WO2010025448A2 - Inhibition of cell proliferation - Google Patents

Inhibition of cell proliferation Download PDF

Info

Publication number
WO2010025448A2
WO2010025448A2 PCT/US2009/055509 US2009055509W WO2010025448A2 WO 2010025448 A2 WO2010025448 A2 WO 2010025448A2 US 2009055509 W US2009055509 W US 2009055509W WO 2010025448 A2 WO2010025448 A2 WO 2010025448A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
aryl
salt
optionally substituted
compound according
Prior art date
Application number
PCT/US2009/055509
Other languages
French (fr)
Other versions
WO2010025448A3 (en
Inventor
Said M. Sebti
Srikumar Chellappan
Nicholas James Lawrence
Original Assignee
University Of South Florida
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of South Florida filed Critical University Of South Florida
Publication of WO2010025448A2 publication Critical patent/WO2010025448A2/en
Publication of WO2010025448A3 publication Critical patent/WO2010025448A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/56—Radicals substituted by sulfur atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/30—Isothioureas
    • C07C335/32—Isothioureas having sulfur atoms of isothiourea groups bound to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C335/00—Thioureas, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C335/30—Isothioureas
    • C07C335/38—Isothioureas containing any of the groups, X being a hetero atom, Y being any atom
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • TECHNICAL FIELD This application relates to compounds, pharmaceutical compositions, and methods for modulating the Rb Raf-1 interaction in vitro or in vivo, and more particularly to treatment of disorders modulated by the Rb Raf-1 interaction, for example, proliferation disorders such as cancer
  • the present disclosure relates to modulators of Rb Raf-1 interactions that are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells
  • the application relates to compounds, pharmaceutical compositions, and methods for modulating cell proliferation and/or Rb Raf-1 interaction in a cell, either in vitro or in vivo
  • disorders that can be treated with the disclosed compounds, compositions, and methods include diseases such as cancer as well as non-cancerous proliferation disorders hi one aspect, there is provided compound according to formula (I)
  • Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl, Y is optionally substituted methylene,
  • X 1 is -O-, -S-, or optionally substituted -NH-,
  • X 3 is -O-, S-, optionally substituted -NH- or optionally substituted methylene
  • X 2 is S or optionally substituted NH
  • X 4 is S or optionally substituted NH
  • X 2 and X 4 are both N and are linked together through an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl ring, and
  • X 5 is an optionally substituted -NH 2 or 3-7 membered heteroaryl or heterocyclyl ⁇ ng, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I,
  • Y is optionally substituted methylene
  • X 1 is -O-, -S-, or optionally substituted -NH-
  • X 2 is S or optionally substituted NH
  • R 6 and R 7 are independently -F, -Cl, -Br, -I, -NO 2 , -CN, -CF 3 , or Ci-C 6 alkoxy, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR a , -C(O)R a , -OC(O)R a , -C(O)OR", -SR a , -C(S)R a , -OC(S)R a , -C(S)OR", -C(O)SR 3 , -C(S)SR a , -S(O)R a , -SO 2 R a , -SO 3 R a , -OSO 2 R a , -OSO 3 R 3 , -PO 2 R a R b ,
  • R 6 and R 7 are not both -Cl and R 6 and R 7 are not both -CF 3
  • R 6 and R 7 are not both -F, R 6 and R 7 are not both -Br, R 6 and R 7 are not both -I, R 6 and R 7 are not both -NO 2 , and R 6 and R 7 are not both -CH 3
  • Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci. 6 alkoxy, Ci- ⁇ haloalkyl, Ci. 6 haloalkoxy,
  • Y is methylene optionally substituted with hydroxyl, Ci_ 6 alkyl, C ⁇ g alkoxy, or Ci 6 alkyl substituted with aryl
  • Y is methylene optionally substituted with Cu alkyl, for example methyl
  • Y is methylene
  • the disclosed compounds are useful in inhibiting the Rb-Raf-1 binding
  • the disclosed compounds are biologically active and therapeutically useful
  • the compounds, pharmaceutical compositions, and methods of treatment descnbed in this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect m the Rb Raf-1 regulatory pathway
  • the disclosed compounds, pharmaceutical compositions, and methods of treatment are therefore believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf-1 binding interactions m the proliferating cells
  • a method of inhibiting proliferation of a cell includes contacting the cell with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • a method of modulating Rb Raf-1 binding in a proliferating cell includes contacting the cell with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • a method of treating or ameliorating a cell proliferation disorder includes contacting proliferating cells with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • a method of treating or ameliorating a cell proliferation disorder includes administering to a subject in need of such treatement an effective amount of a compound according to any one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • a method for inhibiting angiogenic tubule formation m a subject in need thereof includes administering to the subject an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • Rb Raf-1 binding interactions The method includes determining, in the subject or in a sample from the subject, a level of Rb, Raf-1, or Rb bound to Raf-1, wherein treatment with an inhibitor of Rb Raf-1 binding interactions is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal
  • the inhibitor of Rb Raf-1 binding interactions is one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • a method for identifying a subject for therapy includes obtaining a sample from the subject, determining a level of Rb, Raf-1, or Rb bound to Raf-1 in the sample, and identifying the subject for therapy with an inhibitor of Rb Raf-1 binding interactions when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal
  • the inhibitor of Rb Raf-1 binding interactions is one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
  • pharmaceutical compositions including the disclosed compounds, or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable earner
  • the disclosed compounds may be provided for use m of the therapeutic methods described herein
  • FIGURE IA Identification of Rb Raf-1 inhibitors An lmmunoprecipitation- western blot analysis showing the disruption of the Rb Raf-1 interaction by compounds 10b and 10c
  • FIGURE IB BrdU incorporation assay showing that compound 10b arrests wild-type A549 cells, but Rb is required for activity of compound 10b, 5, 10 and 20 ⁇ M of 10b does not inhibit the proliferation of A549 cells over-expressmg shRNA constructs to Rb (sh6 and sh8), but 10b arrests wild-type A549 cells
  • FIGURE 1C BrdU incorporation assay showing that compound 10c arrests wild-type A549 cells, but Rb is required for activity of compound 10c, 5, 10 and 20 ⁇ M of 10c does not inhibit the proliferation of A549 cells over-expressmg shRNA constructs to Rb (sh6 and sh8), but 10c arrests wild-type A549 cells
  • FIGURE ID A BrdU incorporation assay at compound concentrations of 5, 10, 20, 30 and 50 ⁇ M shows dose-dependent inhibition of wild-type A549 cells by compounds 3w, 10a, 10b and 10c
  • FIGURE IE Compounds 10b and 10c inhibit angiogenic tubule formation m mat ⁇ gel in a dose-dependent fashion as shown at concentrations of 20, 50 and 100 ⁇ M For comparison, lack of inhibition of angiogenic tubule formation in mat ⁇ gel is shown for control-no drug, and comparable inhibition is shown by compound 3a at 100 ⁇ M
  • FIGURE IF Compounds 10b and 10c at 150 mg/kg inhibit human tumor growth in nude mice A549 cells xenotransplanted bilaterally into the flanks of athymic nude mice were allowed to grow for 14 days until tumor volume reached 200mm 3 , daily administration of compounds 10b and 10c substantially inhibited tumor growth whereas control tumors grew to almost 1200 mm 3
  • FIGURE IG Compound 10c inhibited the proliferation of a wide range of cancer cells at 20 ⁇ M
  • compound 10c was contacted with a range of cancer cells including PANC-I (human pancreatic carcinoma, epithehal-like), CAPAN-2
  • FIGURE 2 Results of a MTT assay in which U937 myeloid cells were incubated m the absence of compound (control), or with compounds 3a, 10b, or 10c at lO ⁇ M, 20 ⁇ M, or 50 ⁇ M for 24 hours showing dose-dependent reduction in viability of the cancer cells in the presence of the compound
  • FIGURE 3 Results of a MTT assay in which Ramos cells (Burkitt's Lymphoma) were incubated in the absence of compound (control), or with compounds 3a, 10b, or 10c at lO ⁇ M, 20 ⁇ M, or 50 ⁇ M for 24 hours showing dose-dependent reduction m viability of the cancer cells in the presence of the compound
  • FIGURE 4 Results of a BrdU mcoproation assay where cells lacking Raf-1 due to presence of a Raf-inhibitory shRNA or control cells (containing a control shRNA) were incubated in the presence or absence of compounds 3a, 10b and 10c (20 ⁇ M) The compounds inhibit the proliferation of cells having Raf-1 but not the cells lacking Raf-1
  • FIGURE 5A A schematic of the promoters showing the E2F binding site on the genes for MMP2, MMP9 and MMP14
  • FIGURE 5B Results of a QRT-PCR experiment measuring the expression of MMP2, MMP9 and MMP 14 in A549 cells transfected with shRNA to inhibit expression of ECFl or control cells When expression of ECFl is depleted, the expression of MMP9 and MMP14 is reduced
  • FIGURES 6A-D Results of a chromatin immunoprecipitation assay showing the binding of ECFl and the association of Rb with promoters of matrix metalloproemases MMP2 (Figure 6A), MMP9 ( Figure 6B), MMP 14 ( Figure 6C), and MMP 15 ( Figure 6D)
  • FIGURES 7A-D Results of a QRT-PCT experiment performed to measure the effect of compounds 3a, 10b and 10c on the expression of Figures 7A (MMP2), 7B (MMP9), 7C (MMP14) and 7D (MMP15) in MDAMB231 cells (breast cancer) showing expression of MMP9, MMP 14 and MMP 15 inhibited by each of the compounds
  • FIGURE 8A A schematic diagram showing E2F binding sites on the promoters for VEGF receptors, FLTl and KDR
  • FIGURES 8B-D show the results of chromatin immunoprecipitation assay performed using primary endothelial cells human aortic endothelial cells HAEC ( Figure 8B), human umbilical cord vein endothelial cell (HUVEC) ( Figure 8C) and human microvascular endothelial cells from the lung (HMEC-L) ( Figure 8D) Treatment of the primary endothelial cells (human aortic endothelial cells, human umbilical cord vein endothelial cells or human microvascular endothelial cells from the lung) with VEGF induced the binding of E2F1 to the FLTl and KDR promoters
  • FIGURE 9 shows data demonstrating that transient transfection of E2F1 induces FLTl and KDR promoters and that Rb can repress these promoters
  • the transfection assays were performed in both A549 and HUVEC cells
  • FIGURE 10 shows the results of a QRT-PCR expe ⁇ ments performed to measure the effect of compounds 3a, 10b and 10c (50 ⁇ M) on the expression of FLTl and KDR in human aortic endothelial cells Each of the compounds inhibits expression of both FLT and KDR
  • This application relates to compounds, pharmaceutical compositions, and methods for modulating cell proliferation and/or Rb Raf-1 interaction in a cell, either in vitro or in vivo
  • disorders that can be treated with the disclosed compounds, compositions, and methods include diseases such as cancer as well as non-cancerous proliferation disorders
  • the pharmaceutical activity of the disclosed compounds arises, at least in part, to modulation of Rb Raf-1 binding interactions by the disclosed compound, and more particularly to disruption of Rb Raf-1 binding
  • the disclosed compounds are modulators of Rb Raf-1 binding interactions
  • a modulator can change the action or activity of the molecule, enzyme, or system which it targets
  • the disclosed modulators can modulate Rb Raf 1 binding interactions to inhibit, disrupt, prevent, block or antagonize Rb, Raf-1, or Rb Raf-1 binding interactions, or otherwise prevent association or interaction between Rb and Raf-1
  • the disclosed compounds can be inhibitors, disrupters, blockers, or antagonists of Rb or Raf-1 activity, or of Rb Raf-1 binding interactions
  • the compounds, pharmaceutical compositions, and methods of use desc ⁇ bed m this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect in the Rb Raf-1 regulatory pathway
  • the disclosed compounds, pharmaceutical compositions, and methods of use are believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf- 1 binding interactions in the proliferating cells
  • modulators of Rb Raf-1 interactions that are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells
  • modulators of Rb Raf 1 interactions are potent, selective disruptors of Rb Raf-1 binding
  • modulators of Rb Raf 1 interactions are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells, including osteosarcoma, epithelial lung carcinoma, non-small cell lung carcinoma, three different pancreatic cancer cell lines, glioblastoma cell lines, metastatic breast cancer, melanoma, and prostate cancer
  • modulators of Rb Raf 1 interactions effectively disrupt angiogenesis, significantly inhibited anchorage independent tumor and significantly inhibited the growth of human epithelial lung carcinoma in nude mice
  • compounds, pharmaceutical compositions comprising the compounds, methods of inhibiting cell proliferation, methods of treating subjects with cancer, and methods of preparing modulators of Rb Raf 1 interactions are provided herein
  • cell proliferation disorder means a disorder wherein unwanted cell proliferation of one or more subsets of cells in a multicellular organism occurs In some such disorders, cells are made by the organism at an atypically accelerated rate
  • the term includes cancer and non-cancerous cell proliferation disorders
  • the cell proliferation disorder is angiogenesis or the cell proliferation disorder is mediated by angiogenesis
  • an amount of compound or radiation applied in a method refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example an amount that inhibits the abnormal growth or proliferation, or induces apoptosis of cancer cells, resulting m a useful effect
  • treating and “treatment” mean causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, postponing or preventing the further development of a disorder and/or reducing the seventy of symptoms that will or are expected to develop
  • mammals include, for example, humans, non-human primates, e g apes and monkeys, cattle, horses, sheep, rats, mice, pigs, and goats
  • Non-mammals include, for example, fish and birds
  • pharmaceutically acceptable means that the mate ⁇ als (e g , compositions, earners, diluents, reagents, salts, and the like) are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness or gastnc upset B.
  • An aliphatic group is a straight chained, branched non-aromatic hydrocarbon which is completely saturated or which contains one or more units of unsaturation
  • a cycloaliphatic group is an aliphatic group that forms a ⁇ ng Alkyl and cycloalkyl groups are saturated aliphatic and saturated cycloaliphatic groups, respectively
  • a straight chained or branched aliphatic group has from 1 to about 10 carbon atoms, typically from 1 to about 6, and preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, typically from 3 to about 8, and preferably from 3 to about 6
  • An aliphatic group is preferably a straight chained or branched alkyl group, e g , methyl, ethyl, n-propyl, iso-piopyl, n-butyl, sec-butyl, fert-butyl, pentyl,
  • alkyl or "(C x y )alkyl” (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group containing between x and y carbon atoms
  • An alkyl group formally corresponds to an alkane or cycloalkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound
  • An alkyl group may be straight-chained or branched
  • Alkyl groups having 3 or more carbon atoms may be cyclic Cyclic alkyl groups having 7 or more carbon atoms may contain more than one ring and be polycyclic Examples of straight-chained alkyl groups include methyl, ethyl, n-propyl, n-butyl, and n-octyl Examples of branched alkyl groups include (-propyl, ⁇ -butyl, and 2,2-dimethylethyl Examples of cycl
  • alkylene or "(C x y )alkylene” (wherein x and y are integers) refers to an alkylene group containing between x and y carbon atoms
  • An alkylene group formally corresponds to an alkane with two C-H bond replaced by points of attachment of the alkylene group to the remainder of the compound Included are divalent straight hydrocarbon group consisting of methylene groups, such as, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -
  • alkylene or (C 5 y )alkylene may be (Ci 6 )alkylene such as (Ci 3 )alkylene
  • alkenyl or "(C x y ) alkenyl” (wherein x and y are integers) denotes a radical containing x to y carbons, wherein at least one carbon-carbon double bond is present (therefore x must be at least 2)
  • alkoxy or "(C x - y ) alkoxy” (wherein x and y are integers) employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers
  • Embodiments include (Ci 3 )alkoxy, such as ethoxy and methoxy
  • haloalkyl or "(C x y )haloalkyl” (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group or (C x y )alkyl group in which a halogen is substituted for one or more of the hydrogen atoms Examples include t ⁇ fluoromethyl, 2,2,2-tnfluoroethyl and t ⁇ chloromethyl
  • alkylene is a linking alkyl chain represented by -(CHi) n -, wherein n, the number of "backbone” atoms in the chain, is an integer from 1-10, typically 1-6, and preferably 1-4
  • alkenylene is a linking alkyl chain having one or more double bonds, wherein the number of backbone atoms is an integer from 1-10, typically 1-6, and preferably 1-4
  • alkynylene is a linking alkyl chain having one or more triple bonds and optionally one or more double bonds, wherein the number of "backbone” atoms is an integer from 1-10, typically 1-6, and preferably 1-4
  • Heteroalkylene “heteroalkenylene,” and “heteroalkynylene” groups are alkylene, alkenylene, and alkynylene groups, respectively, wherein one or more carbons are replaced with heteroatoms such as N, O, or S
  • heterocyclic group or “heterocyclyl” is a non-aromatic cycloaliphatic group which has from 3 to about 10 ring atoms, typically from 3 to about 8, and preferably from 3 to about 6, wherein one or more of the ⁇ ng atoms is a heteroatom such as N, O, or S m the ring
  • heterocyclic groups include oxazohnyl, thiazolmyl, oxazohdmyl, thiazolidinyl, tetrahydrofuranyl, tetrahyrothiophenyl, morphohno, thiomorpholmo, pyrrolidmyl, piperazinyl, pipendinyl, thiazolidinyl, and the like
  • non-aromatic heterocycles also include monocyclic groups such as azi ⁇ dme, oxirane, th ⁇ rane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidme, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, pipe ⁇ dme, 1,2,3,6-tetrahydropy ⁇ dme, 1,4-dihydropyndme, piperazme, morpholine, thiomorpholme, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazme, homopiperidme, 1,3-dioxepane, 4,7-dihydro-l,3-dioxe
  • aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more ⁇ ngs (typically one, two or three ⁇ ngs), wherein such ⁇ ngs may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene Examples include phenyl, anthracyl, and naphthyl Preferred are phenyl and naphthyl, most preferred is phenyl In some embodiments, the term refers to Ce 14 carbocyclic aromatic groups such as phenyl, biphenyl, and the like Aryl groups also include fused polycyclic aromatic ⁇ ng systems in which a carbocyclic aromatic ⁇ ng is fused to other aryl, cycloalkyl, or cycloaliphatic nngs, such as naphthyl, pyrenyl, anthracyl, 9,10-dihydroanthracyl,
  • aralkyl or "aryl-(C x y )alkyl” means a functional group wherein carbon alkylene chain of x to y carbon atoms is attached to an aryl group, e g , -CH 2 CH 2 -phenyl Examples include is aryl(CH 2 )- (e g benzyl) and aryl(CH(CH 3 ))-
  • substituted aryl-(Ci 3 )alkyl means an aryl-(Ci- 3 )alkyl functional group in which the aryl group is substituted Preferred is substituted aryl(CH 2 )-
  • heteroaryl(Ci 3 )alkyl means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e g , -CH 2 CH 2 -pyndyl P
  • heteroaryl refers to 5-14 membered aryl groups having 1 or more O, S, or N heteroatoms
  • heteroaryl groups include pyridyl, pyrimidyl, pyrazmyl, t ⁇ azmyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-tnzaolyl, 1,2,4-tnazolyl, tetrazolyl, thienyl, thiazoyl, isothiazolyl, furanyl, oxazolyl, isooxazolyl, and the like
  • Heteroaryl groups also include fused polycyclic aromatic ring systems m which a carbocyclic aromatic ⁇ ng or heteroaryl ⁇ ng is fused to one or more other heteroaryl nngs Examples include qumolmyl, isoquinolmyl, qumazolmyl, napthy ⁇ dyl, py ⁇ dopy ⁇ midy
  • heteroaryl groups include py ⁇ dyl, pyrazmyl, pynmidinyl, particularly 2- and 4- ⁇ y ⁇ midinyl, py ⁇ dazmyl, thienyl, furyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-t ⁇ azolyl, 1,2,4-tnazolyl, 1,3,4-t ⁇ azolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1 ,3 ,4-oxadiazolyl
  • polycyclic heterocycles include mdolyl, particularly 3-, 4-, 5-, 6- and 7-mdolyl, indolmyl, qumolyl, tetrahydroqumolyl, lsoqumolyl, particularly 1- and 5-isoqmnolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolmyl, qumoxahnyl, particularly 2- and 5-qumoxalmyl, quinazohnyl, phthalazinyl, 1,5-naphthyndmyl, 1,8-naphthyndmyl, 1 ,4-benzodioxanyl, couma ⁇ n, dihydrocoumann, benzofuryl, particularly 3-, 4-, 5-, 6- and 7-benzofuryl, 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl, particularly 3-, 4-, 5-, 6-, and 7-benzo
  • substituted means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group
  • substituted refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted
  • the substituents are independently selected, and substitution may be at any chemically accessible position
  • valency of a chemical group refers to the number of bonds by which it is attached to other groups of the molecule
  • Suitable optional substituents for a substitutable atom in the preceding groups are those substituents that do not substantially interfere with the pharmaceutical activity of the disclosed compounds
