tumor cell growth and promoting tumor cell death when used in combination with rapamycin, which inhibits the PDK pathway component mTOR. Rapamycin was chosen as the mTOR inhibitor because it is readily available for research purposes and is a marketed drug. Several rapamycin analogs are in development, and temsirolimus has been recently approved for patients with advance renal cell carcinoma (RCC). Several RCC cell lines are available including 786-0 which is widely used in PDK research and has been investigated with LY294002 in an in vivo model. Methods
[0224] Human brain endothelial cell (HBEC) and 786-0 renal carcinoma cells (RCC) (2xlO3) were plated in 50μL RPMI 1640 media with 10% FBS in each well in 96- well plates with five wells per data point. After adding 50μL of SFl 126 solution and other therapeutic agents individually or in combination at various doses in complete media, cells were incubated for 0, 1, 2, or 3 days, and their viability was measured by incubating them for 4 h after adding lOμL WST-I stain (Roche Diagnostics Corporation). To measure the sequential effects of SFl 126 in combination with another drug, the first drug was added on day 1 and the second drug was added 24 hours later. Cells were then incubated for an additional 48 hours. After a total of 3 days of incubation, cell viability was determined by WST staining and OD readings at 450 nm. A Combination Index (CI) was calculated by using CalcuSyn software (Biosoft) based on the method of Chou and Talalay (Trends Pharmacol. ScL 1983;4:450-4), where in general, a value <1 suggests synergy/sensitization, a value equal to 1 suggests additivity, and a value >1 suggests antagonism.
[0225] To determine if the vertical combination of SFl 126 and rapamycin produce better tumor suppression effects than either drug does alone, an efficacy study (SF- V6) was conducted in a 786-0 RCC xenograft model. 786-0 cells (2xlO6) were injected subcutaneously into nude mice in the flank area, and tumor growth was monitored with calipers for external measurement. Tumor volume was calculated using the formula volume = (length x width2)/2. When average tumor volume reached 400mm3, mice were randomly divided up into six groups of six mice each, and treated with rapamycin alone (1.5 mg/kg, i.p.) three times weekly (M, W, F), SFl 126 alone (20 mg/kg, i.v.) three times weekly (M, W, F), or a combination of the two agents in three different sequences: (1) simultaneous treatment combination (simultaneous rapamycin and SFl 126 administration); (2) rapamycin administration followed by SFl 126 one week later; and (3) SFl 126 administration followed by rapamycin one week later. The dose level of rapamycin and SFl 126 in the combination treatment groups was the same as those in the single agent treatment ones. Treatment with SFl 126 lasted four weeks and tumor size was monitored twice weekly. Body weight was monitored once weekly. Mice were euthanized at the end of the study and subcutaneous tumors were harvested, weighted, and fixed in 10% formalin. Means and standard deviations were calculated and the Student t-test was used for statistics. Results and Discussion
[0226] To evaluate the combination of SFl 126 with rapamycin the 786-0 cell line was used in a 3-day proliferation assay using SFl 126 as a single agent, rapamycin as a single agent, and the combination of SFl 126 with rapamycin. Concentrations suitable for Chou-Talalay analysis (fixed ratios) were employed. For the combination studies three sequences were evaluated for the exposure regiment: (1) SFl 126 on day 1 followed by rapamycin exposure on day 2; (2) rapamycin on day 1 with SFl 126 exposure on day 2; and (3) SFl 126 and rapamycin exposure simultaneously on day 1. A clear dependence of the sequence on proliferation was noted (Figure 17). For example, exposure of 786-0 cells to SFl 126 at 1.6 mM and rapamycin at 1.6 nM using simultaneous exposure (sequence 3) resulted in greater proliferation inhibition (76% inhibition versus control) than SFl 126 alone (8%), rapamycin (47%) alone, or the combination in either sequence 1 (48%) or 2 (64%) (p<0.05 for all comparisons except p>0.05 for rapamycin alone versus sequence 1).
[0227] This combination effect was also noted at several other concentrations of these agents in combination. Interestingly, rapamycin exhibited more of a step-function dose response, where above a certain threshold level, the same degree of effect was observed regardless of the dose (Figure 18). Because of the driving role angiogenesis plays in renal cell carcinoma, the effects of these combinations on endothelial cells was also evaluated. Human brain endothelial cells (HBEC) were used as a model endothelial cell line as described previously in a tumor associated angiogenesis model (Cancer Res. 2003:3585-92). Such combinations and sequence effects were evaluated using HBECs (Figures 19 and 20). Simultaneous exposure of HBEC cells to SFl 126 at 4 mM and rapamycin at 4 nM (sequence 3) produced greater proliferation inhibition (49% inhibition vs control) than SFl 126 alone (13% inhibition), rapamycin (18%) alone, or the combination in either sequence 1 (38%) or 2 (41%) (all p<0.05). At lower concentrations of SFl 126 and rapamycin, the effects were less pronounced. Synergies were noted using the Chou- Talaley analysis combination index values shown in Table 4. Table 4
Figure imgf000075_0001
[0228] VEGF is a well known proangiogenic factor and Bv8 (prokineticin-2) is a related factor that has recently been characterized. These two angiogenic stimulants along with IGF were evaluated in 786-0 cells (Figure 21A) and HBECs (Figure 21B) for their effect on PDK pathway signaling. A significant increase in pAkt was noted in HBEC exposed to VEGF (0.1 μg/mL), Bv8 (2.5 μg/mL), and IGF (0.1 μg/mL). A clear dose dependant reduction in pAkt levels in both 786-0 and HBECs was observed with SFl 126 in the presence or absence of these PDK pathway activators.
[0229] Another important aspect in PDK signaling is apoptosis versus cell cycle arrest. Some PDK inhibitors are widely believed to be only cell cycle arrest agents and not capable of inducing apoptosis. The effect of SFl 126 on the level of apoptosis in 786-0 cells was studied using annexin V staining as a marker for early apoptosis detection. Exposure of 786-0 cells to SFl 126 at 5, 10, and 20 mM for 18 hours resulted in a 37%, 64%, and 92% apoptosis increase (respectively) in cells compared to untreated controls (Figure 22). The apoptotic effects of the combination of rapamycin and SFl 126 in 786-0 cells was also evaluated (Figure 23). Rapamycin alone showed significant apoptosis as a single agent at 20 nM (40% of cells undergoing early apoptosis); however, it did not increase apoptosis at higher doses of 50 nM and 100 nM (39%, 37% respectively), indicating a flat dose response. The combination effect achieved by the addition of 5 mM SFl 126 to rapamycin at 20 nM, 50 nM, and 100 nM increased the percentage of cells undergoing early apoptosis to 55%, 55%, and 53%, respectively. Thus, rapamycin shows a flat dose response as a single agent and in combinations with SFl 126. [0230] In vitro cell culture work such as this should ultimately be helpful in guiding clinical decisions such as scheduling of combination agents. The above in vitro results were confirmed in tumor treatment by usingw a 786-0 xenograft tumor model (Figure 24A). Over a four-week period the no-treatment control group showed a 480% increase in tumor volume, SFl 126 alone showed a 328% increase in tumor volume (41% growth inhibition versus control; p<0.05), and rapamycin alone (1.5 mg/kg Lp., 3X per week) showed complete growth inhibition (0% increase in tumor growth). The combination of SFl 126 and rapamycin when administered simultaneously showed a 54% regression of tumor volume versus starting tumor volume. This sequence was significantly better (p<0.001 and p<0.02, respectively) than when rapamycin was administered first followed by SFl 126 (11% regression) or when SFl 126 was administered first followed by rapamycin (7% increase in tumor growth). Similar trends were observed in the excised tumor weights (Figure 24B). No significant changes in body weight were observed in the groups (Figure 24C). These results provide support for simultaneous dosing of SFl 126 with an mTOR inhibitor such as rapamycin in the clinical setting.