  • a "substitutable atom” is an atom that has one or more valences or charges available to form one or more corresponding covalent or ionic bonds with a substituent
  • a carbon atom with two valences available e g , -C(Fk)-
  • a mtrogen-contaming group for example, a heteroaryl or non-aromatic heterocycle, can be substituted with oxygen to form an N-oxide, e g , as m a pyndyl N-oxide, pipe ⁇ dyl N-oxide, and the like
  • a nng nitrogen atom in a nitrogen-containing heterocyclic or heteroaryl group can be substituted to form an N-oxide
  • Suitable substituents for nitrogen atoms having three covalent bonds to other atoms include -OH, alkyl, and alkoxy (preferably Ci $ alkyl and alkoxy)
  • Substituted nng nitrogen atoms that have three covalent bonds to other nng atoms are positively charged, which is balanced by counteranions corresponding to those found in pharmaceutically acceptable salts, such as chlonde, bromide, fluonde, iodide, formate, acetate and the like
  • suitable counteranions are provided in the section below directed to suitable pharmacologically acceptable salts
  • Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl,
  • Y is optionally substituted methylene
  • X 1 is -O-, -S-, or optionally substituted -NH-
  • X 3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene
  • X 2 is S or optionally substituted NH
  • X 4 is S or optionally substituted NH
  • X 2 and X 4 are both N and are linked together through a bond or an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl ⁇ ng
  • X 3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene
  • X 2 is S or optionally substituted NH
  • X 4 is S or optionally substituted NH
  • X 2 and X 4 are both N and are linked together through a bond or an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membere
  • X 5 is an optionally substituted -NH 2 or 3-7 membered heteroaryl or heterocyclyl ⁇ ng, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR a , -C(O)R", -OC(O)R 3 , -C(O)OR a , -SR a , -C(S)R", -OC(S)R a , -C(S)OR a ,
  • each optionally substitutable carbon is optionally substituted with a substituent other than -SR a
  • ⁇ ng A when monosubstituted phenyl is other than 2- t ⁇ fluoromethylphenyl, 3-methoxyphenyl, 3-mtrophenyl, 3-t ⁇ fluoromethylphenyl, 3- vinylphenyl, 4-t-butylphenyl, 4-ehlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4- methylphenyl, 4-mtrophenyl, 4-trifluoromethylphenyl, and/or 4-vmylphenyl
  • nng A when disubstituted phenyl is other than 3,4-dichlorophenyl, 3,5- dit ⁇ fluoromethylphenyl, and/or 2-hydroxy-5-mtrophenyl
  • these provisos apply when X 1 is NH, X 2 is NH, X 3 is NH, X 4 is NH, X 5 is NH 2 , and Y is CH 2 In some embodiments,
  • nng A is monosubstituted phenyl In some embodiments, nng A is 2- or 3- or 4-monosubstituted phenyl In other embodiments, nng A is other than monosubstituted phenyl, or other than 2- or 3- or 4-monosubstituted phenyl In some such embodiments, X 1 is NH, X 2 is NH, X 3 is NH, X 4 is NH, X 5 is NH 2 , and Y is CH 2 hi some embodiments, nng A is disubstituted phenyl In some embodiments, nng A is 2,3- or 2,4- or 2,5- or 2,6- or 3,4- or 3,5-disubstituted phenyl In other embodiments, nng
  • A is other than disubstituted phenyl, or other than 2,3- or 2,4- or 2,5- or 2,6- or 3,4- or 3,5- disubstituted phenyl
  • X 1 is NH
  • X 2 is NH
  • X 3 is NH
  • X 4 is NH
  • X 5 is NH 2
  • Y is CH 2
  • nng A is tnsubstituted phenyl In some embodiments, nng A is 2,3,4- or 2,3,5- or 2,3,6- or 2,4,5- or 2,4,6- or 3,4,5-tnsubstituted phenyl In other embodiments, nng A is other than tnsubstituted phenyl, or other than 2,3,4- or 2,3,5- or 2,3,6- or 2,4,5- or 2,4,6- or 3,4,5-tnsubstituted phenyl In some such embodiments, X 1 is
  • X 2 is NH
  • X 3 is NH
  • X 4 is NH
  • X 5 is NH 2
  • Y is CH 2
  • nng A is tetrasubstituted phenyl hi some embodiments, nng
  • nng A is other than tetrasubstituted phenyl, or other than 2,3,4,5- or 2,3,4,6- or 2,3,5,6-tetrasubstituted phenyl hi some such embodiments,
  • X 1 is NH
  • X 2 is NH
  • X 3 is NH
  • X 4 is NH
  • X 5 is NH 2
  • X 1 is NH
  • X 2 is NH
  • X 3 is NH
  • X 4 is NH
  • X 5 is NH 2
  • nng A is pentasubstituted phenyl In some embodiments, nng A is other than substituted phenyl
  • X 1 is -O-, -S-, or optionally substituted -NH-
  • X 3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene
  • X 2 is S or optionally substituted NH
  • X 4 is S or optionally substituted NH
  • R a is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
  • R b is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
  • R a is other than heterocyclic
  • R c is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
  • R d is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
  • Group A is phenyl substituted in at least the 2-position
  • the phenyl is substituted m the 2-position with halogen
  • the phenyl is substituted in the 2-position with a substituent other than haloalkyl, for example tnfluoromethyl
  • the phenyl is substituted in the 2-position with a substituent other than OH
  • the phenyl is substituted in the 2-position with a substituent other than SR a
  • Group A is phenyl substituted in at least the 2-position
  • the phenyl is substituted in the 2-position with a substituent other than haloalkyl, for example tnfluoromethyl
  • the phenyl is substituted in the 2-position with a substituent other than OH
  • the phenyl is substituted in the 2-position with a substituent other than SR a
  • Group A is phenyl substituted in at least the 4-position
  • the phenyl is substituted in the 4- ⁇ osition with a substituent other than mtro
  • the phenyl is substituted m the 4-position with a substituent other than halogen
  • the phenyl is substituted in the 4- position with a substituent other than halogen unless the nng is further substituted, m some such embodiments the further substituent, if m the 3-position, is other than halogen
  • the phenyl is substituted in the 4-position with a substituent other than SR a
  • the phenyl is substituted in the 2-position with a substituent other than SR"
  • the Group A is substituted phenyl or optionally substituted naphthyl or py ⁇ dyl In some embodiments, in Group A, an unsubstrtuted nng atom is adjacent to the nng atom attached to Y
  • Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci s alkoxy, Ci 6 haloalkyl, Ci s haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic
  • Y is C(O), or methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci ⁇ haloalkoxy, or Ci 6 alkyl substituted with aryl hi some embodiments, Y is methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl
  • Y is methylene optionally substituted with Ci 3 alkyl
  • Y is methylene
  • the compound is represented by the following structural formula (Ia)
  • R 1 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci ⁇ haloalkyl, C] « haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic,
  • R 2 is hydrogen, hydroxyl, Ci 6 alkyl, C] 6 alkoxy, Ci 6 haloalkyl, Ci ⁇ haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic
  • R 3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci s haloalkyl, C] 6 haloalkoxy, Ci ⁇ alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic
  • R 4 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci ⁇ haloalkyl, Ci ⁇ haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic
  • R 4 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci ⁇ halo
  • R 5 is hydrogen, hydroxyl, Ci 6 alkyl, Ci ⁇ alkoxy, Ci ⁇ haloalkyl, Ci ⁇ haloalkoxy, Ci ⁇ alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic
  • R 1 is hydrogen, hydroxyl, Ci ⁇ alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci e haloalkoxy, or Ci 6 alkyl substituted with aryl
  • R 1 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci ⁇ alkyl substituted with aryl
  • R 1 is hydrogen or Ci 3 alkyl, for example methyl
  • R 1 is hydrogen
  • R 2 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci ⁇ haloalkyl, Ci 6 haloalkoxy, or Ci ⁇ alkyl substitute
  • A is optionally substituted naphthyl, for example optionally substituted 1-naphthyl or 2-naphthyl
  • Y is methylene
  • R 1 , R 2 , R 3 , R 4 and R 5 are hydrogen
  • Group A is substituted with a substitutent selected from -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR", -C(O)R", -OC(O)R a , -C(O)OR a , -SR a , -SO 2 R", -SO 3 R a , -OSO 2 R", -OSO 3 R", -N(R a R b ), -C(O)N(R a R b ), -C(O)NR a NR b SO 2 R c , -C(O)NR a SO 2 R c , -SO 2 N(R a R b ), -NR a SO 2 R b , -NR a SO 2 R b , -NR 0 C(O)R", and -NR 0 C(O)OR 3
  • Group A is unsubstituted 2- naphthyl or 1-substituted 2- naphthyl In some embodiments, Group A is naphthyl optionally substituted with one or more of -F, -Cl, -Br, -NO 2 , Ci- 6 alkyl, or -CF 3 . In some embodiments, Group A is naphthyl optionally monosubstituted with -F, -Cl, -Br, -NO 2 , or -CF 3 . In some embodiments, Group A is naphthyl optionally monosubstituted with -F, -Cl, or -Br
  • Particular compounds of interest include the following compounds and salts such as pharmaceutically acceptable salts thereof, particularly the 2,4-dichlorophenyl compound.
  • Y is optionally substituted methylene
  • X 1 is -O-, -S-, or optionally substituted -NH-
  • X 2 is S or optionally substituted NH
  • R 6 and R 7 are independently -F, -Cl, -Br, -I, -NO 2 , -CN, -CF 3 , or C 1 -C 6 alkoxy; wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR a , -C(O)R a , -OC(O)R a , -C(O)OR", -SR a , -C(S)R a , -OC(S)R a , -C(S)OR a , -C(O)SR a , -C(S)SR a , -C(S)SR a , -S(O)SR a , -S(O)R a , -SO 2 R a , -SO 3 R a , -OS
  • R 6 and R 7 are not both -Cl and R 6 and R 7 are not both -CF 3
  • R 6 and R 7 are not both -F
  • R 6 and R 7 are not both -Br
  • R 6 and R 7 are not both -I
  • R 6 and R 7 are not both -NO 2
  • R 6 and R 7 are not both -CH 3
  • this proviso applies when Y is -CH 2 -, X 1 is S and X 2 is NH
  • Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci ⁇ haloalkoxy, Ci- S alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic
  • Y is methylene optionally substituted with hydroxyl, Ci s alkyl, Ci ⁇ alkoxy, or Ci- 6 alkyl substituted with aryl
  • Y is methylene optionally substituted with Ci 3 alkyl, for example methyl
  • Y is methylene
  • the compound of formula II is represented by the following structural formula
  • R 8 is hydrogen, hydroxyl, Ci 6 alkyl, Ci ⁇ alkoxy, Ci ⁇ haloalkyl, Ci ⁇ haloalkoxy, Ci ⁇ alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic In some embodiments thereof, is hydrogen, hydroxyl, C t 6 alkyl, Ci 6 alkoxy, or Ci ⁇ alkyl substituted with aryl In some embodiments, R 8 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R 8 is hydrogen hi the preferred embodiments, Y is methylene and R 8 is hydrogen hi some embodiments, R 6 and R 7 are independently -F, -Cl, -Br, -NO 2 , or -CF 3 Compounds according to formula II of particular interest include those wherein the compound is selected from the group consisting of
  • Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl, Y is optionally substituted methylene,
  • X 1 and X 3 are independently -O-, -S-, or optionally substituted -NH-, or X 3 is optionally substituted methylene,
  • X 2 and X 4 are independently S or optionally substituted NH, or X 2 and X 4 are both N and are linked together through a bond or an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl ⁇ ng,
  • X 5 is an optionally substituted -NH 2 or 3-7 membered heteroaryl or heterocyelyl ring,
  • R 6 and R 7 are independently -F, -Cl, -Br, -I, -NO 2 , -CN, -CF 3 , or Ci-C 6 alkoxy, provided that R 6 and R 7 are not both -Cl and Ri and R 2 are not both -CF 3 In some embodiments, R 6 and R 7 are not both -F In certain embodiments, R 6 and R 7 are independently -F, -Cl, -Br, -NO 2 , or -CF 3 , or m particular embodiments, R 6 and R 7 are independently -F, -Cl, or -NO 2 , each substitutable carbon atom (e g , each optionally substituted carbon) is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR a , -C(O)R", -OC(O)R a , -C(O)OR
  • each substitutable nitrogen (e g , each optionally substituted nitrogen) is optionally substituted with -CN, -NO 2 , -R a , -0R a , -C(O)R", -C(O)R a -aryl, -0C(0)R a , -C(O)OR", -SR a , -S(O)R 3 , -SO 2 R 3 , -SO 3 R 3 , -N(R 3 R"), -C(O)N(R a R b ), -C(O)NR 3 NR b SO 2 R c , -C
  • Each R a -R d is independently H, alkyl, alkoxy, haloalkyl, haloalkoxy, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or, -N(R 3 R b ), taken together, is an optionally substituted heterocyclic group
  • Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy,
  • Y is C(O), or methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, C] $ haloalkoxy, or Ci 6 alkyl substituted with aryl
  • Y is methylene optionally substituted with hydroxyl, Ci ⁇ alkyl, Ci g alkoxy, or Ci 6 alkyl substituted with aryl hi particular embodiments, Y is methylene optionally substituted with Ci 3 alkyl
  • Group A can be substituted phenyl or optionally substituted naphthyl or pyndyl
  • m Group A an unsubstituted ring atom is adjacent to the ⁇ ng atom attached to Y
  • Group A is a phenyl
  • the 6-position of that phenyl can be unsubstituted
  • the compound according to formula Ib is represented by the following structural formula (Ic)
  • each R' is independently hydrogen, hydroxyl, Ci g alkyl, C] (, alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic
  • each R is independently hydrogen, hydroxyl, Ci ⁇ alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci e haloalkoxy, or C] 6 alkyl substituted with aryl
  • each R' is independently hydrogen, hydroxyl, Ci_ 6 alkyl, Ci ⁇ alkoxy, or Ci- 6 alkyl substituted with aryl
  • each R' is independently hydrogen or Ci 3 alkyl
  • the compound according to the formula Ib may be represented by one of the following structural formulae
  • A' is substituted phenyl and A" is optionally substituted naphthyl
  • the compound can be represented by the following structural formula
  • the compound can be represented by the following structural formula
  • one or more substitutable carbons m Group A, Ring A' or Ring A" is substituted with -F, -Cl, -Br, -I, -CN, -NO 2 , -R a , -OR a , -C(O)R", -OC(O)R 3 , -C(O)OR 8 , -SR a , -SO 2 R", -SO 3 R a , -OSO 2 R", -OSO 3 R a , -N(R a R b ), -C(O)N(R a R b ), -C(O)NR a NR b SO 2 R c , -C(O)NR a SO 2 R°, -SO 2 N(R a R b ), -NR a SO 2 R b , -NR c C(O)R a , or -NR 0 C(O
  • Group A or Ring A" is unsubstituted 2- naphthyl or 1- substituted 2- naphthyl In some embodiments, Group A or Ring A" is naphthyl optionally substituted with one or more of -F, -Cl, -Br, -NO 2 , C 1 6 alkyl, or -CF 3 In certain embodiments, Group A or Ring A" is naphthyl optionally monosubstituted with -F, -Cl, -Br, -NO 2 , or -CF 3 In particular embodiments, Group A or Ring A” is naphthyl optionally monosubstituted with -F, -Cl, or -Br In various embodiments, the compound is represented by the following structural formula
  • Y can be as defined in any embodiment herein above In some embodiments, Y is C(O),
  • Y is methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci ⁇ alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloalrphatic
  • Y is methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci ⁇ alkyl substituted with aryl
  • Y is methylene optionally substituted with Ci 3 alkyl
  • the compound is represented by the following structural formula
  • R' can be as defined m any embodiment herein above
  • R' is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, C L 6 haloalkoxy, C 1 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic
  • R' is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl
  • R' is hydrogen or Ci 3 alkyl, for example methyl
  • R' is hydrogen
  • the compounds descnbed above, and any of the embodiments thereof, as well as intermediates used in making the compounds may take the form of salts
  • the compounds, compositions and methods of the present invention include salts of the disclosed compounds, particularly pharmaceutically acceptable salts, and methods and compositions using them
  • the disclosed compounds can have one or more sufficiently acidic protons that can react with a suitable organic or inorganic base to form a base addition salt
  • a compound has a hydrogen atom bonded to an oxygen, nitrogen, or sulfur atom
  • the compound also includes salts thereof where this hydrogen atom has been reacted with a suitable organic or inorganic base to form a base addition salt
  • Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases such as alkoxides, alkyl amides, alkyl and aryl amines, and the like
  • bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like
  • salts embraces addition salts of free acids or free bases which are compounds desc ⁇ bed herein
  • pharmaceutically-acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications, such that the salt is suitable for administration to a subject Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallimty, which may render them useful, for example in processes of synthesis, purification or formulation of compounds desc ⁇ bed herein
  • the useful properties of the compounds described herein do not depend critically on whether the compound is or is not in a salt form, so unless clearly indicated otherwise (such as specifying that the compound should be in "free base” or “free acid” form), reference in the specification to a compound should generally be understood as encompassing salts of the compound, whether or not this is explicitly stated
  • suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, carbonic, sulfuric, phosphoric and nitric acids
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxyhc and sulfonic classes of organic acids, examples of which include p-toluenesulfomc, methanesulfomc, oxalic, p-bromophenyl-sulfomc, carbonic, succinic, citric, benzoic, acetic acid, formic, acetic, propionic, glycolic, gluconic, lactic, malic, tartaric, ascorbic, glucuronic, maleic, fuma ⁇ c, pyruvic, aspartic, glutamic
  • Salts of compounds having an acidic group can be formed by the reaction of the disclosed compounds with a suitable base
  • salts can be formed by the reaction of the disclosed compounds with one equivalent of a suitable base to form a monovalent salt ( ⁇ e , the compound has single negative charge that is balanced by a pharmaceutically acceptable counter cation, e g , a monovalent cation) or with two equivalents of a suitable base to form a divalent salt (e g , the compound has a two-electron negative charge that is balanced by two pharmaceutically acceptable counter cations, e g , two pharmaceutically acceptable monovalent cations or a single pharmaceutically acceptable divalent cation)
  • Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, sodium, potassium, magnesium, calcium and zinc salts
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, JV,JV-dibenzylethylenediatmne, chloroprocame, choline, diethanolamme, ethylenediamme, meglumine (N-methylglucamme) and procaine Salts can also be formed with ammonium compounds, NR t + , wherein each R is independently hydrogen, an optionally substituted aliphatic group (e g , a hydroxyalkyl group, ammoalkyl group or ammomumalkyl group) or optionally substituted aryl group, or two R groups, taken together, form an optionally substituted non-aromatic heterocyclic ⁇ ng optionally fused to an aromatic ring
  • any of the salt forms desc ⁇ bed above can be applied to any of the compounds or embodiments thereof desc ⁇ bed in the Summary or Section II above
  • each one of the salt forms desc ⁇ bed above as being appropnate for compounds containing a base can particularly be applied as being of interest in Section II above
  • each one of the salt forms desc ⁇ bed above as being approp ⁇ ate for compounds containing a base can particularly be applied to each one of the following compounds, and, in particular, the hydrochlonde, hydrobromide, sulfatep-toluenesulfonate, methanesulfonae, succinate, citrate, benzoate, lactate, mahate, tartrate, maleate, fumarate, and benzenesulfonate salts of the disclosed compounds may be mentioned
  • salt forms suitable for use with contaimng a base desc ⁇ bed above are particularly applicable to the 2,4-dichlorophenyl amindinoisothiourea whose structure is provided above
  • salts may be prepared by conventional means from the corresponding compound by reacting the compound with the approp ⁇ ate acid or base
  • the salts are in crystalline form, and preferably prepared by crystallization of the salt from a suitable solvent
  • suitable salts for example, as descnbed in Handbook of Pharmaceutical Salts Properties Selection and Use By P H Stahl and C G Wermuth (Wiley- VCH 2002) IV. Solvate Forms
  • the disclosed compounds, and salts thereof as well as intermediates used in making the compounds may take the form of solvates, including hydrates
  • the compounds include solvate forms for the compound, and the compostitions and methods discosed herein, include compositions and methods wherein the disclosed compound is present or used in the form of a solvate or hydrate, preferably a pharmaceutically acceptable solvate or hydrate
  • solvate means a compound of the present invention or a salt thereof, that further includes a stoichiometric or non-stoichiometnc amount of solvent, e g , water or organic solvent, bound by non-covalent mtermolecular forces, where the solvent is water, the term "hydrate” can be used
  • solvent e g , water or organic solvent
  • the disclosed compounds are intended includes all isomeric forms and racemic mixtures of the disclosed compounds and methods of treating a subject with both pure isomers and mixtures thereof, including racemic mixtures Stereoisomers can be separated and isolated using any suitable method, such as chromatography It will also be understood that certain disclosed compounds can take various tautomeric forms, and the depiction of any compound as a particular tautomer does not preclude other corresponding tautomers of that compound
  • Certain compounds may possess an olefimc double bond
  • the stereochemistry of compounds possessing an olefimc double bond is designated using the nomenclature using E and Z designations
  • the compounds are named according to the Cahn-Ingold-Prelog system, described in the IUPAC 1974 Recommendations, Section E Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc , New York, NY, 4 th ed , 1992, pp 127-38, the entire contents of which are incorporated herein by reference B.
  • Certain compounds may contain one or more chiral centers, and may exist in, and may be isolated as pure enantiomeric or diastereomenc forms or as racemic mixtures
  • the formulae are intended to encompass any possible enantiomers, diastereomers, racemates or mixtures thereof which are biologically active
  • isolated optical isomer means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula
  • the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight
  • Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques According to one such method, a racemic mixture of a compound, or a chiral intermediate in the synthesis thereof, is separated into 99 wt % pure optical isomers by HPLC using a suitable chiral column, such as a member of the senes of DAICEL ® CHIRALP AK* family of columns (Daicel Chemical Industries, Ltd , Tokyo, Japan) The column is operated according to the manufacturer's instructions
  • compositions comprising the disclosed compounds
  • a "pharmaceutical composition” comp ⁇ ses a disclosed compound, typically in conjunction with an acceptable pharmaceutical earner as part of a pharmaceutical composition for administration to a subject
  • the disclosed compounds may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable earner
  • the active ingredient in such formulations may comprise from 0 1 to 99 99 weight percent "Pharmaceutically acceptable earner” means any earner, diluent or excipient which is compatible with the other ingredients of the formulation and not deletenous to the recipient
  • the active agent may be administered with a pharmaceutically acceptable earner selected on the basis of the selected route of administration and standard pharmaceutical practice
  • the active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations See Alphonso Gennaro, ed , Remington The Science and Practice of Pharmacy, 20th Edition (2003), Mack Publishing Co , Easton, PA Suitable dosage forms may compnse, for example, tablets, capsules, solutions, parenteral solutions, troches
  • the active agent may be mixed with a suitable earner or diluent such as water, for example stenle water, an oil (particularly a vegetable oil), ethanol, salme solution (e g physiological salme, bacte ⁇ ostatic saline (saline containing about 0 9% mg/mL benzyl alcohol), phosphate-buffered salme), Hank's solution, Rmger's-lactate, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol Solutions for parenteral administration preferably contain a water soluble salt of the active agent Stabilizing agents, antioxidant agents and preservatives may also be added Suitable antioxidant agents include sulfite, ascorbic acid, citnc acid and its salts, and sodium EDTA Suitable preservatives include benzalkonmm chlonde, methyl- or propyl-paraben, and chlorbut
  • a composition for oral administration can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions
  • the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms
  • the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents
  • carriers which are commonly used include lactose and corn starch Lubricating agents, such as magnesium stearate, are also typically added
  • useful diluents include lactose and d ⁇ ed com starch
  • the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspend
  • a nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in salme, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizmg or dispersing agents known in the art
  • a daily dosage from about 0 05 to about 50 mg/kg/day may be utilized, for example a dosage from about 0 1 to about 10 mg/kg/day Higher or lower doses are also contemplated as it may be necessary to use dosages outside these ranges in some cases
  • the daily dosage may be divided, such as being divided equally into two to four times per day daily dosing
  • the compositions may be formulated in a unit dosage form, each dosage containing from about 1 to about 500mg, more typically, about 10 to about lOOmg of active agent per unit dosage
  • unit dosage form refers to physically discrete units suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active mate ⁇ al calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient
  • compositions desc ⁇ bed herein may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer mat ⁇ ces, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres
  • a controlled-release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time
  • dosage forms provide a supply of a drug to the body during a predetermined pe ⁇ od of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations
  • U S Patent No 5,674,533 discloses controlled-release pharmaceutical compositions in liquid dosage forms for the administration of moguisteme, a potent pe ⁇ pheral antitussive
  • U S Patent No 5,059,595 desc ⁇ bes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances
  • U S Patent No 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-mflammatory agent with potent analgesic properties
  • U S Patent No 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers
  • U S Patent No 5,639,476 discloses a stable solid controlled-release formulation having a coating de ⁇ ved from an aqueous dispersion of a hydropho
  • Formulation of the compound to be administered will vary according to the route of administration selected, e g , parenteral, oral, buccal, epicutaneous, lnhalational, opthalamic, mtraear, intranasal, intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous, intracutaneous, intradermal, intraperitoneal, topically, transdermal, transmucosal, intraarticular, lntrasynovial, mtrastemal, mtralesional, intracranial lnhalational, insufflation, pulmonary, epidural, mtratumoral, intrathecal, vaginal, rectal, or intravitreal administration
  • An "effective amount" to be administered is the quantity of compound in which a beneficial outcome is achieved when the compound is administered to a subject or alternatively, the quantity of compound that possess a desired activity in vivo or in vitro
  • a beneficial clinical outcome includes reduction in the extent or seventy
  • the disclosed compounds can be co-admimstered with anti-cancer agents or chemotherapeutic agents such as alkylating agents, antimetabolites, natural products, hormones, metal coordination compounds, or other anticancer drugs
  • alkylating agents include nitrogen mustards (e g , cyclophosphamide), ethylemmme and methylmelammes (e g- , hexamethlymelamine, thiotepa), alkyl sulfonates (e g , busulfan), nitrosoureas (e g , streptozocm), or tnazenes (decarbazme, etc )
  • antimetabolites include folic acid analogs (e g , methotrexate), pynmidme analogs (e g , fluorouracil), punne analogs (e g , mercaptopunne)
  • natural products include vmca alkaloids (e g ,
  • compounds can be coadmimstered with compounds that can inhibit angiogenesis or inhibit angiogenic tubule formation include, for example, matrix metalloprotemase inhibitors(daltepa ⁇ n, suramin), endothelial cell inhibitors (e g , thalidomide, squalamme, 2-methoxyestradiol), inhibitors of angiogenesis activation (e g , avastatm, endostatm), celecoxib and the like
  • the compounds disclosed herein can be prepared according to the methods desc ⁇ bed m U S Appl Ser No 11/562,903, the entire teachings of which are incorporated herein by reference
  • the methods desc ⁇ bed in U S Appl Ser No 11/562,903 can be modified or augmented by synthetic chemistry functional group transformations known in the art and include, for example, those desc ⁇ bed in R Larock, Comprehensive Organic Transformations, VCH Publishers (1989), L Fieser and M Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994), and L Paquette, ed , Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) Comprehensive Organic Synthesis, Ed B M Trost and I Fleming (Pergamon Press, 1991), Comprehensive Organic Functional Group Transformations, Ed A R Kat ⁇ tzky, O Meth-Cohn, and C W Rees (Pergamon Press, 1996), Comprehensive Organic Functional Group Transformations II, Ed A R Kat ⁇ t
  • Suitable leaving groups LG in the compounds of formula III include halogen, particularly chlorine, bromine, and iodine, and sulfonate groups, particularly methanesulfonate, p-toluenesulfonate, and tnfiuoromethanesulfonate
  • the reactions are typically performed in a solvent at a suitable temperature
  • a base may be used as a catalyst
  • Suitable bases include alkali metal hydroxide or alkoxide salts such as sodium hydroxide or methoxide, and tertiary amines such as t ⁇ ethylamme or 7V,iV-diisopropylethylamme
  • Suitable solvents include alcohols, such as methanol and ethanol, or dichloromethane
  • the reactions may be earned out at a temperature between 0 0 C and the reflux temperature of the solvent, which is typically about 100 0 C
  • the reactions may be performed at a higher temperature by performing the reaction under pressure
  • Suitable leaving groups LQ in the compounds of formula IV include halogen, particularly chlorine, bromine, and iodme, and sulfonate groups, particularly methanesulfonate, p-toluenesulfonate, and tnfluoromethanesulfonate
  • the reactions are typically performed m a solvent at a suitable temperature
  • a base may be used as a catalyst
  • Suitable bases include alkali metal hydroxide or alkoxide salts such as sodium hydroxide or methoxide, and tertiary amines such as tnethylamme or A ⁇ iV-diisopropylemylamme
  • Suitable solvents include alcohols, such as methanol and ethanol, or dichloromethane
  • the reactions may be carried out at a temperature between 0 0 C and the reflux temperature of the solvent, which is typically about 100 °C
  • the reactions may be performed at a higher temperature by performing the reaction under pressure or m
  • the compounds according to formula I or II may be used as isolated compounds
  • isolated compound refers to a preparation of a compound of formula I or II, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds "Isolated” does not necessarily mean that the preparation is technically pure (homogeneous), but can mean that it is sufficiently pure to compound in a form in which it can be used therapeutically
  • isolated compound may refer to a preparation of a compound of formula I which contains the named compound or mixture of compounds according to formula I in an amount of at least 10 percent by weight of the total weight, at least 50 percent by weight of the total weight, at least 80 percent by weight of the total weight, at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation
  • the compounds of formula I and II and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystalhzation or chromatography, including flash column chromatography, or HPLC
  • the preferred method for purification of the compounds according to formula I and II or salts thereof composes crystallizing the compound or salt from a solvent to form, preferably, a crystalline form of the compounds or salts thereof Following crystallization, the crystallization solvent is removed by a process other than evaporation, for example filtration or decanting, and the crystals are then preferably washed using pure solvent (or a mixture of pure solvents)
  • Suitable solvents for crystallization include water, alcohols, particularly alcohols containing up to four carbon atoms such as methanol, ethanol, isopropanol, and butan-1-ol, butan-2-ol, and 2-methyl-2- propanol, ethers, for example diethyl ether, d
  • protecting groups which would be sensitive to the reaction conditions may be protected by protecting groups
  • a protecting group is a de ⁇ vative of a chemical functional group which would otherwise be incompatible with the conditions required to perform a particular reaction which, after the reaction has been earned out, can be removed to re-generate the original functional group, which is thereby considered to have been "protected”
  • Any chemical functionality that is a structural component of any of the reagents used to synthesize compounds desc ⁇ bed herein may be optionally protected with a chemical protecting group if such a protecting group is useful in the synthesis of compounds desc ⁇ bed herein
  • protecting groups are indicated, how to select such groups, and processes that can be used for selectively introducing and selectively removing them, because methods of selecting and using protecting groups have been extensively documented in the chemical literature
  • suitable protecting groups and strategies for protecting and deprotectmg functional groups using protecting groups useful in synthesizing the disclosed compounds are known in the art and include, for example, those
  • Suitable solvents are those that facilitate the intended reaction but do not react with the reagents or the products of the reaction Suitable solvents can include, for example ethereal solvents such as diethyl ether or tetrahydrofuran, ketone solvents such as acetone or methyl ethyl ketone, halogenated solvents such as dicloromethane, chloroform, carbon tetrachlonde, or tnchloroethane, aromatic solvents such as benzene, toluene, xylene, or pyridine, polar aprotic organic solvents such as acetomt ⁇ le, dimethyl sulfoxide, dimethyl formamide, N-methyl pyrrohdone, hexamefhyl phosphoramide, nitromethane, nitrobenzene, or the like, polar protic solvents such
  • Reactions or reagents which are water sensitive may be handled under anhydrous conditions
  • Reactions or reagents which are oxygen sensitive may be handled under an inert atmosphere, such as nitrogen, helium, neon, argon, and the like
  • Reactions or reagents which are light sensitive may be handled in the dark or with suitably filtered illumination
  • Reactions or reagents which are temperature-sensitive, e g , reagents that are sensitive to high temperature or reactions which are exothermic may be conducted under temperature controlled conditions For example, reactions that are strongly exothermic may be conducted while being cooled to a reduced temperature
  • Reactions that are not strongly exothermic may be conducted at higher temperatures to facilitate the intended reaction, for example, by heating to the reflux temperature of the reaction solvent Reactions can also be conducted under microwave irradiation conditions
  • the first and second reagents are reacted together under microwave irradiation
  • Reactions may also be conducted at atmospheric pressure, reduced pressure compared to atmospheric, or elevated pressure compared to atmospheric pressure
  • a reduction reaction may be conducted in the presence of an elevated pressure of hydrogen gas in combination with a hydrogenation catalyst
  • Reactions may be conducted at stoichiometric ratios of reagents, or where one or more reagents are in excess
  • the disclosed compounds can be assayed for binding and biological activity by any means desc ⁇ bed herein or known to the art
  • the disclosed compounds can be screened for binding activity in an ELISA assay (see Methods)
  • the IC 50 values of the disclosed compounds can be determined by in vitro binding assays (see Methods)
  • the binding selectivity of the disclosed compounds can be measured in competitive ELISA assays
  • the ability of the disclosed compounds to disrupt Rb Raf-1 in vitro or in vivo can be assayed
  • the disclosed compounds can be tested for their ability to kill or inhibit the growth of tumor cells or angiogenic tubules
  • Suitable assays include, for example, (a) tumor cell m anchorage/independent growth (soft agar assays), (b) tumor cell in anchorage- dependent growth (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue and DNA synthesis assays), (c) tumor
  • the level of Rb, Raf-1, or Rb bound to Raf-1 can be determined in the subject or in a sample from the subject, e g , a subject with a cell proliferation disorder Treatment with the disclosed compounds is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal "Elevated compared to normal" means that the levels are higher than in a reference sample of cells of the same type that are healthy
  • the level of Rb, Raf-1, or Rb bound to Raf-1 in cells from a non-small cell lung cancer tumor can be compared to the level of Rb, Raf-1, or Rb bound to Raf-1 in normal, noncancerous cells
  • Enzyme Linked Immunosorbent Assay ELISA
  • ELISA Enzyme Linked Immunosorbent Assay
  • the assay can be embodied in a
  • methods relating to cells can be conducted on cells in vitro or in vivo, particularly wherein the cell is in vivo, i e , the cell is located in a subject
  • a "subject" can be any animal with a proliferative disorder, for example, mammals, birds, reptiles, or fish
  • the animal is a mammal More preferably, the mammal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, mice, non- human p ⁇ mates, and humans Most preferably, the mammal is a human
  • the disclosed compounds are useful in inhibiting the Rb-Raf-1 binding
  • the disclosed compounds are biologically active and therapeutically useful
  • the Ras/Raf/Mek/MAPK cascade is a proliferative pathway induced by a wide array of growth factors and is activated in many human tumors It has been shown that signaling pathways through the MAP kinase cascade do not proceed in a linear fashion, but rather that they have been found to have substrates outside the cascade as well
  • the Rb protein appears to be an important cellular target of the Raf-1 kinase outside the MAP kinase cascade
  • the binding of Raf-1 to Rb was found to occur only in proliferating cells and contributed to cell cycle progression Further, it was found that the level of Rb Raf-1 interaction was elevated in NSCLC tissue, suggesting that it may have cont ⁇ minded to the oncogenic process
  • the compounds, pharmaceutical compositions, and methods of treatment desc ⁇ bed in this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect in the Rb Raf-1 regulatory pathway
  • the disclosed compounds, pharmaceutical compositions, and methods of treatment are therefore believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf-1 binding interactions in the proliferating cells
  • the disclosed compounds can participate in a protem-ligand complex
  • a protein hgand complex includes a compound and at least one protem selected from the group consisting of retinoblastoma tumor suppressor protein and serine-threonine kinase Raf-1
  • the complex can include a disclosed compound, retinoblastoma tumor suppressor protein, and serme-threonme kinase Raf-1
  • a method of inhibiting proliferation of a cell includes contacting the cell with an effective amount of the disclosed compounds or compositions Typically, regulation of proliferation in the cell is mediated by at least one protem selected from the group consisting of retinoblastoma tumor suppressor protem and serme-threonine kinase Raf-1
  • the cells have an elevated level of Rb, Raf-1, or Rb bound to Raf-1
  • the method includes assaying the level of Rb, Raf-1, or Rb bound to Raf- I m the cell
  • a method of modulating the Rb Raf-1 interaction in a proliferating cell includes contacting the cell with an effective amount of the disclosed compounds or compositions
  • a method of modulating the Rb Raf-1 interaction in a proliferating cell includes contacting the cell with a modulator of the Rb Raf-1 interaction that is suitable for oral administration In some embodiments, the modulator of the Rb Raf-1 interaction is orally administered
  • a method of treating or ameliorating a cell proliferation disorder includes contacting the proliferating cells with an effective amount of the disclosed compounds or compositions.
  • regulation of cell proliferation in the disorder can be mediated by at least one protem selected from the group consisting of retinoblastoma tumor suppressor protein and se ⁇ ne-threonme kinase Raf-1
  • the regulation of proliferation in the cells may be mediated by the interaction between retinoblastoma rumor suppressor protem and serme-threomne kinase Raf-1
  • the cell proliferation disorder may be cancer or a non-cancerous cell proliferation disorder
  • the cell proliferation disorder may include angiogenesis or the cell proliferation disorder may be mediated by angiogenesis
  • a method of treating or ameliorating a cell proliferation disorder may also include administering the compound, or a pharmaceutically acceptable salt thereof, to a patient in need of such treatment
  • the cell proliferation disorder is or the proliferating cells are de ⁇ ved from a cancererous or a non-cancerous cell proliferation disorder
  • cancererous and non-cancerous cell proliferation disorders include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewmg's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma
  • the preceding methods of treating subjects or cells can also include coadministration of an anticancer drug or a compound that modulates angiogenic tubule formation, particularly coadministration of a compound that inhibits angiogenic tubule formation
  • chemotherapeutic agents include any of abarehx, aldesleukin, alemtuzumab, ahtretmoin, allopunnol, altretamine, anastrozole, arsenic t ⁇ oxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabme, carboplatm, carmustine, cetuximab, chlorambucil, cisplatm, cladnbme, clofarabine
  • Rb, Raf-1, or Rb bound to Raf-1 wherein treatment with an inhibitor of Rb Raf-1 binding interactions is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal
  • a method of identifying a subject for therapy includes the steps of providing a sample from the subject, determining a level of Rb, Raf-1, or Rb bound to Raf-1 in the sample, and identifying the subject for therapy with an inhibitor of Rb Raf-1 binding interactions when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal
  • a kit includes an antibody specific for Rb, Raf-1, or Rb bound to Raf-1, and instructions for determining the level of Rb, Raf-1, or Rb bound to Raf-1 m a sample using the antibody specific for Rb, Raf-1, or Rb bound to Raf-1
  • methods relating to cells can be conducted on cells in vitro or in vivo, particularly wherein the cell is in vivo in a subject
  • the subject can be, for example, a bird, a fish, or a mammal, e g , a human
  • the compounds according to the invention may be administered to individuals (mammals, including animals and humans) afflicted with a cell proliferation disorder such as cancer, malignant and benign tumors, blood vessel proliferative disorders, autoimmune disorders, and fibrotic disorders
  • a cell proliferation disorder such as cancer, malignant and benign tumors, blood vessel proliferative disorders, autoimmune disorders, and fibrotic disorders
  • the compounds are believed effective against a broad range of tumor types, including but not limited to the following ovarian cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, lung cancer, renal cancer, colorectal cancer, skm cancer, bram cancer, leukemia, including acute myeloid leukemia, chrome myeloid leukemia, acute lymphoid leukemia, and chrome lymphoid leukemia
  • cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endot
  • Cancers may be solid tumors that may or may not be metastatic Cancers may also occur, as in leukemia, as a diffuse tissue
  • tumor cell includes a cell afflicted by any one of the above identified disorders
  • the compounds are also believed useful in the treatment of non-cancer cell proliferation disorders, that is, cell proliferation disorders which are characterized by benign indications Such disorders may also be known as "cytoprohferative” or “hyperproliferative” in that cells are made by the body at an atypically elevated rate
  • the non-cancerous cell proliferation disorder includes cells that have a mutation or defect in the Rb Raf-1 pathway
  • Non-cancer cell proliferation disorders believed treatable by compounds according to the invention include, for example, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy, cardiac hyperplasia, benign prostatic hyperplasia, ovarian
  • the compounds disclosed or claimed herein are provided for use m medicine
  • the compounds are also provided for use in the therapeutic methods desc ⁇ bed or claimed herein, and for manufactunng a medicament for carrying out the therapeutic methods desc ⁇ bed or claimed herein
  • the human promyelocyte leukemia cell line U937 was cultured in RPMI (Mediatech, Hernden, VA) containing 10% fetal bovine serum (FBS, Mediatech) U2-OS, Saos-2, MCF7, PANCl and MDA-MB-231 cell lines were cultured in Dulbecco modified Eagle Medium (DMEM, Mediatech) containing 10% FBS A549 cells and A549 shRNA Rb cell lines were maintained m Ham F-12K supplemented with 10% FBS ShRNA cells lines were maintained in media containing 0 5 ⁇ g/mL puromycm Hl 650, PC-9 and Aspcl cell line were cultured in RPMI (Gibco/Invitrogen, Carlsbad, CA) contaimng 10% FBS PANCl and CAPAN2 pancreatic cell lines and the A375 Melanoma cell line was grown m DMEM supplemented with 10% FBS Human aortic endothelial cells (HAECs, Clonetics, San Diego,
  • Glutathione S-transferase (GST) fusion of Rb, Raf-1, E2F1, and MEKl have been previously described (Dasgupta P, Sun J, Wang S, et al MoI Cell Biol 2004,24(21) 9527-9541)
  • 200 ⁇ g of the U937 lysates were incubated with glutathione beads carrying an equal amount of the GST fusion proteins in 200 ⁇ l of protein binding buffer (20 mM T ⁇ s [pH 7 5], 50 mM KCL, 0 5 mM EDTA, 1 mM dithiothreitol, 0 5% NP-40, 3mg of bovme serum albumm/mL) at 4 0 C for 2h (Wang S
  • Matrigel Assays Matrigel (Collaborative Biomedical Products) was used to promote the differentiation of HAECs into capillary tube-like structures (Dasgupta P, Sun J, Wang S, et al MoI Cell Biol 2004,24(21) 9527-9541) A total of lOO ⁇ l of thawed Mat ⁇ gel was added to 96-well tissue culture plates, followed by incubation at 37°C for 60 minutes to allow polymerization Subsequently, 1 X lO 4 HAECs were seeded on the gels in EGM medium supplemented with 5% FBS in the presence or absence of 20 ⁇ M concentrations of the indicated compounds, followed by incubation for 24 hours at 37°C Capillary tube formation assessed by using a Leica DMIL phase contrast microscope Lysate preparation, immunoprecipitation, and Western blotting.