Example 25 Combined Effects of SFl 126 and docetaxel
[0231] The effects of SFl 126 were studied in combination with docetaxel, an approved chemotherapeutic drug, to see if there is an enhanced effect in nude mice bearing PC-3 human prostate cancer. First, different scheduling regimens of SFl 126 and docetaxel treatment were tested on tumor growth inhibition. PC-3 tumors were generated in nude mice by subcutaneous inoculation of 3 x 106 cells at the right flank. Mice were randomly divided into 6 groups of 5 when average tumor volume was 300 mm3. Mice were either not treated as control (Group 1), treated only with SFl 126 at 50 mg/kg dose s.c administration 3 times weekly for 3 weeks (Group 2), or treated only with docetaxel at 12 mg/kg dose iv administration on MWF of the first week (Group 3). Groups 4 and 5 of mice were treated with SFl 126 s.c administration 3 times weekly for the whole period of 3 weeks and docetaxel was administered during the second week to group 4 mice (MWF) and during the first week (MWF) to group 5 mice. Mice in group 6 received docetaxel during the first week (MWF) and SFl 126 was given in the following 2 weeks thereafter. Tumor growth was monitored twice weekly and tumor volume was calculated using the formula V=A x B2 / 2, where A is the length and B is the width of the tumor. [0232] Three different schedules for the combination of SFl 126 with docetaxel (Taxotere®) were studied in a PC-3 s.c. xenograft mouse model (Figure 25). PC-3 was chosen because PC-3 cells exhibit Bcl-xL overexpression that protects them from PI3K inhibition induced apoptosis (J. Biol. Chem. 278(28), pp25872-25878, 2003). Generally, the most activity observed using a PI3K inhibitor with PC-3 is complete growth inhibition but not regression. In this preliminary experiment a trend toward better efficacy was observed when SFl 126 was given shortly after docetaxel on the same dosing day versus the two other schedules where either SFl 126 or docetaxel was administered for a week followed by the other agent the following week.
Example 26 SFl 126 inhibits PC3 tumor growth and sensitizes tumor response to docetaxel
[0233] This example shows that SFl 126 inhibits PC3 tumor growth and sensitizes tumor response to paclitaxel in a nuce mouse xenograft model. Athymic nude mice bearing subcutaneous PC-3 tuors were randomly divided in groups of six when tumor volume reached 300mm3. Mice were treated with either docetaxel or SFl 126 alone or in combination. Docetaxel was intravenously administered at 6 mg/kg dose every other day for a total of three doses. SFl 126 was subcutaneously administered at a 50 mg/kg dose three times weekly for six weeks. Tumor size was monitored twice a week using calipers for external measurement and tumor volume was calculated using the formula V=AxB2/2, where A is length and B is the width of the tumor. Mice were sacrificed when tumor volume in the untreated control group exceeded 2000 mm3.
[0234] Both SFl 126 alone at 50 mg/kg three times weekly for six weeks and docetaxel alone at 6 mg/kg dose 3x BOD significantly inhibited PC-3 tumor growth in nude mice (with p values less than 0.01 and 0.001, respectively) (Figure 26). The combination treatment was significantly more efficient at inhibition of tumor growth compared with either SFl 126 (p<0.005) or docetaxel (p<0.01) treatment alone. Compared with 70% tumor growth inhibition by SFl 126 alone and 84% by docetaxel alone, the combination treatment resulted in 100% turmor growth inhibition with an additional 67% tumor regression without causing any observed toxicities. [0235] In another experiment to investigate the combination effect of SFl 126 with docetaxel, PC3 cells were plated in 50 μL RPMI 1640 media with 10% FBS in each well of 96 well plates in hexaplicated settings. After adding 50 ul of SFl 126 and docetaxel as individual or in combination in complete media to various doses, cells were incubated for 0, 1, 2 or 3 days followed by measuring viability using WST staining (adding 10 μL WST solution [Roche Diagnostics] for 4 hours). Treatment of cells with either SFl 126 or docetaxel alone resulted in significant growth inhibition reflected by a decrease in the optical density reading after WST staining of viable cells. Combining the two agents at the same concentration showed further statistically significant inhibition of cell growth (Figure 27).