  • Lysates from cells treated with different agents were prepared by NP-40 lysis as desc ⁇ bed earlier (Wang 1998) Tumor lysates were prepared with T-Per tissue lysis buffer (Pierce) and a Fischer PowerGen 125 dounce homogemzer Physical interaction between proteins in vivo was analyzed by immunoprecipitation- Western blot analyses with 200 ⁇ g of lysate with l ⁇ g of the indicated antibody as previously desc ⁇ bed (Wang 1998) Polyclonal E2F1 and Cyclin D were obtained from Santa Cruz Biotechnology Monoclonal Rb and Raf-1 were supplied by BD Transduction laboratories (San Jose, CA) Polyclonal antibodies to phospho-Rb (807,811) phospho- MEK1/2, MEK1/2, phospho-Erkl/2 and ERK1/2 were supplied by Cell Signaling (Danvers, MA) Chromatin Immunoprecipitation (ChIP) assay.
  • phospho-Rb 807,811) phospho- MEK1/2, MEK1/2, phospho
  • A549 cells were rendered quiescent by serum starvation and re-stimulated with serum for 2h or 16h m the presence or absence of RRD 251 at 20 ⁇ M Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature Subsequently, the cells were harvested and lysates were prepared Immunoprecipitations were analyzed for the presence of E2F1, Rb, Raf-1, Brgl, HPl, and HDACl by PCR as previously descnbed (Dasgupta 2004) Rabbit anti-mouse secondary antibody was used as the control for all reactions
  • the sequences of the PCR primers used in the PCRs were as follows Cdc6 promoter (forward p ⁇ mer), 5'- GGCCTCACAG CGACTCTAAGA-3', and Cdc6 promoter (reverse p ⁇ mer),
  • Bromodeoxyu ⁇ dme (BrdU) labeling kits were obtained from Roche Biochemicals (Indianapolis, IN) Cells were plated m poly-D-lysine coated chamber slides at a density of 10,000 cells per well and rendered quiescent by serum starvation for 24 hours Cells were then re-stimulated with serum in the presence or absence of the indicated drugs for 18h S- ⁇ hase cells were visualized by microscopy and quantitated by counting 3 fields of 100 m quadruplicate
  • Soft Agar assay Soft agar assays were done in triplicate in 12-well plates (Corning, Corning NY) First, the bottom layer of agar (0 6%) was allowed to solidify at room temperature Next the top layer of agar was (0 3%) was mixed with 5,000 cells per well and the indicated drug The drugs were added twice weekly in complete media to the agar wells Colonies were quantified by staining with MTT
  • NSC-35400 (1 ) NSC-35950 (2)
  • Benzyhsothiourea de ⁇ vatives 3 lacking substitution at the ⁇ benzyhc position, are prepared in good yields by reaction of thiourea with the appropriate benzyl hahde (Scheme 3, Table 1) (Yong 1997)
  • the desired benzyl halides are obtained from the corresponding benzyl alcohols (prepared when necessary by NaBFL t reduction of the corresponding aldehyde) followed by reaction with thionyl chloride to generate the corresponding benzyl chloride
  • the corresponding benzylisothiourea de ⁇ vatives 3 are usually obtained in good to quantitative yields
  • Reagents and Conditions i ethanol, 100 °C, 1-2 hours, or microwave irradiation, 100 °C, 10 minutes, 100 Watts
  • Benzylisothiouronium de ⁇ vatives 4 bea ⁇ ng an alkyl group at the benzyhc position may be prepared by the reaction of thiourea with the appropriate ⁇ -substituted benzyl halides
  • the ⁇ -substituted benzyl halides may be prepared by addition of an alkylmagnesmm bromide to the approp ⁇ ate benzaldehyde, followed by treatment of the intermediate alcohol with thionyl chlo ⁇ de
  • Substituted amidinoisothiourea compounds may be prepared by analogous methods
  • Reagents and Conditions i ethanol, 100 0 C, 1-2 hours, or microwave irradiation, 100 0 C, 10 minutes, 100 Watts, ii RCHbMgBr, tetrahydrofuran or diethyl ether, reflux, 1 hour, m Toluene, thionyl chloride, 100 0 C, 2-10 hours
  • Benzylguamdmium salts 6 may be obtained via the reaction between di-tert butoxycarbonyl thiourea and the appropnate benzylamme, (Yong 1997) followed by deprotection of the corresponding di-tert-butoxycarbonyl guamdme product with tm(rV) chlo ⁇ de (Miel 1997) or tnfluoroacetic acid, (Guisado 2002)
  • a microwave reaction tube (2 mL) is charged with a mixture of ethanol (0 5-1 mL), the appropnate benzyl chlo ⁇ de (1-2 mmol) and thiourea or guanylthiourea (1 molar eq )
  • the tube is capped and heated in a microwave reactor (Biotage Initiator I) at 110-120 0 C for 30-45 minutes
  • the reactions are momtored by thm layer chromatography (ethyl acetate hexane, 1 4, v v)
  • the reaction mixture was concentrated under vacuum and the residue is washed with hexane
  • the solid product is filtered and dried under high vacuum to give the product Typical Reaction Conditions for Synthesis of Compounds 3.
  • a lO milliliter (mL) microwave reaction tube is charged with the benzyl halide (1 0 milhmole, mmol) and thiourea (76 mg, 1 0 mmol) in ethanol (1 5 mL)
  • the tube is capped and irradiated in the microwave reactor (single-mode CEM DiscoverTM system, CEM,
  • the compounds were screened for Rb Raf-1 binding inhibitory properties using a
  • the most active compounds tended to possess a monosustituted or disubstituted benzene ring, bearing at least one hahde in either one or both of the positions ortho, meta, or para to the carbon bound to the isothiouronmm group
  • Example 17 Modulators of Rb:Raf 1 interactions Disrupt Rb:Raf-l In Intact Cells.
  • U937 cells were serum starved serum starved for 48 hours and subsequently serum stimulated for 2 hours in the presence or absence of 20 ⁇ M of the compounds Compounds 10b and 10c significantly inhibited the binding of Raf-1 to Rb, as seen by lmmunoprecipitation-Western blot analysis (FIG. IA) Raf-1 peptide conjugated to penetratm was used as a positive control Thus it appears that these two compounds were capable of disrupting the Rb Raf-1 interaction
  • Example 18 Compounds 10b & 10c Inhibited Epithelial Lung Cancer Cells.
  • A549 cells human epithelial lung carcinoma
  • sh6 and sh8 two different shRNA constructs
  • A549 cells stably expressing the Rb shRNAs had significantly less Rb protein compared to parental A549 cells
  • Compounds 10b and 10c were very effective at inhibiting S-phase entry m parental A549 cells but had little or no effect on cells stably expressing sh6 and sh8, which lacked Rb This result confirms that compounds 10b and 10c arrest the proliferation of epithelial lung cancer cells in a Rb dependent manner
  • Example 19 Dose-Dependent Inhibition of Cancer Cells by 3w, 10a, 10b and 10c.
  • angiogenic tubule formation could be inhibited by compounds 10b and 10c Human aortic endothelial cells (HAECs) were grown in matngel in the presence or absence of 20, 50 and 100 ⁇ M of 10b or 10c, or 100 ⁇ M of compound 3a It was found that while angiogenic tubules formed in control (no drug) wells, compounds 10b and 10c significantly inhibited angiogenic tubule formation in a dose dependent fashion, and showed inhibition comparable to that of compound 3a at 100 ⁇ M (FIG IE)
  • Example 21 Modulators of Rb:Raf 1 interactions 3a & 9a Significantly Inhibited Human Tumor Line in vivo.
  • Experiments were performed to assess whether compounds 10b and 10c could inhibit human tumor growth in vivo using a nude mice xenograft model Athymic nude mice were implanted with IXlO 7 A549 cells bilaterally and the tumors were allowed to reach 200mm 3 in size before treatment began FIG IF shows that tumors from vehicle treated mice grew to an average size of over 1200 mm 3 . In contrast, tumors treated with compounds 10b and 10c at at 150 mg/kg were substantially inhibited
  • Compound 10c inhibited the proliferation of a wide range of cancer cells at 20 ⁇ M as shown in FIG. IG
  • compound 10c was contacted with a range of cancer cells including PANC-I (human pancreatic carcinoma, epithelial-hke), CAPAN-2 (human pancreatic ductal adenocarcinoma), Mel-5 (human malignant melanoma), MCF-7 (human breast adenocarcinoma), LNCAP (androgen-sensitive human prostate adenocarcinoma), A549 (human epithelial lung carcinoma), and PC-3 (human prostate adenocarcinoma), and compared to Rb-deficient cancer cells (A549 cells stably transfected with two different shRNA constructs (sh6 and sh8) to knock down Rb expression, and the Rb-deficient prostate cancer cell line DU145)
  • PANC-I human pancreatic carcinoma, epithelial-hke
  • CAPAN-2 human pancreatic ductal adenocar
  • Example 23 Compounds 3a, 10b and 10c Reduce the Viability of U937 Myeloid Cells
  • Example 24 Compounds 3a, 10b and 10c Reduce the Viability of Ramos Burkitt's Lymphoma Cells
  • Ramos cells (Burkitt's Lymphoma) were incubated in the absence of compound (control), or with compounds 3a, 10b, or 10c at lO ⁇ M, 20 ⁇ M, or 50 ⁇ M for 24 hours Cell viability was assessed by an MTT assay, a coloromet ⁇ c assay which measures the number of cells by measu ⁇ ng the activity of enzymes that reduce 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazohum bromide The results are shown m Figure 3 A dose-dependent reduction m cell number was seen with each of the compounds, demonstrating that they reduce cell viability significantly Example 25. Evidence that Inhibition of Cell Proliferation By Compounds of the Invention is Mediated by Raf-1
  • A549 cells lacking Raf-1 were generated by stably transfectmg a shRNA to Raf-1
  • Control cells were generated by stably transfectmg A549 cells with a control shRNA
  • the cells were incubated m the presence or absence of ccompounds 3a, 10b and 10c (20 ⁇ M) and S-phase entry was assessed by measuring BrdU incorporation
  • the results are shown in Figure 4 Relative to controls incubated in the absence of compound, proliferation of the cells with control shRNA (having Raf-1) was inhibited by each of the compounds
  • proliferation of the cells lacking Raf-1 was not inhibited by the compound
  • This experiment provides evidence that inhibition of cell proliferation by compounds of the invention is mediated by Raf-1 as well as Rb and Raf-1
  • Example 26 Evidence that the Rb-E2F Pathway Regulates the Expression of Matrix Metalloproteinase (MMP) Genes
  • Figure 9A hows a schematic of the promoters showing the E2F binding site on the genes for MMP2, MMP9 and MMP14 Using A549 cells transfected with an shRNA to inhibit expression of E2F1, QRT-PCR expe ⁇ ments were perfomed to measure the expression of matrix metalloprotemases, MMP2, MMP9 and MMP 14 The results are shown in Figure 5 and show that when A549 cells are depleted of E2F1, the expression of MMP9 and MMP 14 is reduced This expe ⁇ ment provides evidence that the Rb-E2F pathway can regulate the expression of matrix metalloprotemases (MMPs)
  • MMPs matrix metalloprotemases
  • Figure 10 shows the results of chromatin immunoprecipitation assays showing the binding of E2F1 as well as the association of Rb with the promoters of matrix proteases
  • MMP9 Figure 6A
  • MMP2 Figure 6B
  • MMP14 Figure 6C
  • MMP15 Figure 6D
  • Figure 8A Figures 8B-D show the results of chromatin immunoprecipitation assay performed using p ⁇ mary endothelial cells human aortic endothelial cells HAEC ( Figure 8B), human umbilical cord vein endothelial cell
  • Figure 8D Treatment of the primary endothelial cells (human aortic endothelial cells, human umbilical cord vein endothelial cells or human microvascular endothelial cells from the lung) with VEGF induced the binding of E2F1 to the FLTl and KDR promoters This provides evidence that these promoters can be regulated by the Rb-E2F pathway and could possibly be targeted by the Rb-Raf-1 disruptors
  • Example 30 Evidence that Compounds of the Invention Inhibit The Expression of FLTl and KDR.
  • Chellappan S, Kraus VB, Kroger B, et al Adenovirus ElA, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product Proc Natl Acad Sa USA 1992,89 4549-53
  • Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevms JR The E2F transcription factor is a cellular target for the RB protem Cell 1991,65(6) 1053-61
  • Lam EW, La TN DP and E2F proteins coordinating transcription with cell cycle progression Curr Opm Cell Biol 1994,6(6) 859-66
  • Welch PJ Wang JY Disruption of retmoblastoma protein function by coexpression of its C pocket fragment Genes Dev 1995,9(1) 31-46 Welch PJ, Wang JYJ A C terminal protem-bindmg domain m the retmoblastoma protein regulates nuclear c-Abl tyrosine in the cell cycle Cell 1993,75 779-90

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oncology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hospice & Palliative Care (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Communicable Diseases (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Compounds of formula (I) and (II) are provided as modulators of Rb:Raf-1 interactions which are potent, selective disruptors of Rb:Raf-1 binding. Therapeutic methods of using the compounds, for example for treating or ameliorating a cell proliferation disorder such as cancer, are provided.

Description

INHIBITION OF CELL PROLIFERATION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U S Provisional Application 61/093,287 filed August 29, 2008, which is incorporated herein by reference m its entirety GOVERNMENT SUPPORT
This invention was made with government support under grant numbers CA063136 and CAl 18210 awarded by the National Institutes of Health The Government has certain rights in the invention
TECHNICAL FIELD This application relates to compounds, pharmaceutical compositions, and methods for modulating the Rb Raf-1 interaction in vitro or in vivo, and more particularly to treatment of disorders modulated by the Rb Raf-1 interaction, for example, proliferation disorders such as cancer
BACKGROUND Cellular proliferative orders such as cancer are among the most common causes of death in developed countries For diseases for which treatments exist, despite continuing advances, the existing treatments often have undesirable side effects and limited efficacy Identifying new effective drugs for cell proliferation disorders, including cancer, is a continuing focus of medical research SUMMARY
The mactivation of the retinoblastoma tumor suppressor protem Rb by cell cycle regulatory kinases is disrupted in almost all cancers In normal cells, mactivation of Rb is necessary for the Gl to S phase progression of the cell cycle Raf-1 signaling kinase is known to play a role m promoting cancer, and studies have shown that Rb Raf-1 binding facilitates cell proliferation
The present disclosure relates to modulators of Rb Raf-1 interactions that are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells The application relates to compounds, pharmaceutical compositions, and methods for modulating cell proliferation and/or Rb Raf-1 interaction in a cell, either in vitro or in vivo For example, disorders that can be treated with the disclosed compounds, compositions, and methods include diseases such as cancer as well as non-cancerous proliferation disorders hi one aspect, there is provided compound according to formula (I)
Figure imgf000003_0001
or a salt thereof, wherein
Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl, Y is optionally substituted methylene,
X1 is -O-, -S-, or optionally substituted -NH-,
X3 is -O-, S-, optionally substituted -NH- or optionally substituted methylene, X2 is S or optionally substituted NH, X4 is S or optionally substituted NH, or X2 and X4 are both N and are linked together through an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl ring, and
X5 is an optionally substituted -NH2 or 3-7 membered heteroaryl or heterocyclyl πng, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I,
-CN, -NO2, -Ra, -ORa, -C(O)Ra, -OC(O)Ra, -C(O)OR3, -SRa, -C(S)R", -OC(S)R3, -C(S)OR3, -C(O)SRa, -C(S)SR3, -S(O)Ra, -SO2R3, -SO3R8, -OSO2R3, -OSO3Ra, -PO2RaRb, -OPO2R3R", -PO3R3Rb, -OPO3RaRb, -N(RaRb), -C(O)N(R3Rb), -C(0)NRaNRbS02Rc, -C(O)NR1SO2R0, -C(O)NR3CN, -S02N(RaRb), -NR3SO2Rb, -NR0C(O)R3, -NR0C(O)OR3, -NR°C(0)N(RaRb), -C(NR°)-N(RaRb), -NRd-C(NRc)-N(R3Rb), -NRaN(RaRb), -CR°=CR3Rb, -C≡CR3, O, =S, =CRaRb, =NRa, =NORa, or =NNR\ or two optionally substitutable carbons are linked with C1 3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -ORa, -C(O)Ra, -C(O)Ra-aryl, -OC(O)Ra, -C(0)0Ra, -SRa, -S(O)Ra, -SO2Ra, -SO3Ra, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2Rc, -C(O)NRaCN, -S02N(RaRb), -NRaSO2Rb, -NR0C(O)R", -NRcC(0)0Ra, ~NR°C(O)N(RaRb), or oxygen to form an N-oxide, and is optionally protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable countenon, and wherein each of Ra, Rb, Rc and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, or in any occurrence of -N(RaRb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group with the proviso that when X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2, then πng A is other than 2-tπfluoromethylphenyl, 3-methoxyphenyl, 3-mtrophenyl, 3-tnfluoromethylphenyl, 3-vmylphenyl, 4-t-butylphenyl, 4-chlorophenyl, 4-fluorophenyl, 4- methoxyphenyl, 4-methylphenyl, 4-mtrophenyl, 4-tπfluoromethylphenyl, 4-vmylphenyl, 3,4- dichlorophenyl, 3,5-ditnfluoromethylphenyl, and 2-hydroxy-5-mtrophenyl
In another aspect, there is provided a compound according to formula (II)
Figure imgf000004_0001
or a salt thereof, wherein
Y is optionally substituted methylene, X1 is -O-, -S-, or optionally substituted -NH-, and X2 is S or optionally substituted NH,
R6 and R7 are independently -F, -Cl, -Br, -I, -NO2, -CN, -CF3, or Ci-C6 alkoxy, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)Ra, -OC(O)Ra, -C(O)OR", -SRa, -C(S)Ra, -OC(S)Ra, -C(S)OR", -C(O)SR3, -C(S)SRa, -S(O)Ra, -SO2Ra, -SO3Ra, -OSO2Ra, -OSO3R3, -PO2RaRb, -OPO2RaRb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2R°, -C(O)NRaCN, -SO2N(RaRb), -NRaSO2Rb, -NRcC(O)Ra, -NRcC(0)0Ra, -NRcC(0)N(RaRb), -C(NRc)-N(RaRb), -NRd-C(NRc)-N(RaRb), -NRaN(RaRb), -CRc=CRaRb, -C≡CRa, =O, =S, =CR3Rb, =NR3, =NORa, or =NNRa, or two optionally substitutable carbons are linked with Cu alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -ORa, -C(O)Ra, -C(O)Ra-aryl, -OC(O)R3, -C(O)ORa, -SRa, -S(O)Ra, -SO2R3, -SO3R3, -N(R3Rb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(R3R"), -NRaS02Rb, -NR0C(O)R", -NRcC(O)ORa, -NR°C(O)N(R3Rb), or oxygen to form an N-oxide, and optionally is protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable counteπon, and wherein each of Ra, Rb, R° and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, or in any occurrence of -N(RaRb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group
Pn some embodiments of the compounds of formula II, R6 and R7 are not both -Cl and R6 and R7 are not both -CF3
In some embodiments of the compounds of formula II, when Y is -CH2-, X1 is S and X2 is NH, then R6 and R7 are not both -F, R6 and R7 are not both -Br, R6 and R7 are not both -I, R1 and R2 are not both -NO2, and R6 and R7 are not both -CH3,
In some embodiments of the compounds of formula II, R6 and R7 are not both -F, R6 and R7 are not both -Br, R6 and R7 are not both -I, R6 and R7 are not both -NO2, and R6 and R7 are not both -CH3
In some embodiments of the compounds of formula II, Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci^ alkyl, Ci.6 alkoxy, Ci-β haloalkyl, Ci.6 haloalkoxy,
Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic In some embodiments, Y is methylene optionally substituted with hydroxyl, Ci_6 alkyl, Cμg alkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, Y is methylene optionally substituted with Cu alkyl, for example methyl In some embodiments, Y is methylene
Also provided are methods of using the disclosed compounds The disclosed compounds are useful in inhibiting the Rb-Raf-1 binding The disclosed compounds are biologically active and therapeutically useful
The compounds, pharmaceutical compositions, and methods of treatment descnbed in this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect m the Rb Raf-1 regulatory pathway The disclosed compounds, pharmaceutical compositions, and methods of treatment are therefore believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf-1 binding interactions m the proliferating cells
A method of inhibiting proliferation of a cell is provided The method includes contacting the cell with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof A method of modulating Rb Raf-1 binding in a proliferating cell is provided The method includes contacting the cell with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
A method of treating or ameliorating a cell proliferation disorder is provided The method includes contacting proliferating cells with an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
A method of treating or ameliorating a cell proliferation disorder is provided The method includes administering to a subject in need of such treatement an effective amount of a compound according to any one of the disclosed compounds, or a pharmaceutically acceptable salt thereof A method is provided for inhibiting angiogenic tubule formation m a subject in need thereof The method includes administering to the subject an effective amount of one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
A method is provided for assessing a subject for treatment with an inhibitor of
Rb Raf-1 binding interactions The method includes determining, in the subject or in a sample from the subject, a level of Rb, Raf-1, or Rb bound to Raf-1, wherein treatment with an inhibitor of Rb Raf-1 binding interactions is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal The inhibitor of Rb Raf-1 binding interactions is one of the disclosed compounds, or a pharmaceutically acceptable salt thereof
A method is provided for identifying a subject for therapy The method includes obtaining a sample from the subject, determining a level of Rb, Raf-1, or Rb bound to Raf-1 in the sample, and identifying the subject for therapy with an inhibitor of Rb Raf-1 binding interactions when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal The inhibitor of Rb Raf-1 binding interactions is one of the disclosed compounds, or a pharmaceutically acceptable salt thereof Also provided are pharmaceutical compositions including the disclosed compounds, or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable earner
The disclosed compounds may be provided for use m of the therapeutic methods described herein
Also provided is the use of the disclosed compounds, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for carrying out the therapeutic methods described herein
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the descnption below Other features, objects, and advantages of the invention will be apparent from the descnption and drawings, and from the claims DESCRIPTION OFDRAWINGS
FIGURE IA Identification of Rb Raf-1 inhibitors An lmmunoprecipitation- western blot analysis showing the disruption of the Rb Raf-1 interaction by compounds 10b and 10c
FIGURE IB BrdU incorporation assay showing that compound 10b arrests wild-type A549 cells, but Rb is required for activity of compound 10b, 5, 10 and 20 μM of 10b does not inhibit the proliferation of A549 cells over-expressmg shRNA constructs to Rb (sh6 and sh8), but 10b arrests wild-type A549 cells
FIGURE 1C BrdU incorporation assay showing that compound 10c arrests wild-type A549 cells, but Rb is required for activity of compound 10c, 5, 10 and 20 μM of 10c does not inhibit the proliferation of A549 cells over-expressmg shRNA constructs to Rb (sh6 and sh8), but 10c arrests wild-type A549 cells FIGURE ID A BrdU incorporation assay at compound concentrations of 5, 10, 20, 30 and 50 μM shows dose-dependent inhibition of wild-type A549 cells by compounds 3w, 10a, 10b and 10c
FIGURE IE Compounds 10b and 10c inhibit angiogenic tubule formation m matπgel in a dose-dependent fashion as shown at concentrations of 20, 50 and 100 μM For comparison, lack of inhibition of angiogenic tubule formation in matπgel is shown for control-no drug, and comparable inhibition is shown by compound 3a at 100 μM
FIGURE IF Compounds 10b and 10c at 150 mg/kg inhibit human tumor growth in nude mice A549 cells xenotransplanted bilaterally into the flanks of athymic nude mice were allowed to grow for 14 days until tumor volume reached 200mm3, daily administration of compounds 10b and 10c substantially inhibited tumor growth whereas control tumors grew to almost 1200 mm3
FIGURE IG Compound 10c inhibited the proliferation of a wide range of cancer cells at 20 μM In a BrdU incorporation assay, compound 10c was contacted with a range of cancer cells including PANC-I (human pancreatic carcinoma, epithehal-like), CAPAN-2
(human pancreatic ductal adenocarcinoma), Mel-5 (human malignant melanoma), MCF-7
(human breast adenocarcinoma), LNCAP (androgen-sensitive human prostate adenocarcinoma), A549 (human epithelial lung carcinoma), and PC-3 (human prostate adenocarcinoma), and compared to Rb-deficient cancer cells (A549 cells stably transfected with two different shRNA constructs (sh6 and sh8) to knock down Rb expression, and the
Rb-deficient prostate cancer cell line DU145) This result confirms that compound 10c arrests the proliferation of a wide vaπety of cancer cells in a Rb dependent manner
FIGURE 2 Results of a MTT assay in which U937 myeloid cells were incubated m the absence of compound (control), or with compounds 3a, 10b, or 10c at lOμM, 20μM, or 50μM for 24 hours showing dose-dependent reduction in viability of the cancer cells in the presence of the compound
FIGURE 3 Results of a MTT assay in which Ramos cells (Burkitt's Lymphoma) were incubated in the absence of compound (control), or with compounds 3a, 10b, or 10c at lOμM, 20μM, or 50μM for 24 hours showing dose-dependent reduction m viability of the cancer cells in the presence of the compound FIGURE 4 Results of a BrdU mcoproation assay where cells lacking Raf-1 due to presence of a Raf-inhibitory shRNA or control cells (containing a control shRNA) were incubated in the presence or absence of compounds 3a, 10b and 10c (20μM) The compounds inhibit the proliferation of cells having Raf-1 but not the cells lacking Raf-1 FIGURE 5A A schematic of the promoters showing the E2F binding site on the genes for MMP2, MMP9 and MMP14
FIGURE 5B Results of a QRT-PCR experiment measuring the expression of MMP2, MMP9 and MMP 14 in A549 cells transfected with shRNA to inhibit expression of ECFl or control cells When expression of ECFl is depleted, the expression of MMP9 and MMP14 is reduced
FIGURES 6A-D Results of a chromatin immunoprecipitation assay showing the binding of ECFl and the association of Rb with promoters of matrix metalloproemases MMP2 (Figure 6A), MMP9 (Figure 6B), MMP 14 (Figure 6C), and MMP 15 (Figure 6D)
FIGURES 7A-D Results of a QRT-PCT experiment performed to measure the effect of compounds 3a, 10b and 10c on the expression of Figures 7A (MMP2), 7B (MMP9), 7C (MMP14) and 7D (MMP15) in MDAMB231 cells (breast cancer) showing expression of MMP9, MMP 14 and MMP 15 inhibited by each of the compounds
FIGURE 8A A schematic diagram showing E2F binding sites on the promoters for VEGF receptors, FLTl and KDR FIGURES 8B-D show the results of chromatin immunoprecipitation assay performed using primary endothelial cells human aortic endothelial cells HAEC (Figure 8B), human umbilical cord vein endothelial cell (HUVEC) (Figure 8C) and human microvascular endothelial cells from the lung (HMEC-L) (Figure 8D) Treatment of the primary endothelial cells (human aortic endothelial cells, human umbilical cord vein endothelial cells or human microvascular endothelial cells from the lung) with VEGF induced the binding of E2F1 to the FLTl and KDR promoters
FIGURE 9 shows data demonstrating that transient transfection of E2F1 induces FLTl and KDR promoters and that Rb can repress these promoters The transfection assays were performed in both A549 and HUVEC cells FIGURE 10 shows the results of a QRT-PCR expeπments performed to measure the effect of compounds 3a, 10b and 10c (50μM) on the expression of FLTl and KDR in human aortic endothelial cells Each of the compounds inhibits expression of both FLT and KDR
DETAILED DESCRIPTION This application relates to compounds, pharmaceutical compositions, and methods for modulating cell proliferation and/or Rb Raf-1 interaction in a cell, either in vitro or in vivo For example, disorders that can be treated with the disclosed compounds, compositions, and methods include diseases such as cancer as well as non-cancerous proliferation disorders Without wishing to be bound by any theory, it is believed that the pharmaceutical activity of the disclosed compounds arises, at least in part, to modulation of Rb Raf-1 binding interactions by the disclosed compound, and more particularly to disruption of Rb Raf-1 binding
In various embodiments, the disclosed compounds are modulators of Rb Raf-1 binding interactions A modulator can change the action or activity of the molecule, enzyme, or system which it targets For example, the disclosed modulators can modulate Rb Raf 1 binding interactions to inhibit, disrupt, prevent, block or antagonize Rb, Raf-1, or Rb Raf-1 binding interactions, or otherwise prevent association or interaction between Rb and Raf-1 Thus, the disclosed compounds can be inhibitors, disrupters, blockers, or antagonists of Rb or Raf-1 activity, or of Rb Raf-1 binding interactions Thus, the compounds, pharmaceutical compositions, and methods of use descπbed m this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect in the Rb Raf-1 regulatory pathway In particular, the disclosed compounds, pharmaceutical compositions, and methods of use are believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf- 1 binding interactions in the proliferating cells
The mactivation of the retinoblastoma tumor suppressor protein Rb by cell cycle regulatory kinases is disrupted m almost all cancers In normal cells, mactivation of Rb is necessary for the Gl to S phase progression of the cell cycle Rb controls entry into the S phase by repressing the transcriptional activity of the E2F family of transcription factors, especially E2Fs 1, 2, and 3 Rb is inactivated through multiple phosphorylation events mediated by kinases associated with D and E type cyclms in the Gl phase of the cell cycle It was found that the signaling kinase Raf-1 initiates the phosphorylation events, Raf-1 signaling kinase is known to play a role in promoting cancer, and studies have shown that Rb Raf-1 binding facilitates cell proliferation It has also been found that the Rb Raf-1 interaction is elevated in human tumors compared to adjacent normal tissue m 80% of samples examined Because Raf-1 is persistently activated in many tumors, a few attempts have been made to selectively inhibit tumors by modulating Rb and/or Raf-1 activity with Raf-1 antisense oligonucleotides, the multikmase inhibitor Sorafenib, and a peptide fragment of Raf-1 coupled to a earner peptide However, there is still a need for effective modulators of the Rb Raf-1 interaction
Without being bound by any theory, it has been found that modulators of Rb Raf-1 interactions that are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells For example, modulators of Rb Raf 1 interactions are potent, selective disruptors of Rb Raf-1 binding Also, modulators of Rb Raf 1 interactions are surprisingly effective in inhibiting the tumor growth and survival of a wide variety of cancer cells, including osteosarcoma, epithelial lung carcinoma, non-small cell lung carcinoma, three different pancreatic cancer cell lines, glioblastoma cell lines, metastatic breast cancer, melanoma, and prostate cancer Moreover, modulators of Rb Raf 1 interactions effectively disrupt angiogenesis, significantly inhibited anchorage independent tumor and significantly inhibited the growth of human epithelial lung carcinoma in nude mice Accordingly, compounds, pharmaceutical compositions comprising the compounds, methods of inhibiting cell proliferation, methods of treating subjects with cancer, and methods of preparing modulators of Rb Raf 1 interactions are provided herein
I. Definitions A. General
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
The term "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system The term "cell proliferation disorder" means a disorder wherein unwanted cell proliferation of one or more subsets of cells in a multicellular organism occurs In some such disorders, cells are made by the organism at an atypically accelerated rate The term includes cancer and non-cancerous cell proliferation disorders In some embodiments, the cell proliferation disorder is angiogenesis or the cell proliferation disorder is mediated by angiogenesis
The expression "effective amount", when used to describe an amount of compound or radiation applied in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example an amount that inhibits the abnormal growth or proliferation, or induces apoptosis of cancer cells, resulting m a useful effect
The terms "treating" and "treatment" mean causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, postponing or preventing the further development of a disorder and/or reducing the seventy of symptoms that will or are expected to develop
As used herein, "individual" (as in the subject of the treatment) means both mammals and non-mammals Mammals include, for example, humans, non-human primates, e g apes and monkeys, cattle, horses, sheep, rats, mice, pigs, and goats Non-mammals include, for example, fish and birds As used herein, the term "pharmaceutically acceptable" means that the mateπals (e g , compositions, earners, diluents, reagents, salts, and the like) are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness or gastnc upset B. Chemical In the following paragraphs some of the definitions include examples The examples are intended to be illustrative, and not limiting When a term defined below is used in the specification, it is to be understood that the term includes the embodiments encompassed by the term, including the exemplary embodiments descnbed herein
An aliphatic group is a straight chained, branched non-aromatic hydrocarbon which is completely saturated or which contains one or more units of unsaturation A cycloaliphatic group is an aliphatic group that forms a πng Alkyl and cycloalkyl groups are saturated aliphatic and saturated cycloaliphatic groups, respectively Typically, a straight chained or branched aliphatic group has from 1 to about 10 carbon atoms, typically from 1 to about 6, and preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, typically from 3 to about 8, and preferably from 3 to about 6 An aliphatic group is preferably a straight chained or branched alkyl group, e g , methyl, ethyl, n-propyl, iso-piopyl, n-butyl, sec-butyl, fert-butyl, pentyl, hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbon atoms Ci 6 straight chained or branched alkyl or alkoxy groups or a C3 8 cyclic alkyl or alkoxy group (preferably Ci 6 straight chained or branched alkyl or alkoxy group) are also referred to as a "lower alkyl" or "lower alkoxy" groups, such groups substituted with -F, -Cl, -Br, or -I are "lower haloalkyl" or "lower haloalkoxy" groups, a "lower hydroxyalkyl" is a lower alkyl substituted with -OH, and the like
The term "alkyl" or "(Cx y)alkyl" (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group containing between x and y carbon atoms An alkyl group formally corresponds to an alkane or cycloalkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound An alkyl group may be straight-chained or branched Alkyl groups having 3 or more carbon atoms may be cyclic Cyclic alkyl groups having 7 or more carbon atoms may contain more than one ring and be polycyclic Examples of straight-chained alkyl groups include methyl, ethyl, n-propyl, n-butyl, and n-octyl Examples of branched alkyl groups include (-propyl, ^-butyl, and 2,2-dimethylethyl Examples of cyclic alkyl groups include cyclopentyl, cyclohexyl, cyclohexylmethyl, and 4-methylcyclohexyl Examples of polycyclic alkyl groups include bicyclo[2 2 ljheptanyl, norbornyl, and adamantyl Examples of alkyl and (Cx y)alkyl groups are (Ci 6)alkyl such as (Ci^alkyl, for example methyl and ethyl