[0236] In a further experiment the combined effect of SFl 126 in combination with docetaxel at different dose levels on tumor growth was studied. To better understand if the tumor-inhibiting effect is related to the administration sequence, an efficacy study (SF-R6) was conducted in PC3 prostate cancer xenograft model. 3 x 106 of PC3 cells were inoculated subcutaneously into nude mice at the flank area and tumor growth was monitored with calipers for external measurement. Tumor volume was calculated using the formula Volume = A x B2 / 2, where A is the length and B is the width of the tumor. When average tumor volume reached 300 mm3, mice were randomly divided up into 3 groups and treated with either SFl 126 alone as a positive control at 20 mg/kg dose intravenously administered three times weekly (M, W, F) or SFl 126 in combination with a single 10 mg/kg dose of docetaxel given at different schedules. Docetaxel was given either at the same day when SFl 126 treatment was started or administered one week after the starting of SFl 126 treatment. There was a 3 hour interval between SFl 126 injection and docetaxel administration when administered on the same day. Treatment with SFl 126 lasted for 6 weeks and tumor size was monitored three times weekly.
[0237] Compared with SFl 126 alone, the docetaxel and SFl 126 simultaneous treatment group started to show significant tumor growth inhibition on day 37 after tumor cell inoculation (9 days after docetaxel was administered) and the inhibitory effect was statistically significant versus SFl 126 alone all the way through the study (Figure 28). In the group where docetaxel was given one week after SFl 126 dosing there was statistically significant tumor growth inhibition on day 48 after tumor cell inoculation (13 days after docetaxel was administered). These results support the in vitro observations suggesting that simultaneous administration of docetaxel could be the preferred schedule. When comparing tumor growth inhibition at the end of the study, there was a 76% inhibition for the simultaneous treatment group (p<0.005 vs. control group) and 86% for the group that docetaxel was administered one week later (p<0.001 vs. control group) but no statistical significance between the two docetaxel containing groups.
Example 27 Combined effect of SFl 126 and docetaxel on various prostate cancer cell lines
[0238] This examples shows that SFl 126 acts synergistically with docetaxel and rapamycin on tumor cells. To investigate the combination effect of SFl 126 with docetaxel (Taxotere, TXT) and rapamycin, 2 x 103 prostate cancer PC3, LNCaP or DU145 cells were plated in 50 ul RPMI 1640 media with 10% FBS in each well of 96 well plates in hexaplicated settings. After adding 50μL of SFl 126, docetaxel and rapamycin as individual or in combination in complete media to various doses, cells were incubated for 0, 1, 2 or 3 days followed by measuring viability using WST staining (adding lOμL WST solution [Roche Diagnostics] for 4 hours). To investigate sequential effects in combination treatment, the first drug was added on day 1 and the second drug was added 24 hours later. Cells were incubated for additional 48 hours. After a total of 3 days incubation, viability of cells was determined by WST staining and OD readings at 450 nm were obtained. A Combination Index (CIIC50) was calculated using CalcuSyn software based on the method of Chou & Talalay, where, in general, a value <1 suggests synergy/sensitization, a value =1 suggests additivity, and a value >1 suggests antagonism.
[0239] The TXT results tend to show the best effect at low SFl 126 doses and with simultaneous treatments (Figure 29). Detailed mechanisms underlying the different inhibitory effects by variable combinational options are not known yet. However, it is reasonable to think that better therapeutic effect should be achieved for the treatment of tumors if two different targets at different location in the same signaling pathway can be hit at the same time. In support of this, almost all of the Rapamycin combinations were synergistic or at least additive regardless of scheduling effects (Figure 29).
[0240] Based on the results, another efficacy study (SF-T6-A) was conducted to test the combination effects of SFl 126 and docetaxel in nude mice bearing tumors of a different prostate cancer, DU145. In this study, mice were implanted with 2 x 106 of DU- 145 cells and randomly divided into 4 groups of 5 when average tumor volume reached 400 mm3. One group was untreated control, two other groups were treated with SFl 126 (20 mg/kg iv 3x weekly for 4 weeks) or docetaxel (6 mg/kg iv QOD on first week) as single agent. The last group was SFl 126 and docetaxel combination with the same dose and frequency.
[0241] SFl 126 at 20 mg/kg dose 3X weekly for 5 weeks significantly inhibited DU145 human prostate cancer cells growth in nude mice (73% inhibition vs. the no-treatment control; (<0.01) (Figure 30). Three total doses of docetaxel at 6 mg/kg level administered on the first week of treatment significantly inhibited DU145 human prostate cancer cell growth in nude mice (95% inhibition vs. the control with p<0.001). The combination treatment with the same dose and frequency (simultaneous treatment) resulted in 100% inhibition of tumor growth in this experiment (p<0.001). There is no statistical difference between the two docetaxel treatment groups.
[0242] In a further study the combinational effect of SFl 126 and docetaxel was tested on relatively big DU 145 tumors. Tumor volume was about 1200 mm3 when treatment was started. Mice were administered with 3 every-other-day doses (10 mg/kg) of docetaxel on the starting week with or without SFl 126 (20 mg/kg dose 3x weekly for 3 weeks). Mice were euthanized at the end of the study and subcutaneous tumors were harvested, weighed and frozen in OCT medium. Means and standard deviations were calculated and the Student t test was used for statistics.
[0243] The combination treatment resulted in significant reduction of tumor volume compared with docetaxel alone (p<0.05) (Figure 31). Tumor volume of the combination group was also significantly smaller than that at the beginning of the treatment (27% regression, p<0.05). As seen in Figures 31A and B, the excised tumor weight in the combination group was significantly less than that of docetaxel only group (p<0.05).
Example 28 Alternative Synthesis of Compound 1126
(all compound numbers refer to this example only) A) Preparation of tert-Butyl chloromethyl succinate (2)
Figure imgf000080_0001
[0244] To a vigorously stirred, room temperature solution of mono-tert-butyl succinate (3.50 g, 20 mmol), sodium bicarbonate (6.75 g, 80 mmol), and tetrabutylammonium bisulfate (682 mg, 2 mmol) in water (20 mL) was added dichloromethane (20 mL) followed by the dropwise addition of a solution of chloromethylchloro sulfate (2.44 mL, 24.1 mmol) in dichloromethane (5 mL). After stirring at room temperature for 1 hour, the dichloromethane layer was separated, washed with 5% aqueous sodium bicarbonate solution (1 x 50 mL), dried over magnesium sulfate, filtered, and concentrated in-vacuo to give tert-butyl chloromethyl succinate (2, 4.55g, 20.4 mmol, 102% yield) as lot A139-045, a clear oil, which was used without further purification in the next step.
B) Preparation of 4-((4-tert-Butoxy-4-oxobutanoyloxy)methoxy)-2-morpholino-8- phenylchromenylium iodide (4)
Figure imgf000081_0001
[0245] 2-Morpholino-8-phenyl-4H-chromen-4-one (3, 1.84g, 6.0 mmol) and sodium iodide (1.35g, 9.0 mmol) were added to a room temperature solution of tert-butyl chloromethyl succinate (2, 2.00g, 9.0 mmol) in acetonitrile (20 mL), and the mixture was stirred for 16 hours at 60 0C. The mixture was concentrated in-vacuo, the residue dissolved in water (60 mL), and the aqueous solution was extracted with dichloromethane (3 x 60 mL). The dichloromethane extracts were combined, dried over magnesium sulfate, filtered, and concentrated in-vacuo to give a yellow solid. The yellow solid was dissolved in hot acetonitrile (10 mL) and precipitated with cooling at -20 0C. The precipitate was filtered and dried in-vacuo to give 4-((4-tert-butoxy- 4-oxobutanoyloxy)methoxy)-2-morpholino-8-phenylchromenylium iodide (4, 1.95g, 3.14 mmol, 52% yield) as lot A139-046 as a yellow solid. ESI-MS: m/z = 308 (M - CH2OCOCH2CH2COOtBu). This material (lot A 139-046) was recrystallized using acetonitrile and the crystals were analyzed by X-ray crystallography to give a crystal structure indicating O- alkylation as depicted for another batch of SFl 110 in Figure 10. C) Preparation of 4-((4-Chloro-4-oxobutanoyloxy)methoxy)-2-morpholino-8- phenylchromenylium chloride (5)