The term "alkylene" or "(Cx y)alkylene" (wherein x and y are integers) refers to an alkylene group containing between x and y carbon atoms An alkylene group formally corresponds to an alkane with two C-H bond replaced by points of attachment of the alkylene group to the remainder of the compound Included are divalent straight hydrocarbon group consisting of methylene groups, such as, -CH2-, -CH2CH2-, -CH2CH2CH2- In some embodiments, alkylene or (C5 y)alkylene may be (Ci 6)alkylene such as (Ci 3)alkylene The term "alkenyl" or "(Cx y) alkenyl" (wherein x and y are integers) denotes a radical containing x to y carbons, wherein at least one carbon-carbon double bond is present (therefore x must be at least 2) Some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons Both E and Z isomers are embraced by the term "alkenyl " Furthermore, the term "alkenyl" includes di- and tn- alkenyls Accordingly, if more than one double bond is present then the bonds may be all E or Z or a mixtures of E and Z Examples of an alkenyl include vinyl, allyl, 2-butenyl, 3- butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl, 2,4- hexadienyl and the like The term "alkynyl" or "(Cx y) alkynyl" (wherein x and y are integers) denotes a radical containing 2 to 6 carbons and at least one carbon-carbon triple bond, some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons Examples of an alkynyl include ethynyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like The term "alkynyl" includes di- and tn-ynes
The term "alkoxy" or "(Cx-y) alkoxy" (wherein x and y are integers) employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers Embodiments include (Ci 3)alkoxy, such as ethoxy and methoxy
The term "haloalkyl" or "(Cx y)haloalkyl" (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group or (Cx y)alkyl group in which a halogen is substituted for one or more of the hydrogen atoms Examples include tπfluoromethyl, 2,2,2-tnfluoroethyl and tπchloromethyl
An "alkylene" group is a linking alkyl chain represented by -(CHi)n-, wherein n, the number of "backbone" atoms in the chain, is an integer from 1-10, typically 1-6, and preferably 1-4 An "alkenylene" group is a linking alkyl chain having one or more double bonds, wherein the number of backbone atoms is an integer from 1-10, typically 1-6, and preferably 1-4 An "alkynylene" group is a linking alkyl chain having one or more triple bonds and optionally one or more double bonds, wherein the number of "backbone" atoms is an integer from 1-10, typically 1-6, and preferably 1-4
"Heteroalkylene," "heteroalkenylene," and "heteroalkynylene" groups are alkylene, alkenylene, and alkynylene groups, respectively, wherein one or more carbons are replaced with heteroatoms such as N, O, or S
A "heterocyclic group" or "heterocyclyl" is a non-aromatic cycloaliphatic group which has from 3 to about 10 ring atoms, typically from 3 to about 8, and preferably from 3 to about 6, wherein one or more of the πng atoms is a heteroatom such as N, O, or S m the ring Examples of heterocyclic groups include oxazohnyl, thiazolmyl, oxazohdmyl, thiazolidinyl, tetrahydrofuranyl, tetrahyrothiophenyl, morphohno, thiomorpholmo, pyrrolidmyl, piperazinyl, pipendinyl, thiazolidinyl, and the like
Examples of non-aromatic heterocycles also include monocyclic groups such as aziπdme, oxirane, thπrane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidme, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, pipeπdme, 1,2,3,6-tetrahydropyπdme, 1,4-dihydropyndme, piperazme, morpholine, thiomorpholme, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazme, homopiperidme, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethyleneoxide The term "aromatic" refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i e having (4n + 2) delocalized π (pi) electrons where n is an integer)
The term "aryl", employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more πngs (typically one, two or three πngs), wherein such πngs may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene Examples include phenyl, anthracyl, and naphthyl Preferred are phenyl and naphthyl, most preferred is phenyl In some embodiments, the term refers to Ce 14 carbocyclic aromatic groups such as phenyl, biphenyl, and the like Aryl groups also include fused polycyclic aromatic πng systems in which a carbocyclic aromatic πng is fused to other aryl, cycloalkyl, or cycloaliphatic nngs, such as naphthyl, pyrenyl, anthracyl, 9,10-dihydroanthracyl, fluorenyl, and the like
The term "aralkyl" or "aryl-(Cx y)alkyl" means a functional group wherein carbon alkylene chain of x to y carbon atoms is attached to an aryl group, e g , -CH2CH2-phenyl Examples include is aryl(CH2)- (e g benzyl) and aryl(CH(CH3))- The term "substtitued aralkyl" or "substituted aryl-(Ci 3)alkyl" means an aryl-(Ci-3)alkyl functional group in which the aryl group is substituted Preferred is substituted aryl(CH2)- Similarly, the term "heteroaryl(Ci 3)alkyl" means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e g , -CH2CH2-pyndyl Preferred is heteroaryl(CH2)- The term "substituted heteroaryl-(Ci 3)alkyl" means a heteroaryl-(Ci C3)alkyl functional group in which the heteroaryl group is substituted Preferred is substituted heteroaryl(CH2)-
The term "heteroaryl" refers to 5-14 membered aryl groups having 1 or more O, S, or N heteroatoms Examples of heteroaryl groups include pyridyl, pyrimidyl, pyrazmyl, tπazmyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, 1,2,3-tnzaolyl, 1,2,4-tnazolyl, tetrazolyl, thienyl, thiazoyl, isothiazolyl, furanyl, oxazolyl, isooxazolyl, and the like Heteroaryl groups also include fused polycyclic aromatic ring systems m which a carbocyclic aromatic πng or heteroaryl πng is fused to one or more other heteroaryl nngs Examples include qumolmyl, isoquinolmyl, qumazolmyl, napthyπdyl, pyπdopyπmidyl, benzothienyl, benzothiazolyl, benzoisotmazolyl, thienopyπdyl, thiazolopyπdyl, isothiazolopyπdyl, benzofuranyl, benzooxazolyl, benzoisooxazolyl, furanopyridyl, oxazolopyridyl, lsooxazolopyridyl, mdolyl, lsomdolyl, benzimidazolyl, benzopyrazolyl, pyrrolopyπdyl, isopyrrolopyndyl, lmidazopyπdyl, pyrazolopyndyl, and the like A nng recited as a substituent herein can be bonded via any substitutable atom m the πng
Examples of heteroaryl groups include pyπdyl, pyrazmyl, pynmidinyl, particularly 2- and 4-ρyπmidinyl, pyπdazmyl, thienyl, furyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-tπazolyl, 1,2,4-tnazolyl, 1,3,4-tπazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1 ,3 ,4-oxadiazolyl
Examples of polycyclic heterocycles include mdolyl, particularly 3-, 4-, 5-, 6- and 7-mdolyl, indolmyl, qumolyl, tetrahydroqumolyl, lsoqumolyl, particularly 1- and 5-isoqmnolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolmyl, qumoxahnyl, particularly 2- and 5-qumoxalmyl, quinazohnyl, phthalazinyl, 1,5-naphthyndmyl, 1,8-naphthyndmyl, 1 ,4-benzodioxanyl, coumaπn, dihydrocoumann, benzofuryl, particularly 3-, 4-, 5-, 6- and 7-benzofuryl, 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl, particularly 3-, 4-, 5-, 6-, and 7-benzothienyl, benzoxazolyl, benzthiazolyl, particularly 2-benzothiazolyl and 5-benzothiazolyl, puπnyl, benzimidazolyl, particularly 2-benzimidazolyl, benztπazolyl, thioxanthmyl, carbazolyl, carbolmyl, acπdmyl, pyrrolizidmyl, and quinohzidmyl
The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting The term "substituted" means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group For aryl and heteroaryl groups, the term "substituted", unless otherwise indicated, refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted The substituents are independently selected, and substitution may be at any chemically accessible position
The "valency" of a chemical group refers to the number of bonds by which it is attached to other groups of the molecule
Suitable optional substituents for a substitutable atom in the preceding groups, e g , alkyl, cycloalkyl, aliphatic, cycloahphatic, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocyclic, aryl, and heteroaryl groups, are those substituents that do not substantially interfere with the pharmaceutical activity of the disclosed compounds A "substitutable atom" is an atom that has one or more valences or charges available to form one or more corresponding covalent or ionic bonds with a substituent For example, a carbon atom with one valence available (e g , -C(-H)=) can form a single bond to an alkyl group (e g , -C(-alkyl)=), a carbon atom with two valences available (e g , -C(Fk)-) can form one or two smgle bonds to one or two substituents (e g , -C(alkyl)(H)-, -C(alkyl)(Br))-,) or a double bond to one substituent (e g , -C(-O)-), and the like Substitutions contemplated herein include only those substitutions that form stable compounds For example, suitable optional substituents for substitutable carbon atoms include -F, -Cl, -Br, -I, -CN, -NO2, -ORa, -C(O)Ra, -OC(O)Ra, -C(O)OR3, -SRa, -C(S)Ra, -OC(S)R2, -C(S)ORa, -C(O)SR", -C(S)SRa, -S(O)R", -SO2Ra, -SO3Ra, -OSO2R3, -OSO3R3, -PO2RaRb, -OPO2R3Rb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2Rc, -C(O)NR3CN, -SO2N(R3R11), -SO2N(RaRb), -NRcC(0)Ra, -NR0C(O)OR3, -NR°C(O)N(RaRb), -C(NR°)-N(RaRb), -NRd-C(NRc)-N(RaRb), -NRaN(RaRb), -CRc=CR3Rb, -C≡ CR3, =O, =S, =CR3Rb, =NRa, =NORa, =NNRa, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, and optionally substituted heteroaryl, wherein R3-R are each independently — H or an optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, or optionally substituted heteroaryl, or, -N(RaRb), taken together, is an optionally substituted heterocyclic group Suitable substituents for nitrogen atoms having two covalent bonds to other atoms include, for example, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, -NO2, -ORa, -C(O)R8, -OC(O)R3, -C(O)ORa, -SRa, -S(O)R3, -SO2R3, -SO3R3, -N(R3Rb), -C(O)N(R3R"), -C(O)NR3NRbSO2Rc, -C(O)NR3SO2R0, -C(0)NRaCN, -SO2N(R3R"), -SO2N(R3R"), -NR0C(O)R3, -NR0C(O)OR3, -NRcC(0)N(RaRb), and the like
A mtrogen-contaming group, for example, a heteroaryl or non-aromatic heterocycle, can be substituted with oxygen to form an N-oxide, e g , as m a pyndyl N-oxide, pipeπdyl N-oxide, and the like For example, in various embodiments, a nng nitrogen atom in a nitrogen-containing heterocyclic or heteroaryl group can be substituted to form an N-oxide
Suitable substituents for nitrogen atoms having three covalent bonds to other atoms include -OH, alkyl, and alkoxy (preferably Ci $ alkyl and alkoxy) Substituted nng nitrogen atoms that have three covalent bonds to other nng atoms are positively charged, which is balanced by counteranions corresponding to those found in pharmaceutically acceptable salts, such as chlonde, bromide, fluonde, iodide, formate, acetate and the like Examples of other suitable counteranions are provided in the section below directed to suitable pharmacologically acceptable salts
II. Compounds
It is appreciated that certain features of the invention, which are, for clarity, descnbed in the context of separate embodiments, may also be provided in combination in a single embodiment Conversely, various features of the invention which are, for brevity, descnbed m the context of a single embodiment, may also be provided separately or m any suitable subcombination
In one aspect, there is provided a compound according to formula (I)
or a salt such as a pharmaceutically acceptable salt thereof, wherein
Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl,
Y is optionally substituted methylene, X1 is -O-, -S-, or optionally substituted -NH-,
X3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene, X2 is S or optionally substituted NH, X4 is S or optionally substituted NH, or X2 and X4 are both N and are linked together through a bond or an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl πng, and
X5 is an optionally substituted -NH2 or 3-7 membered heteroaryl or heterocyclyl πng, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R", -OC(O)R3, -C(O)ORa, -SRa, -C(S)R", -OC(S)Ra, -C(S)ORa,
-C(O)SR3, -C(S)SRa, -S(O)Ra, -SO2Ra, -SO3R8, -OSO2R3, -OSO3Ra, -PO2RaRb, -OPO2RaRb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2R°, -C(O)NR11SO2R0, -C(O)NR2CN, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R", -NR0C(O)OR3, -NR°C(O)N(RaRb), -C(NR°)-N(RaRb), -NRd-C(NR°)-N(RaRb), -NRaN(RaRb), -CRc=CRaRb, -C≡CRa, =O, =S, =CRaRb, =NRa, =NORa, or =NNRa, or two optionally substitutable carbons are linked with Ci 3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -0Ra, -C(0)Ra, -C(O)Ra-aryl, -OC(O)R3, -C(O)OR3, -SRa, -S(O)R", -SO2Ra, -SO3Ra, -N(RaRb), -C(0)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NR3SO2R0, -C(0)NRaCN, -SO2N(RaRb), -NR3SO2R", -NR°C(0)Ra, -NR0C(O)OR3, -NR°C(0)N(RaRb), or oxygen to form an N-oxide, and is optionally protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable counteπon, and wherem each of Ra, Rb, R° and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or in any occurrence of -N(RaRb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group hi some embodiments, when X2 and X4 are both N and are linked together, they are linked together through an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 6-7 membered heteroaryl or heterocyclyl πng
In some embodiments, each optionally substitutable carbon is optionally substituted with a substituent other than -SRa
In some embodiments, πng A when monosubstituted phenyl is other than 2- tπfluoromethylphenyl, 3-methoxyphenyl, 3-mtrophenyl, 3-tπfluoromethylphenyl, 3- vinylphenyl, 4-t-butylphenyl, 4-ehlorophenyl, 4-fluorophenyl, 4-methoxyphenyl, 4- methylphenyl, 4-mtrophenyl, 4-trifluoromethylphenyl, and/or 4-vmylphenyl In some embodiments, nng A when disubstituted phenyl is other than 3,4-dichlorophenyl, 3,5- ditπfluoromethylphenyl, and/or 2-hydroxy-5-mtrophenyl In some embodiments, these provisos apply when X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2 In some embodiments, nng A when substituted phenyl is other than 2- haloalkylphenyl, 3-alkoxyphenyl, 3-mtrophenyl, 3-haloalkylphenyl, 3-vmylphenyl, 4- alkenylphenyl, 4-alkylphenyl, 4-haloalkylphenyl, 4-halophenyl, 4-alkoxyphenyl, and/or 4- mtrophenyl In some embodiments, nng A when disubstituted phenyl is other than 3,4- dihalophenyl, 3,5-haloalkylρhenyl, and/or 2-hydroxy-5-mtroρhenyl In some embodiments, these provisos apply when X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2
In some embodiments, nng A is monosubstituted phenyl In some embodiments, nng A is 2- or 3- or 4-monosubstituted phenyl In other embodiments, nng A is other than monosubstituted phenyl, or other than 2- or 3- or 4-monosubstituted phenyl In some such embodiments, X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2 hi some embodiments, nng A is disubstituted phenyl In some embodiments, nng A is 2,3- or 2,4- or 2,5- or 2,6- or 3,4- or 3,5-disubstituted phenyl In other embodiments, nng
A is other than disubstituted phenyl, or other than 2,3- or 2,4- or 2,5- or 2,6- or 3,4- or 3,5- disubstituted phenyl In some such embodiments, X1 is NH, X2 is NH, X3 is NH, X4 is NH,
X5 is NH2, and Y is CH2
In some embodiments, nng A is tnsubstituted phenyl In some embodiments, nng A is 2,3,4- or 2,3,5- or 2,3,6- or 2,4,5- or 2,4,6- or 3,4,5-tnsubstituted phenyl In other embodiments, nng A is other than tnsubstituted phenyl, or other than 2,3,4- or 2,3,5- or 2,3,6- or 2,4,5- or 2,4,6- or 3,4,5-tnsubstituted phenyl In some such embodiments, X1 is
NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2
In some embodiments, nng A is tetrasubstituted phenyl hi some embodiments, nng
A is 2,3,4,5- or 2,3,4,6- or 2,3,5,6-tetrasubstituted phenyl In other embodiments, nng A is other than tetrasubstituted phenyl, or other than 2,3,4,5- or 2,3,4,6- or 2,3,5,6-tetrasubstituted phenyl hi some such embodiments, X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and
Y is CH2
In some embodiments, nng A is pentasubstituted phenyl In some embodiments, nng A is other than substituted phenyl
In some embodiments, X1 is -O-, -S-, or optionally substituted -NH-, X3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene, X2 is S or optionally substituted NH, and X4 is S or optionally substituted NH
In some embodiments Ra is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
In some embodiments Rb is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic In some embodiments, Ra is other than heterocyclic In some embodiments Rc is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
In some embodiments Rd is other than -H, is other than alkyl, is other than haloalkyl, is other than aralkyl, is other than aryl, is other than heteroaryl, is other than heterocyclyl, or is other than cycloaliphatic
In some embodiments, Group A is phenyl substituted in at least the 2-position In some such embodiments, the phenyl is substituted m the 2-position with halogen In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than haloalkyl, for example tnfluoromethyl In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than OH In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than SRa
In some embodiments, Group A is phenyl substituted in at least the 2-position In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than haloalkyl, for example tnfluoromethyl In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than OH In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than SRa
In some embodiments, Group A is phenyl substituted in at least the 4-position In some such embodiments, the phenyl is substituted in the 4-ρosition with a substituent other than mtro In some such embodiments, the phenyl is substituted m the 4-position with a substituent other than halogen In some such embodiments, the phenyl is substituted in the 4- position with a substituent other than halogen unless the nng is further substituted, m some such embodiments the further substituent, if m the 3-position, is other than halogen In some such embodiments, the phenyl is substituted in the 4-position with a substituent other than SRa In some such embodiments, the phenyl is substituted in the 2-position with a substituent other than SR"
In some embodiments, the Group A is substituted phenyl or optionally substituted naphthyl or pyπdyl In some embodiments, in Group A, an unsubstrtuted nng atom is adjacent to the nng atom attached to Y
In some embodiments, Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci β alkyl, Ci s alkoxy, Ci 6 haloalkyl, Ci s haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic In some embodiments, Y is C(O), or methylene optionally substituted with hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci ^ haloalkoxy, or Ci 6 alkyl substituted with aryl hi some embodiments, Y is methylene optionally substituted with hydroxyl, Ci β alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, Y is methylene optionally substituted with Ci 3 alkyl In some embodiments, Y is methylene
In some embodiments, the compound is represented by the following structural formula (Ia)
Figure imgf000023_0001
or a salt such as a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci β haloalkyl, C] « haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic,
R2 is hydrogen, hydroxyl, Ci 6 alkyl, C] 6 alkoxy, Ci 6 haloalkyl, Ci β haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, R3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci s haloalkyl, C] 6 haloalkoxy, Ci β alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, R4 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci β haloalkyl, Ci β haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, and
R5 is hydrogen, hydroxyl, Ci 6 alkyl, Ci β alkoxy, Ci β haloalkyl, Ci β haloalkoxy, Ci β alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic In some embodiments, R1 is hydrogen, hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci e haloalkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, R1 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci β alkyl substituted with aryl In some embodiments, R1 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R1 is hydrogen In some embodiments, R2 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci β haloalkyl, Ci 6 haloalkoxy, or Ci β alkyl substituted with aryl In some embodiments, R2 is hydrogen, hydroxyl, Ci β alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, R2 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R2 is hydrogen In some embodiments, R3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci β haloalkyl, Ci 6 haloalkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, R3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci β alkoxy, or Ci <; alkyl substituted with aryl In some embodiments, R3 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R3 is hydrogen In some embodiments, R4 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci g haloalkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, R4 is hydrogen, hydroxyl, Ci β alkyl, Ci β alkoxy, or Ci β alkyl substituted with aryl In some embodiments, R4 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R4 is hydrogen In some embodiments, R5 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci « haloalkoxy, or Ci β alkyl substituted with aryl In some embodiments, R5 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl In some embodiments, R5 is hydrogen or Ci 3 alkyl, for example methyl hi some embodiments, R5 is hydrogen In some embodiments of the compounds of formula Ia, A is substituted phenyl In particular embodiments thereof, Y is methylene, R1, R2, R3, R4 and R5 are hydrogen
In some embodiments of the compounds of formula Ia, A is optionally substituted naphthyl, for example optionally substituted 1-naphthyl or 2-naphthyl In particular embodiments thereof, Y is methylene, and R1, R2, R3, R4 and R5 are hydrogen
In the compounds of formula I, and Ia, and the embodiments thereof, m some embodiments, Group A is substituted with a substitutent selected from -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -OR", -C(O)R", -OC(O)Ra, -C(O)ORa, -SRa, -SO2R", -SO3Ra, -OSO2R", -OSO3R", -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2Rc, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R", and -NR0C(O)OR3, or two substitutable carbons are linked with Ci 3 alkylenedioxy For example, in some embodiments, one, two or three substitutable carbons m Group A may be substituted with a substituent independently selected from -F, -Cl, -Br, -I, -CN, -NO2, Ci e alkyl, Ci β alkoxy, -CF3, and Ci 6 haloalkoxy, or two substitutable carbons may be linked with Ci 2 alkylenedioxy In some embodiments, Group A is phenyl, wherein one, two or three substitutable carbons of the phenyl are substituted with a substituent independently selected from -F, -Cl, -Br, -I, -CN, -NO2, Ci 6 alkyl, Ci 6 alkoxy, -CF3, and Ci 6 haloalkoxy, or two substitutable carbons are linked with Ci 2 alkylenedioxy In some embodiments, Group A is phenyl unsubstituted at its 6-position In some embodiments, Group A is 2,4-substituted phenyl In some embodiments, Group A is 2,4-disubstituted phenyl substituted at at least the 2-position, or m at least the 4-position, or in both the 2- and 4-positions with halogen, m some such embodiments, one of the halogens may be chlorine In some embodiments, Group A or is phenyl monsubstituted at its 2, 3, or 4 positions or independently disubstituted at its 2,3, 2,4, 2,5 or 3,4 positions with -F, -Cl, -Br, -NO2, Ci 6 alkyl, or -CF3 In some embodiments, Group A is phenyl independently disubstituted at its 2,3, 2,4, 3,4, or 2,5 positions with -NO2, -Cl, -F or -CF3 In some embodiments, Group A is phenyl monosubstituted at its 2, 3, or 4 position with -NO2, -Cl or -F In some embodiments, Group A is phenyl independently disubstituted at its 2,4 positions with -NO2, -Cl or -F
In some embodiments, Group A is unsubstituted 2- naphthyl or 1-substituted 2- naphthyl In some embodiments, Group A is naphthyl optionally substituted with one or more of -F, -Cl, -Br, -NO2, Ci-6 alkyl, or -CF3. In some embodiments, Group A is naphthyl optionally monosubstituted with -F, -Cl, -Br, -NO2, or -CF3. In some embodiments, Group A is naphthyl optionally monosubstituted with -F, -Cl, or -Br
Particular compounds of interest include the following compounds and salts such as pharmaceutically acceptable salts thereof, particularly the 2,4-dichlorophenyl compound.