Figure imgf000082_0001
[0246] A mixture of 4-((4-tert-butoxy-4-oxobutanoyloxy)methoxy)-2-morpholino-8- phenylchromenylium iodide (4, 1.24g, 2.0 mmol) in thionyl chloride (4 rnL) was stirred for 4 hours at 65 0C. The solution was concentrated in-vacuo to give 4-((4-chloro-4- oxobutanoyloxy)methoxy)-2-morpholino-8-phenylchromenylium chloride (5, 0.98g, 1.98 mmol, 99% yield) assigned lot A139-047 as a yellow solid, which was used without further purification. D) Preparation and isolation of SFl 126 as Acetate Salt [4-((8S,14S,17S)-17-Carboxy-14- (carboxymethyl)-8-(3-guanidinopropyl)- 18-hydroxy-3,6, 9,12, 15-pentaoxo-2-oxa-7, 10, 13,16- tetraazaoctadecyloxy)-2-morpholino-8-phenylchromenylium acetate] (SFl 126, 6)
Figure imgf000082_0002
[0247] A solution of 4-((4-chloro-4-oxobutanoyloxy)methoxy)-2-morpholino-8-phenyl- chromenylium chloride (5, 500 mg, 0.86 mmol) was added to a stirred solution of H-Arg(Pbf)- Gly-Asp(OtBu)-Ser(tBu)-OtBu (730 mg, 0.86 mmol) at 0 0C in dichloromethane (4 mL) and pyridine (4 mL)[the RGDS protected tetrapeptide was prepared using standard solution phase peptide synthesis]. The solution was allowed to warm to room temperature and stirred for 1 hour. The solution was diluted with dichloromethane (50 mL) and washed with 0.1% aqueous citric acid solution (3 x 50 mL), 5% aqueous sodium bicarbonate solution (3 x 50 mL), and brine solution (2 x 50 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated in-vacuo to give a yellow residue (798 mg). The yellow residue (317 mg) was dissolved in dichloromethane (5 niL), anisole (0.1 niL), and trifluoroacetic acid (5 niL), and stirred at room temperature for 3 hours. The solution was concentrated in-vacuo and immediately purified by preparative high-pressure liquid chromatography. The fractions containing the desired compound were combined and lyophilized to give SFl 126 as the acetate salt [4-((8S,14S,17S)-17-carboxy-14-(carboxymethyl)-8-(3-guanidinopropyl)-18-hydroxy- 3,6,9, 12, 15-pentaoxo-2-oxa-7, 10,13, 16-tetraazaoctadecyloxy)-2-morpholino-8- phenylchromenylium acetate] (6, 15 mg, 0.016 mmol, 7% yield) as a white solid. ESI-MS: m/2z = 427 (M+H / 2).

Claims

1. A compound of the formula:
Figure imgf000084_0001
or a pharmaceutically acceptable salt thereof, wherein,
Ring A is benzo;
Z1 is S or O;
Z2 is S or O;
R1 and R2 independently are H, optionally substituted Ci-24aliphatic, optionally substituted aryl, hydroxyl, halogen, Ci_24alkoxy, C3_12heterocycle, cyano, amino, or, are taken together to form an optionally substituted C3_12cycloaliphatic or optionally substituted aryl;
R3 represents H, optionally substituted Ci_24aliphatic, and optionally substituted aryl;
R4 and R5 independently are H, optionally substituted Ci_12aliphatic, optionally substituted aryl,
C3-12heterocycle, aryloxy, carboxy, or, are taken together to form an optionally substituted C3-
12heterocycle or optionally substituted heteroaryl;
R6 represents H, optionally substituted Ci_24aliphatic, optionally substituted aryl, alkoxy, carboxy, amino, C3-12 heterocycle, aryloxy, any of which may be optionally substituted with a targeting agent, selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate; and
L represents a linker group selected from oxygen, sulphur, -NH-, -CH2-, -C(O)-, -C(O)NH- or saturated or unsaturated aliphatic group of up to 6 carbon atoms wherein one or two saturated carbons of the chain are optionally replaced by -C(O)-, -C(O)C(O)-, -CONH-, -CONHNH-, -
C(O)O-, -OC(O)-, -NHCO2-, -0-, -NHCONH-, -OC(O)NH-, -NHNH-, -NHCO-, -S-, -SO-, -
SO2-, -NH-, -SO2NH- or NHSO2-; wherein the bond between Z1 and L of the compound is hydrolyzable.
2. The compound or salt thereof of claim 1 having the formula
Figure imgf000085_0001
wherein,
Z3 and Z4 independently are S or O; and
R7 represents -CH2-, -CH(CH3)-, -CH(Ph)-, -C(CH3)(COOH)- or CH(CH(CH3)2).
3. The compound or salt thereof of claim 2, wherein R1-RnIgA-R2 is selected from the group consisting of
Figure imgf000085_0002
Figure imgf000086_0001
Figure imgf000086_0002
wherein R4-N-R5 are selected from the group consisting of
Figure imgf000086_0003
wherein R6 is selected from the group consisting of
Figure imgf000087_0001
Figure imgf000087_0002
Figure imgf000087_0003
Figure imgf000087_0004
Figure imgf000087_0005
Figure imgf000087_0006
4. The compound or salt thereof of claim 2 having the formula:
Figure imgf000088_0001
5. The compound or salt thereof of claim 4 and salts thereof having the formula
Figure imgf000088_0002
wherein
T (targeting agent) is selected from a carbohydrate, vitamin, peptide or peptidomimetic, protein, nucleoside, nucleotide, nucleic acid, liposome, lipid, bone-seeking agent, cartilage- seeking agent, diazepine, glucose, galactose, mannose or mannose-6-phosphate.
6. The compound or salt thereof of claim 5 wherein R6-T is selected from the group consisting of
Figure imgf000089_0001
7. The compound or salt thereof of claim 5 having the formula:
Figure imgf000090_0001
and salts thereof.
8. The compound or salt thereof of claim 5, wherein T is a vitamin.
9. The compound or salt thereof of claim 8, wherein the vitamin is selected from folate or vitamin C.
10. The compound or salt thereof of claim 5, wherein T is a peptide.
11. The compound or salt thereof of claim 10, wherein the peptide is an RGD- containing peptide selected from the group consisting of RGDs, c(RGDfK), vitronectin, fibronectin, somatostatin-receptor agonists and somatostatin-receptor antagonists.
12. The compound or salt thereof of claim 5, wherein the targeting agent is a protein.
13. The compound or salt thereof of claim 12, wherein the protein is a tumor- specific monoclonal antibody or fragment thereof.
14. The compound or salt thereof of claim 5, wherein the targeting agent is a bone- seeking agent.
15. The compound or salt thereof of claim 14, wherein the bone-seeking agent is selected from the group consisting of a phosphonate, phosphonic acid, aminomethylphosphonic acid, phosphate, polyphosphate, and hydroxyapatite-binding polypeptides.
16. The compound or salt thereof of claim 14, wherein the bone-seeking agent is EDTMP, DOTMP, ABDTMP, BAD, MTX-BP, CF-BP, (Asp)6, (GIu)6, alendronate, pamidronate, 4-aminobutylphosphonic acid, l-hydroxyethane-l,l-diphosphonic acid, aminomethylenebisphosphonic acid, phytic acid, or N,N-bis(methylphosphono)-4-amino-benzoic acid.
17. A method of treating a PI3K-related cancer in a patient in need thereof comprising administering an effective amount of a compound or salt thereof according to any one of claims 1-16.