Figure imgf000026_0001
In another aspect, there is provided a compound according to formula (II):
Figure imgf000026_0002
or a salt such as a pharmaceutically acceptable salt thereof, wherein: Y is optionally substituted methylene; X1 is -O-, -S-, or optionally substituted -NH-; and X2 is S or optionally substituted NH;
R6 and R7 are independently -F, -Cl, -Br, -I, -NO2, -CN, -CF3, or C1-C6 alkoxy; wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)Ra, -OC(O)Ra, -C(O)OR", -SRa, -C(S)Ra, -OC(S)Ra, -C(S)ORa, -C(O)SRa, -C(S)SRa, -S(O)Ra, -SO2Ra, -SO3Ra, -OSO2Ra, -OSO3R3, -PO2RaRb, -OPO2RaRb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(R3R"), -NRaSO2Rb, -NR0C(O)R3, -NRcC(O)ORa, -NRcC(O)N(RaRb), -C(NRc)-N(RaRb), -NRd-C(NRc)-N(RaRb), -NRaN(RaRb), -CRc=CRaRb, -C≡CR3, -O, =S, =CR3Rb, =NRa, =N0Ra, or =NNRa, or two optionally substitutable carbons are linked with Cu alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -OR3, -C(O)Ra, -C(O)Ra-aryl, -OC(O)R3, -C(O)OR3, -SRa, -S(O)R3, -SO2R3, -SO3R", -N(RaRb), -C(O)N(R3R"), -C(O)NRaNRbSO2Rc, -C(O)NR3SO2R", -C(O)NR3CN, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R3, -NR0C(O)OR3, -NR°C(0)N(RaRb), or oxygen to form an N-oxide, and optionally is protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable counteπon, and wherein each of Ra, Rb, R0 and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or in any occurrence of -N(RaRb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group
In some embodiments of the compounds of formula II, R6 and R7 are not both -Cl and R6 and R7 are not both -CF3 In some embodiments of the compounds of formula II, R6 and R7 are not both -F, R6 and R7 are not both -Br, R6 and R7 are not both -I, R6 and R7 are not both -NO2, and R6 and R7 are not both -CH3 In some embodiments, this proviso applies when Y is -CH2-, X1 is S and X2 is NH
In some embodiments of the compounds of formula II, Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci β haloalkoxy, Ci-S alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic In some embodiments, Y is methylene optionally substituted with hydroxyl, Ci s alkyl, Ci β alkoxy, or Ci-6 alkyl substituted with aryl In some embodiments, Y is methylene optionally substituted with Ci 3 alkyl, for example methyl In some embodiments, Y is methylene In some embodiments, the compound of formula II is represented by the following structural formula
Figure imgf000028_0001
or a salt such as a pharmaceutically acceptable salt thereof, wherein R8 is hydrogen, hydroxyl, Ci 6 alkyl, Ci β alkoxy, Ci β haloalkyl, Ci β haloalkoxy, Ci β alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic In some embodiments thereof, is hydrogen, hydroxyl, Ct 6 alkyl, Ci 6 alkoxy, or Ci β alkyl substituted with aryl In some embodiments, R8 is hydrogen or Ci 3 alkyl, for example methyl In some embodiments, R8 is hydrogen hi the preferred embodiments, Y is methylene and R8 is hydrogen hi some embodiments, R6 and R7 are independently -F, -Cl, -Br, -NO2, or -CF3 Compounds according to formula II of particular interest include those wherein the compound is selected from the group consisting of
Figure imgf000028_0002
and salts such as pharmaceutically acceptable salts thereof
In another aspect, compounds are included which are represented by one of the following structural formulae (Ib) and (lib)
Figure imgf000028_0003
Figure imgf000029_0001
wherein
Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl, Y is optionally substituted methylene,
X1 and X3 are independently -O-, -S-, or optionally substituted -NH-, or X3 is optionally substituted methylene,
X2 and X4 are independently S or optionally substituted NH, or X2 and X4 are both N and are linked together through a bond or an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl πng,
X5 is an optionally substituted -NH2 or 3-7 membered heteroaryl or heterocyelyl ring,
R6 and R7 are independently -F, -Cl, -Br, -I, -NO2, -CN, -CF3, or Ci-C6 alkoxy, provided that R6 and R7 are not both -Cl and Ri and R2 are not both -CF3 In some embodiments, R6 and R7 are not both -F In certain embodiments, R6 and R7 are independently -F, -Cl, -Br, -NO2, or -CF3, or m particular embodiments, R6 and R7 are independently -F, -Cl, or -NO2, each substitutable carbon atom (e g , each optionally substituted carbon) is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R", -OC(O)Ra, -C(O)ORa, -SRa, -C(S)R", -OC(S)R3, -C(S)ORa, -C(O)SRa, -C(S)SRa, -S(O)Ra, -SO2Ra, -SO3R3, -OSO2Ra,
-OSO3Ra, -P02RaRb, -OPO2RaRb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb),
-C(O)NRaNRbSO2Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(RaRb), -NRaS02Rb,
-NRcC(O)Ra, -NRcC(0)0Ra, -NR°C(0)N(RaRb), -C(NRc)-N(RaRb), -NRd-C(NRc)-N(RaRb),
-NRaN(RaRb), -CRc=CRaRb, -C≡CRa, =O, =S, =CRaRb, =NR", =N0R", or =NNRa, or two substitutable carbons are linked with Ci 3 alkylenedioxy, each substitutable nitrogen (e g , each optionally substituted nitrogen) is optionally substituted with -CN, -NO2, -Ra, -0Ra, -C(O)R", -C(O)Ra-aryl, -0C(0)Ra, -C(O)OR", -SRa, -S(O)R3, -SO2R3, -SO3R3, -N(R3R"), -C(O)N(RaRb), -C(O)NR3NRbSO2Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(RaRb), -NRaS02Rb, -NR0C(O)R3, -NR0C(O)OR3, -NRcC(0)N(RaRb), or oxygen to form an N-oxide and each nitrogen can also be optionally protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable countenon, and
Each Ra-Rd is independently H, alkyl, alkoxy, haloalkyl, haloalkoxy, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or, -N(R3Rb), taken together, is an optionally substituted heterocyclic group
In various embodiments of the compounds Ib and Ha, Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy,
Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic In some embodiments, Y is C(O), or methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, C] $ haloalkoxy, or Ci 6 alkyl substituted with aryl In certain embodiments, Y is methylene optionally substituted with hydroxyl, Ci β alkyl, Ci g alkoxy, or Ci 6 alkyl substituted with aryl hi particular embodiments, Y is methylene optionally substituted with Ci 3 alkyl
In the compounds of formula lib, Group A can be substituted phenyl or optionally substituted naphthyl or pyndyl In some embodiments, m Group A, an unsubstituted ring atom is adjacent to the πng atom attached to Y For example, when Group A is a phenyl, the 6-position of that phenyl can be unsubstituted
In some embodiments, the compound according to formula Ib is represented by the following structural formula (Ic)
Figure imgf000030_0001
wherein each R' is independently hydrogen, hydroxyl, Ci g alkyl, C] (, alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic In some embodiments, each R is independently hydrogen, hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci e haloalkoxy, or C] 6 alkyl substituted with aryl In certain embodiments, each R' is independently hydrogen, hydroxyl, Ci_6 alkyl, Ci β alkoxy, or Ci-6 alkyl substituted with aryl In particular embodiments, each R' is independently hydrogen or Ci 3 alkyl
In various embodiments, the compound according to the formula Ib may be represented by one of the following structural formulae
Figure imgf000031_0001
wherein A' is substituted phenyl and A" is optionally substituted naphthyl In some embodiments, the compound can be represented by the following structural formula
Figure imgf000031_0002
In some embodiments, the compound can be represented by the following structural formula
Figure imgf000031_0003
In various embodiments of the compounds of formula Ib, one or more substitutable carbons m Group A, Ring A' or Ring A" is substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R", -OC(O)R3, -C(O)OR8, -SRa, -SO2R", -SO3Ra, -OSO2R", -OSO3Ra, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2R°, -SO2N(RaRb), -NRaSO2Rb, -NRcC(O)Ra, or -NR0C(O)OR", or two substitutable carbons are linked with C1 3 alkylenedioxy In some embodiments, in Group A, Ring A' or Ring A" one, two or three substitutable carbons are substituted with -F, -Cl, -Br, -I, -CN, -NO2, Ci 6 alkyl, C1 6 alkoxy, -CF3, or C1 6 haloalkoxy, or two substitutable carbons are linked with C1 2 alkylenedioxy In various embodiments of the compounds of formula Ib, Group A or Ring A' is phenyl unsubstituted at its 6-position In some embodiments, Group A or Ring A' is 2,4- substituted phenyl In certain embodiments, Group A or Ring A' is phenyl monsubstituted at its 2, 3, or 4 positions or independently disubstituted at its 2,3, 2,4, 2,5 or 3,4 positions with -F, -Cl, -Br, -NO2, Ci 6 alkyl, or -CF3 hi particular embodiments, Group A or Ring A' is phenyl independently disubstituted at its 2,3, 2,4, 3,4, or 2,5 positions with -NO2, -Cl, -F or -CF3 In some embodiments, Group A or Ring A' is phenyl monosubstituted at its 2, 3, or 4 position with NO2, -Cl or -F In certain embodiments, Group A or Ring A' is phenyl independently disubstituted at its 2,4 positions with -NO2, -Cl or -F
In various embodiments, Group A or Ring A" is unsubstituted 2- naphthyl or 1- substituted 2- naphthyl In some embodiments, Group A or Ring A" is naphthyl optionally substituted with one or more of -F, -Cl, -Br, -NO2, C1 6 alkyl, or -CF3 In certain embodiments, Group A or Ring A" is naphthyl optionally monosubstituted with -F, -Cl, -Br, -NO2, or -CF3 In particular embodiments, Group A or Ring A" is naphthyl optionally monosubstituted with -F, -Cl, or -Br In various embodiments, the compound is represented by the following structural formula
Figure imgf000032_0001
Y can be as defined in any embodiment herein above In some embodiments, Y is C(O),
C(S), or methylene optionally substituted with hydroxyl, C1 β alkyl, Ci 6 alkoxy, Ci ^ haloalkyl, Ci β haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloalrphatic In certain embodiments, Y is methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci β alkyl substituted with aryl In particular embodiments, Y is methylene optionally substituted with Ci 3 alkyl
In vaπous embodiments, the compound is represented by the following structural formula
Figure imgf000033_0001
R' can be as defined m any embodiment herein above In some embodiments, R' is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, CL 6 haloalkoxy, C1 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic In certain embodiments, R' is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl In particular embodiments, R' is hydrogen or Ci 3 alkyl, for example methyl In particular embodiments, R' is hydrogen
Also included are pharmaceutically acceptable salts, solvates, hydrates, tautomers, stereoisomers and diasteromers of the compounds The compounds can be modulators of Rb Raf 1 interactions It is to be understood that other embodiments of the invention will combine the features of embodiments explicitly descnbed above Embodiments defined by such combinations are contemplated as embodiments of the invention
III. Salts
The compounds descnbed above, and any of the embodiments thereof, as well as intermediates used in making the compounds may take the form of salts The compounds, compositions and methods of the present invention include salts of the disclosed compounds, particularly pharmaceutically acceptable salts, and methods and compositions using them
The disclosed compounds can have one or more sufficiently acidic protons that can react with a suitable organic or inorganic base to form a base addition salt When it is stated that a compound has a hydrogen atom bonded to an oxygen, nitrogen, or sulfur atom, it is contemplated that the compound also includes salts thereof where this hydrogen atom has been reacted with a suitable organic or inorganic base to form a base addition salt Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases such as alkoxides, alkyl amides, alkyl and aryl amines, and the like Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like
The term "salts" embraces addition salts of free acids or free bases which are compounds descπbed herein The term "pharmaceutically-acceptable salt" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications, such that the salt is suitable for administration to a subject Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallimty, which may render them useful, for example in processes of synthesis, purification or formulation of compounds descπbed herein In general the useful properties of the compounds described herein do not depend critically on whether the compound is or is not in a salt form, so unless clearly indicated otherwise (such as specifying that the compound should be in "free base" or "free acid" form), reference in the specification to a compound should generally be understood as encompassing salts of the compound, whether or not this is explicitly stated
When the disclosed compounds contain a basic group, such as an amine, suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, carbonic, sulfuric, phosphoric and nitric acids Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxyhc and sulfonic classes of organic acids, examples of which include p-toluenesulfomc, methanesulfomc, oxalic, p-bromophenyl-sulfomc, carbonic, succinic, citric, benzoic, acetic acid, formic, acetic, propionic, glycolic, gluconic, lactic, malic, tartaric, ascorbic, glucuronic, maleic, fumaπc, pyruvic, aspartic, glutamic, anthramlic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), ethanesulfomc, benzenesulfomc, pantothenic, tnfluoromethanesulfonic, 2-hydroxyethanesulfomc, sulfamlic, cyclohexylammosulfomc, steaπc, algimc, β-hydroxybutync, salicylic, galactaric and galacturomc acid Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dimtrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2-sulfonate, mandelate, and the like In certain embodiments, the disclosed compound forms a pharmaceutically acceptable salt with HCl, HF, HBr, HI, tnfluoracetic acid, or sulfuric acid In particular embodiments, the disclosed compounds form a pharmaceutically acceptable salt with sulfuric acid Examples of acids which form pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates
Salts of compounds having an acidic group can be formed by the reaction of the disclosed compounds with a suitable base For example, salts can be formed by the reaction of the disclosed compounds with one equivalent of a suitable base to form a monovalent salt (ι e , the compound has single negative charge that is balanced by a pharmaceutically acceptable counter cation, e g , a monovalent cation) or with two equivalents of a suitable base to form a divalent salt (e g , the compound has a two-electron negative charge that is balanced by two pharmaceutically acceptable counter cations, e g , two pharmaceutically acceptable monovalent cations or a single pharmaceutically acceptable divalent cation)
Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, lithium, sodium, potassium, magnesium, calcium and zinc salts Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, JV,JV-dibenzylethylenediatmne, chloroprocame, choline, diethanolamme, ethylenediamme, meglumine (N-methylglucamme) and procaine Salts can also be formed with ammonium compounds, NRt +, wherein each R is independently hydrogen, an optionally substituted aliphatic group (e g , a hydroxyalkyl group, ammoalkyl group or ammomumalkyl group) or optionally substituted aryl group, or two R groups, taken together, form an optionally substituted non-aromatic heterocyclic πng optionally fused to an aromatic ring Generally, the pharmaceutically acceptable cation is Li+, Na+, K+, NH3(C2H5OH)+ or N(CHs)3(C2H5OH)+
Where applicable, any of the salt forms descπbed above can be applied to any of the compounds or embodiments thereof descπbed in the Summary or Section II above Any of the salt forms appropπate for compounds containing a basic group can be applied to any of the compounds having a basic nitrogen - such as the isothiourea compounds and amidinoisothiourea compounds descπbed above hi particular, the hydrochloπde, hydrobromide, sulfatep-toluenesulfonate, methanesulfonae, succinate, citrate, benzoate, lactate, mahate, tartrate, maleate, fumarate, and benzenesulfonate salts of the disclosed compounds may be mentioned
The salt forms descπbed above as being appropnate for compounds containing a base can particularly be applied as being of interest in Section II above In particular, each one of the salt forms descπbed above as being appropπate for compounds containing a base can particularly be applied to each one of the following compounds, and, in particular, the hydrochlonde, hydrobromide, sulfatep-toluenesulfonate, methanesulfonae, succinate, citrate, benzoate, lactate, mahate, tartrate, maleate, fumarate, and benzenesulfonate salts of the disclosed compounds may be mentioned
Figure imgf000036_0001
The salt forms suitable for use with contaimng a base descπbed above are particularly applicable to the 2,4-dichlorophenyl amindinoisothiourea whose structure is provided above
All of these salts may be prepared by conventional means from the corresponding compound by reacting the compound with the appropπate acid or base Preferably the salts are in crystalline form, and preferably prepared by crystallization of the salt from a suitable solvent The person skilled m the art will know how to prepare and select suitable salts for example, as descnbed in Handbook of Pharmaceutical Salts Properties Selection and Use By P H Stahl and C G Wermuth (Wiley- VCH 2002) IV. Solvate Forms
The disclosed compounds, and salts thereof as well as intermediates used in making the compounds may take the form of solvates, including hydrates Thus, the compounds include solvate forms for the compound, and the compostitions and methods discosed herein, include compositions and methods wherein the disclosed compound is present or used in the form of a solvate or hydrate, preferably a pharmaceutically acceptable solvate or hydrate The term "solvate" means a compound of the present invention or a salt thereof, that further includes a stoichiometric or non-stoichiometnc amount of solvent, e g , water or organic solvent, bound by non-covalent mtermolecular forces, where the solvent is water, the term "hydrate" can be used In general, the useful properties of the compounds described herein are not believed to depend critically on whether the compound or salt thereof is or is not in the form of a solvate
V. Stereochemistry, Tautomerism, and Conformational Isomerism
It will also be understood that certain disclosed compounds can be obtained as different stereoisomers (e g , diastereomers and enantiomers) and tautomers
The disclosed compounds are intended includes all isomeric forms and racemic mixtures of the disclosed compounds and methods of treating a subject with both pure isomers and mixtures thereof, including racemic mixtures Stereoisomers can be separated and isolated using any suitable method, such as chromatography It will also be understood that certain disclosed compounds can take various tautomeric forms, and the depiction of any compound as a particular tautomer does not preclude other corresponding tautomers of that compound
A. Geometrical Isomerism
Certain compounds may possess an olefimc double bond The stereochemistry of compounds possessing an olefimc double bond is designated using the nomenclature using E and Z designations The compounds are named according to the Cahn-Ingold-Prelog system, described in the IUPAC 1974 Recommendations, Section E Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc , New York, NY, 4th ed , 1992, pp 127-38, the entire contents of which are incorporated herein by reference B. Optical Isomerism
Certain compounds may contain one or more chiral centers, and may exist in, and may be isolated as pure enantiomeric or diastereomenc forms or as racemic mixtures The formulae are intended to encompass any possible enantiomers, diastereomers, racemates or mixtures thereof which are biologically active
The isomers resulting from the presence of a single chiral center compπse a pair of non-supeπmposable isomers that are called "enantiomers " Single enantiomers of a pure compound are optically active, z e , they are capable of rotating the plane of plane polarized light Single enantiomers are designated according to the Cahn-Ingold-Prelog system The formulae encompasses diastereomers as well as their racemic and resolved, diastereomeπcally and enantiomencally pure forms and salts thereof Diastereomenc pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization
"Isolated optical isomer" means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula Preferably, the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight
Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques According to one such method, a racemic mixture of a compound, or a chiral intermediate in the synthesis thereof, is separated into 99 wt % pure optical isomers by HPLC using a suitable chiral column, such as a member of the senes of DAICEL® CHIRALP AK* family of columns (Daicel Chemical Industries, Ltd , Tokyo, Japan) The column is operated according to the manufacturer's instructions
C. Conformational Isomerism Due to chemical properties such as resonance lending some double bond character to a C-N bond, it is possible that individual conformers of certain compounds descrbied above may be observable and even separable under certain circumstances The compounds therefore includes any possible stable rotamers which are biologically active D. Tautomerism
Certain of the compounds descπbed above may exist in tautomeric forms, which differ by the location of a hydrogen atom and typically are m rapid equilibrium In such circumstances, molecular formulae drawn will typically only represent one of the possible tautomers even though equilibration of these tautomeπc forms will occur in equilibrium in the compound Examples include keto-enol tautomeπsm and amide-imidic acid tautomerism Tautomensm is frequently also seen in heterocyclic compounds AU tautomeric forms of the compounds are to be understood as being included within the scope of the formulae depicted V. Pharmaceutical Compositions and Formulations
Also included are pharmaceutical compositions comprising the disclosed compounds A "pharmaceutical composition" compπses a disclosed compound, typically in conjunction with an acceptable pharmaceutical earner as part of a pharmaceutical composition for administration to a subject The disclosed compounds may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable earner The active ingredient in such formulations may comprise from 0 1 to 99 99 weight percent "Pharmaceutically acceptable earner" means any earner, diluent or excipient which is compatible with the other ingredients of the formulation and not deletenous to the recipient The active agent may be administered with a pharmaceutically acceptable earner selected on the basis of the selected route of administration and standard pharmaceutical practice The active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations See Alphonso Gennaro, ed , Remington The Science and Practice of Pharmacy, 20th Edition (2003), Mack Publishing Co , Easton, PA Suitable dosage forms may compnse, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositones, suspensions, injection compositions, infusion compositions, topical administration solutions, emulsions, capsules, creams, ointments, tablets, pills, lozenges, suppositones, depot preparations, implanted reservoirs, lntravagmal nngs, coatings on implantable medical devices (eg , a stent), impregnation in implantable medical devices, and the like Suitable pharmaceutical earners may contain inert ingredients which do not interact with the compound
For parenteral administration, the active agent may be mixed with a suitable earner or diluent such as water, for example stenle water, an oil (particularly a vegetable oil), ethanol, salme solution (e g physiological salme, bacteπostatic saline (saline containing about 0 9% mg/mL benzyl alcohol), phosphate-buffered salme), Hank's solution, Rmger's-lactate, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol Solutions for parenteral administration preferably contain a water soluble salt of the active agent Stabilizing agents, antioxidant agents and preservatives may also be added Suitable antioxidant agents include sulfite, ascorbic acid, citnc acid and its salts, and sodium EDTA Suitable preservatives include benzalkonmm chlonde, methyl- or propyl-paraben, and chlorbutanol The composition for parenteral administration may take the form of an aqueous or non-aqueous solution, dispersion, suspension or emulsion For example, a stenle injectable composition such as a stenle injectable aqueous or oleaginous suspension, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents The stenle injectable preparation can also be a stenle injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol Other examples of acceptable vehicles and solvents include mannitol, water, Ringer's solution and isotonic sodium chlonde solution In addition, stenle, fixed oils are conventionally employed as a solvent or suspending medium (e g , synthetic mono- or diglycendes) Fatty acids, such as oleic acid and its glycende denvatives can be useful in the preparation of mjectables, as well as natural pharmaceutically-acceptable oils, such as olive oil or castor oil, for example in their polyoxyethylated versions Oil solutions or suspensions can also contain a long-cham alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents
A composition for oral administration, for example, can be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions The active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms For example, the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch Lubricating agents, such as magnesium stearate, are also typically added For oral administration in a capsule form, useful diluents include lactose and dπed com starch When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents If desired, certain sweetening, flavoring, or coloπng agents can be added According to one tablet embodiment, the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, manmtol and starch, and then formed into tablets by conventional tabletmg methods Methods for encapsulating compositions (such as m a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al , "Controlled Release of Biological Active Agents", John Wiley and Sons, 1986)
A nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation and can be prepared as solutions in salme, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizmg or dispersing agents known in the art
The specific dose of a compound according required to obtain therapeutic benefit in the methods of treatment described herein will, of course, be determined by the particular circumstances of the individual patient including the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease disorder, and the route of administration of the compound