18. The method according to claim 17 wherein the cancer is selected from the group consisting of brain cancer, lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, ovary cancer, prostate cancer, testes cancer, gastric cancer, genitourinary tract cancer, lymphatic cancer, rectum cancer, larynx cancer, pancreas cancer, esophagus cancer, stomach cancer, gall bladder cancer, cervix cancer, thyroid cancer, skin cancer, hematopoietic cancer, mesenchymal cancer, thyroid cancer, follicular cancer, multiple myeloma, and nervous system cancer.
19. A method of inhibiting PD kinase in a cancer cell comprising administering to a patient in need thereof an effective amount of a compound or salt thereof according to any one of claims 1-16.
20. A method of inhibiting tumor growth comprising administering to a patient in need thereof an effective amount of a compound or salt thereof according to any one of claims 1- 16.
21. A method for treating a PD-K related non-cancer disease comprising administering to a patient in need thereof an effective amount of a compound or salt thereof according to any one of claims 1-16.
22. The method of claim 21, wherein the disease is selected from the group consisting of inflammatory disease, pancreatitis, ulcers, age-related macular degeneration, hypertension, autoimmune disease, graft versus host disease, rheumatoid arthritis, atherosclerosis, thrombosis, PTEN-related disease, and diabetes.
23. The method of claim 22, wherein the PTEN-related disease is Cowden's disease.
24. A method for treating a cancer in a patient, comprising administering to a patient in need thereof a first agent in combination with a second agent, wherein the first agent is an anti-cancer agent and the second agent is a compound or salt thereof according to claims 1-16.
25. The method of claim 24, wherein the anti-cancer agent is selected from the group consisting of: a MEK inhibitor, a MAP kinase pathway inhibitor, mTOR inhibitor, CCI-779, paclitaxel, docetaxel, trastuzumab, carboplatin, cetuximab, sunitib, lapatinib, imatinib mesylate, an EGFR inhibitor, bortezumib, and sorafanib.
26. The method of claim 25, wherein the mTOR inhibitor is rapamycin or a rapamycin analog.
27. The method of claim 26, wherein the rapamycin analog is temsirolimus or RAD- 001.
28. The method of claim 24, wherein the cancer is selected from the group consisting of: a solid tumor, multiple myeloma, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, a gastrointestinal stromal tumor, endometrial cancer, and breast cancer.
29. The method of claim 28, wherein the cancer is renal cell cancer.
30. The method of claim 24, wherein the anti-cancer agent is administered prior to or simultaneously with the second agent.
31. The method of claim 24, wherein the patient is positive for a PI3K/PTEN pathway biomarker.
32. The method of claim 31, wherein the PI3K/PTEN pathway biomarker is elevated expression of a marker selected from the group consisting of: stathmin, pAkt, and pS6.
33. The method of claim 24, wherein the second agent is a compound having the formula:
Figure imgf000092_0001
or salts thereof.
34. A composition comprising a first agent in combination with a second agent, wherein the first agent is an anti-cancer agent and the second agent is a compound or salt thereof according to any one of claims 1-16.
35. The composition of claim 34, wherein the anti-cancer agent is selected from the group consisting of: a MEK inhibitor, a MAP kinase pathway inhibitor, a mTOR inhibitor, CCI- 779, paclitaxel, docetaxel, trastuzumab, carboplatin, cetuximab, sunitib, lapatinib, imatinib mesylate, an EGFR inhibitor, bortezumib, and sorafanib.
36. The composition of claim 35, wherein mTOR inhibitor is rapamycin or a rapamycin analog.
37. The composition of claim 36, wherein the rapamycin analog is temsirolimus or RAD-OOl.
38. The composition of claim 34, wherein the second agent is a compound having the formula:
Figure imgf000093_0001
or salts thereof.
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US11253614B2 (en) 2016-08-23 2022-02-22 University Of Maryland, Baltimore Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014036483A1 (en) * 2012-08-31 2014-03-06 University Of Maryland, Baltimore Methods and compositions for detecting drusen and predicting age-related macular degeneration
US11253614B2 (en) 2016-08-23 2022-02-22 University Of Maryland, Baltimore Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease

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