For example, a daily dosage from about 0 05 to about 50 mg/kg/day may be utilized, for example a dosage from about 0 1 to about 10 mg/kg/day Higher or lower doses are also contemplated as it may be necessary to use dosages outside these ranges in some cases The daily dosage may be divided, such as being divided equally into two to four times per day daily dosing The compositions may be formulated in a unit dosage form, each dosage containing from about 1 to about 500mg, more typically, about 10 to about lOOmg of active agent per unit dosage The term "unit dosage form" refers to physically discrete units suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active mateπal calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient
The pharmaceutical compositions descπbed herein may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matπces, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres
In general, a controlled-release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time Such dosage forms provide a supply of a drug to the body during a predetermined peπod of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations
U S Patent No 5,674,533 discloses controlled-release pharmaceutical compositions in liquid dosage forms for the administration of moguisteme, a potent peπpheral antitussive U S Patent No 5,059,595 descπbes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances U S Patent No 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-mflammatory agent with potent analgesic properties U S Patent No 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers U S Patent No 5,073,543 descπbes controlled-release formulations containing a trophic factor entrapped by a ganglioside-lrposome vehicle U S Patent No 5,639,476 discloses a stable solid controlled-release formulation having a coating deπved from an aqueous dispersion of a hydrophobic acrylic polymer Biodegradable microparticles are known for use in controlled-release formulations U S Patent No 5,354,566 discloses a controlled-release powder that contains the active ingredient U S Patent No 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions The controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds Vaπous mechanisms of drug release exist For example, in one embodiment, the controlled-release component may swell and form porous openings large enough to release the active ingredient after administration to a patient The term "controlled-release component" means a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres that facilitate the controlled-release of the active ingredient in the pharmaceutical composition In another embodiment, the controlled-release component is biodegradable, induced by exposure to the aqueous environment, pH, temperature, or enzymes in the body In another embodiment, sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e g , at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade) Particularly for human consumption, the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U S Food and Drug Administration For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U S Food and Drug Administration
VI. Mode of Administration
Formulation of the compound to be administered will vary according to the route of administration selected, e g , parenteral, oral, buccal, epicutaneous, lnhalational, opthalamic, mtraear, intranasal, intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intraosseous, intracutaneous, intradermal, intraperitoneal, topically, transdermal, transmucosal, intraarticular, lntrasynovial, mtrastemal, mtralesional, intracranial lnhalational, insufflation, pulmonary, epidural, mtratumoral, intrathecal, vaginal, rectal, or intravitreal administration An "effective amount" to be administered is the quantity of compound in which a beneficial outcome is achieved when the compound is administered to a subject or alternatively, the quantity of compound that possess a desired activity in vivo or in vitro In the case of cell proliferation disorders, a beneficial clinical outcome includes reduction in the extent or seventy of the symptoms associated with the disease or disorder and/or an increase m the longevity and/or quality of life of the subject compared with the absence of the treatment The precise amount of compound admimstered to a subject will depend on the type and seventy of the disease or condition and on the charactenstics of the subject, such as general health, age, sex, body weight and tolerance to drugs It will also depend on the degree, seventy and type of disorder The skilled artisan will be able to determine appropnate dosages depending on these and other factors The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is descnbed, for example, in Freireich et al , (1966) Cancer Chemother Rep 50 219 Body surface area may be approximately determined from height and weight of the patient See, e g , Scientific Tables, Geigy Pharmaceuticals, Ardley, N Y, 1970, 537 An effective amount of the disclosed compounds can range from about 0 001 mg/kg to about 1000 mg/kg, more preferably 0 01 mg/kg to about 500 mg/kg, more preferably 1 mg/kg to about 200 mg/kg Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other agents
The disclosed compounds can be co-admimstered with anti-cancer agents or chemotherapeutic agents such as alkylating agents, antimetabolites, natural products, hormones, metal coordination compounds, or other anticancer drugs Examples of alkylating agents include nitrogen mustards (e g , cyclophosphamide), ethylemmme and methylmelammes (e g- , hexamethlymelamine, thiotepa), alkyl sulfonates (e g , busulfan), nitrosoureas (e g , streptozocm), or tnazenes (decarbazme, etc ) Examples of antimetabolites include folic acid analogs (e g , methotrexate), pynmidme analogs (e g , fluorouracil), punne analogs (e g , mercaptopunne) Examples of natural products include vmca alkaloids (e g , vmcnstine), epipodophyllotoxms (e g , etoposide), antibiotics (e g , doxorubicin,), enzymes (e g , L-asparagmase), or biological response modifiers (e g , interferon alpha) Examples of hormones and antagonists include adrenocorticosteroids (e g , prednisone), progestins (e g , hydroxyprogesterone), estrogens (e g , diethlystilbestrol), antiestrogen (e g , tamoxifen), androgens (e g , testosterone), antiandrogen (e g , flutamide), and gonadotropin releasing hormone analog (e g , leuprolide) Other agents that can be used in the methods and with the compositions of the invention for the treatment or prevention of cancer include platinum coordination complexes (e g , cisplatm, carboblatin), anthracenedione (e g , mitoxantrone), substituted urea (e g , hydroxyurea), methyl hydrazine deπvative (e g , procarbazine), or adrenocortical suppressants (e g , mitotane)
In various embodiments compounds can be coadmimstered with compounds that can inhibit angiogenesis or inhibit angiogenic tubule formation include, for example, matrix metalloprotemase inhibitors(daltepaπn, suramin), endothelial cell inhibitors (e g , thalidomide, squalamme, 2-methoxyestradiol), inhibitors of angiogenesis activation (e g , avastatm, endostatm), celecoxib and the like
VII. Methods of Preparation Processes for preparing compounds the disclosed compounds and intermediates that are useful in the preparation of such compounds, and processes for preparing such intermediates are also provided herein
The compounds disclosed herein can be prepared according to the methods descπbed m U S Appl Ser No 11/562,903, the entire teachings of which are incorporated herein by reference The methods descπbed in U S Appl Ser No 11/562,903 can be modified or augmented by synthetic chemistry functional group transformations known in the art and include, for example, those descπbed in R Larock, Comprehensive Organic Transformations, VCH Publishers (1989), L Fieser and M Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994), and L Paquette, ed , Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) Comprehensive Organic Synthesis, Ed B M Trost and I Fleming (Pergamon Press, 1991), Comprehensive Organic Functional Group Transformations, Ed A R Katπtzky, O Meth-Cohn, and C W Rees (Pergamon Press, 1996), Comprehensive Organic Functional Group Transformations II, Ed A R Katπtzky and R J K Taylor (Editor) (Elsevier, 2nd Edition, 2004), Comprehensive Heterocyclic Chemistry, Ed A R Katntzky and C W Rees (Pergamon Press, 1984), and Comprehensive Heterocyclic Chemistry II, Ed A R Katπtzky, C W Rees, and E F V Scnven (Pergamon Press, 1996) The entire teachings of these documents are are incorporated herein by reference
Compounds of formula I may be prepared by the reaction compounds of formula III, wherein LG represents a suitable leaving group, by reaction with a compound of formula IV
Figure imgf000046_0001
Suitable leaving groups LG in the compounds of formula III include halogen, particularly chlorine, bromine, and iodine, and sulfonate groups, particularly methanesulfonate, p-toluenesulfonate, and tnfiuoromethanesulfonate The reactions are typically performed in a solvent at a suitable temperature In some cases a base may be used as a catalyst Suitable bases include alkali metal hydroxide or alkoxide salts such as sodium hydroxide or methoxide, and tertiary amines such as tπethylamme or 7V,iV-diisopropylethylamme Suitable solvents include alcohols, such as methanol and ethanol, or dichloromethane The reactions may be earned out at a temperature between 0 0C and the reflux temperature of the solvent, which is typically about 100 0C The reactions may be performed at a higher temperature by performing the reaction under pressure or in a sealed vessel Microwave heating may be used In a typical procedure, the components are reacted in by performing microwave heating, for example in ethanol at a temperature from about 80 to about 120 0C
Compounds of formula III are either commercially available, known in the art, or may be prepared by methods known to one skilled in the art For example, -CH- groups alpha to an aromatic ring can be readily halogenated under free radical conditions Alternatively, appropπate leaving groups could be introduced by conversion of the corresponding alcohol (by conversion of OH to halogen, or treatment with a sulfonyl chloride such as p-toluenesulfonyl chloride), which can be prepared by a variety of methods, for example, reduction of a aromatic carboxyhc acid or an aromatic aldehyde or ketone Compounds of formula IV are either commercially available, known in the art, or may be prepared by methods known to one skilled in the art For example, amidinothiourea (2-immo-4-thiobmret) (CAS registry no 2114-02-5) is commercially available from Sigma- Aldπch and other suppliers
Compounds of formula II may be prepared by the reaction compounds of formula V, wherein LG represents a suitable leaving group, by reaction with a compound of formula VI
Figure imgf000047_0001
Scheme 2 Suitable leaving groups LQ in the compounds of formula IV include halogen, particularly chlorine, bromine, and iodme, and sulfonate groups, particularly methanesulfonate, p-toluenesulfonate, and tnfluoromethanesulfonate The reactions are typically performed m a solvent at a suitable temperature In some cases a base may be used as a catalyst Suitable bases include alkali metal hydroxide or alkoxide salts such as sodium hydroxide or methoxide, and tertiary amines such as tnethylamme or AζiV-diisopropylemylamme Suitable solvents include alcohols, such as methanol and ethanol, or dichloromethane The reactions may be carried out at a temperature between 0 0C and the reflux temperature of the solvent, which is typically about 100 °C The reactions may be performed at a higher temperature by performing the reaction under pressure or m a sealed vessel Microwave heating may be used In a typical procedure, the components are reacted in by performing microwave heating, for example in ethanol at a temperature from about 80 to about 120 0C
Compounds of formula V, such as benzyl hahdes, are either commercially available, known in the art, or may be prepared by methods known to one skilled m the art For example, -CH- groups alpha to benzene πng can be readily halogenated under free radical conditions Alternatively, appropπate leaving groups could be introduced by conversion of the corresponding alcohol (by conversion of OH to halogen, or treatment with a sulfonyl chloride such as £>-toluenesulfonyl chloride), which can be prepared by a variety of methods, for example, reduction of a benzoic acid or a benzaldehyde or phenyl ketone
Compounds of formula VI are either commercially available, known in the art, or may be prepared by methods known to one skilled m the art For example, thiourea (CAS registry no 62-56-6) is commercially available from Sigma- Aldπch and other suppliers
The above-described reactions, unless otherwise noted, are usually conducted at a pressure of about one to about three atmospheres, such as at ambient pressure (about one atmosphere)
In some embodiments, the compounds according to formula I or II may be used as isolated compounds The expression "isolated compound" refers to a preparation of a compound of formula I or II, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds "Isolated" does not necessarily mean that the preparation is technically pure (homogeneous), but can mean that it is sufficiently pure to compound in a form in which it can be used therapeutically The term "isolated compound" may refer to a preparation of a compound of formula I which contains the named compound or mixture of compounds according to formula I in an amount of at least 10 percent by weight of the total weight, at least 50 percent by weight of the total weight, at least 80 percent by weight of the total weight, at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation
The compounds of formula I and II and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystalhzation or chromatography, including flash column chromatography, or HPLC The preferred method for purification of the compounds according to formula I and II or salts thereof composes crystallizing the compound or salt from a solvent to form, preferably, a crystalline form of the compounds or salts thereof Following crystallization, the crystallization solvent is removed by a process other than evaporation, for example filtration or decanting, and the crystals are then preferably washed using pure solvent (or a mixture of pure solvents) Suitable solvents for crystallization include water, alcohols, particularly alcohols containing up to four carbon atoms such as methanol, ethanol, isopropanol, and butan-1-ol, butan-2-ol, and 2-methyl-2- propanol, ethers, for example diethyl ether, dnsopropyl ether, ^-butyl methyl ether, 1,2- dimethoxyethane, tetrahydrofuran and 1,4-dioxane, carboxylic acids, for example formic acid and acetic acid, and hydrocarbon solvents, for example peπtane, hexane, toluene, and mixtures thereof, particularly aqueous mixtures such as aqueous ethanol Pure solvents, preferably at least analytical grade, and more preferably pharmaceutical grade are preferably used In a preferred embodiment of the processes, the products are so isolated In the some embodiments of compounds according to formula I and II or salts thereof, and pharmaceutical compositions thereof, the compound according to formula I and II or salt thereof is in or prepared from a crystalline form, which may be prepared by crystallization according to such a process
It will be appreciated by one skilled m the art that certain aromatic substituents m the compounds of formula I and II, intermediates used in the processes described above, or precursors thereto, may be introduced by employing aromatic substitution reactions to introduce or replace a substituent, or by using functional group transformations to modify an existing substituent, or a combination thereof Such reactions may be effected either prior to or immediately following the processes mentioned above The reagents and reaction conditions for such procedures are known in the art Specific examples of procedures which may be employed include, but are not limited to, electrophilic functionalization of an aromatic πng, for example via nitration, halogenation, or acylation, transformation of a mtro group to an amino group, for example via reduction, such as by catalytic hydrogenation, acylation, alkylation, or sulfonylation of an ammo or hydroxyl group, replacement of an amino group by another functional group via conversion to an intermediate diazomum salt followed by nucleophilic or free radical substitution of the diazomum salt, or replacement of a halogen by another group, for example via nucleophilic or organometallically-catalyzed substitution reactions
In implementing preparations of the disclosed compounds functional groups which would be sensitive to the reaction conditions may be protected by protecting groups A protecting group is a deπvative of a chemical functional group which would otherwise be incompatible with the conditions required to perform a particular reaction which, after the reaction has been earned out, can be removed to re-generate the original functional group, which is thereby considered to have been "protected" Any chemical functionality that is a structural component of any of the reagents used to synthesize compounds descπbed herein may be optionally protected with a chemical protecting group if such a protecting group is useful in the synthesis of compounds descπbed herein The person skilled in the art knows when protecting groups are indicated, how to select such groups, and processes that can be used for selectively introducing and selectively removing them, because methods of selecting and using protecting groups have been extensively documented in the chemical literature As used herein, "suitable protecting groups" and strategies for protecting and deprotectmg functional groups using protecting groups useful in synthesizing the disclosed compounds are known in the art and include, for example, those descπbed in T W Greene and P G M Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons (2nd Ed 1991) or 4th Ed (2006), the entire teachings of which are incorporated herein by reference For example, suitable hydroxyl protecting groups include, but are not limited to substituted methyl ethers (e g , methoxymethyl, benzyloxym ethyl) substituted ethyl ethers (e g- , ethoxymefhyl, ethoxyethyl) benzyl ethers (benzyl, mtrobenzyl, halobenzyl) silyl ethers (e g , tπmethylsilyl), esters, and the like Examples of suitable amine protectmg groups include benzyloxycarbonyl, tert-butoxycarbonyl, tert-butyl, benzyl and fluorenylmethyloxy-carbonyl (Fmoc) Examples of suitable thiol protecting groups include benzyl, tert-butyl, acetyl, methoxymethyl and the like
The reactions descnbed herein may be conducted in any suitable solvent for the reagents and products m a particular reaction Suitable solvents are those that facilitate the intended reaction but do not react with the reagents or the products of the reaction Suitable solvents can include, for example ethereal solvents such as diethyl ether or tetrahydrofuran, ketone solvents such as acetone or methyl ethyl ketone, halogenated solvents such as dicloromethane, chloroform, carbon tetrachlonde, or tnchloroethane, aromatic solvents such as benzene, toluene, xylene, or pyridine, polar aprotic organic solvents such as acetomtπle, dimethyl sulfoxide, dimethyl formamide, N-methyl pyrrohdone, hexamefhyl phosphoramide, nitromethane, nitrobenzene, or the like, polar protic solvents such as methanol, ethanol, propanol, butanol, ethylene glycol, tetraethylene glycol, or the like, nonpolar hydrocarbons such as pentane, hexane, cyclohexane, cyclopentane, heptane, octance, or the like, basic amine solvents such as pyridine, tπethyleamine, or the like, and other solvents known to the art
Reactions or reagents which are water sensitive may be handled under anhydrous conditions Reactions or reagents which are oxygen sensitive may be handled under an inert atmosphere, such as nitrogen, helium, neon, argon, and the like Reactions or reagents which are light sensitive may be handled in the dark or with suitably filtered illumination
Reactions or reagents which are temperature-sensitive, e g , reagents that are sensitive to high temperature or reactions which are exothermic may be conducted under temperature controlled conditions For example, reactions that are strongly exothermic may be conducted while being cooled to a reduced temperature
Reactions that are not strongly exothermic may be conducted at higher temperatures to facilitate the intended reaction, for example, by heating to the reflux temperature of the reaction solvent Reactions can also be conducted under microwave irradiation conditions For example, in vaπous embodiments of the method, the first and second reagents are reacted together under microwave irradiation
Reactions may also be conducted at atmospheric pressure, reduced pressure compared to atmospheric, or elevated pressure compared to atmospheric pressure For example, a reduction reaction may be conducted in the presence of an elevated pressure of hydrogen gas in combination with a hydrogenation catalyst
Reactions may be conducted at stoichiometric ratios of reagents, or where one or more reagents are in excess
VIII. Assay Methods
The disclosed compounds can be assayed for binding and biological activity by any means descπbed herein or known to the art For example, the disclosed compounds can be screened for binding activity in an ELISA assay (see Methods), the IC50 values of the disclosed compounds can be determined by in vitro binding assays (see Methods), the binding selectivity of the disclosed compounds can be measured in competitive ELISA assays, and the ability of the disclosed compounds to disrupt Rb Raf-1 in vitro or in vivo can be assayed Further, the disclosed compounds can be tested for their ability to kill or inhibit the growth of tumor cells or angiogenic tubules Suitable assays include, for example, (a) tumor cell m anchorage/independent growth (soft agar assays), (b) tumor cell in anchorage- dependent growth (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), trypan blue and DNA synthesis assays), (c) tumor cell survival (TUNEL, PARP cleavage, caspace activation and other apoptosis assays), (d) tumor cell invasion and metastasis, (e) endothelial cell migration, invasion and angiogenesis, (f) tumor cell proliferation inhibition assays, (g) anti-tumor activity assays in animal models, and other such assays known to the art Certain assays can be used to assess a subject for treatment with an inhibitor of
Rb Raf-1 binding interactions or to identify a subject for therapy The level of Rb, Raf-1, or Rb bound to Raf-1 can be determined in the subject or in a sample from the subject, e g , a subject with a cell proliferation disorder Treatment with the disclosed compounds is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal "Elevated compared to normal" means that the levels are higher than in a reference sample of cells of the same type that are healthy For example, the level of Rb, Raf-1, or Rb bound to Raf-1 in cells from a non-small cell lung cancer tumor can be compared to the level of Rb, Raf-1, or Rb bound to Raf-1 in normal, noncancerous cells For example, Enzyme Linked Immunosorbent Assay (ELISA) can be used in combination with antibodies to Rb, Raf-1, or Rb bound to Raf-1 (see Methods, In vitro library screening assays) The assay can be embodied in a kit For example, a kit includes a reagent or indicator, such as an antibody, that is specific for Rb, Raf-1, or Rb bound to Raf-1 The kit can also include instructions for determining the level of Rb, Raf-1, or Rb bound to Raf-1 in a sample using the reagent or indicator, such as an antibody, that is specific for Rb, Raf-1, or Rb bound to Raf-1 In vitro/in vivo
In various embodiments, methods relating to cells can be conducted on cells in vitro or in vivo, particularly wherein the cell is in vivo, i e , the cell is located in a subject A "subject" can be any animal with a proliferative disorder, for example, mammals, birds, reptiles, or fish Preferably, the animal is a mammal More preferably, the mammal is selected from the group consisting of dogs, cats, sheep, goats, cattle, horses, pigs, mice, non- human pπmates, and humans Most preferably, the mammal is a human
IX. Therapeutic Methods and Uses of the Compounds
Described herein are methods of using the disclosed compounds The disclosed compounds are useful in inhibiting the Rb-Raf-1 binding The disclosed compounds are biologically active and therapeutically useful
Evidence for the therapeutic utility of inhibitors of Rb-Raf-1 binding was presented in WO2007/062222, which is incorporated herein by reference in its entirety, particularly the results descπbed m Examples 5 to 20 and in Figures 1-4 A of that application, which are also incorporated herein by reference In that application, compounds which modulated Rb Raf-1 modulators selectively over Rb E2F1 were descπbed The molecules were able disrupt Rb Raf-1 in vitro as well as in intact cells Compound 3a was found to inhibit the proliferation of Rb-expressmg osteocarcoma cells (U2-OS), human epithelial lung carcinoma cells (A549), non-small cell lung cancer cells (Hl 650), pancreatic cancer cells (Aspcl, PANCl, and CAPAN2), glioblastoma cells (U87MG and U251MG), metastatic breast cancer cells (MDA-MB-231), melanoma cells (A375), prostate cancer cells (LNCaP and PC3) The compounds also inhibited the adherence-independent growth of various types of cancer cells A549 (human epithelial lung carcinoma), Hl 650 (NSCLC), SK-MEL-5, SK-MEL-28 (melanoma), and PANCl (pancreatic) cells in soft agar The compounds were belived to exert their anti-cancer effects through disruption of the Rb Raf-1 mteration The inhibitors of Rb Raf-1 binding also disrupted angiogenesis Inhibitors of Rb-Raf-1 binding were also shown to inhibit proliferation of a human tumor cell line (A549) in vivo in a nude mouse xenografts model
The Ras/Raf/Mek/MAPK cascade is a proliferative pathway induced by a wide array of growth factors and is activated in many human tumors It has been shown that signaling pathways through the MAP kinase cascade do not proceed in a linear fashion, but rather that they have been found to have substrates outside the cascade as well Without wishing to be bound by theory, in this context, the Rb protein appears to be an important cellular target of the Raf-1 kinase outside the MAP kinase cascade The binding of Raf-1 to Rb was found to occur only in proliferating cells and contributed to cell cycle progression Further, it was found that the level of Rb Raf-1 interaction was elevated in NSCLC tissue, suggesting that it may have contπbuted to the oncogenic process These observations support the hypothesis that targeting the Rb Raf-1 interaction with the disclosed compounds is a viable method to develop anticancer drugs The cell-permeable, orally available, and target specific small molecule compound
3a, can maintain the tumor suppressor functions of Rb The in vitro results indicate that compound 3a selectively inhibits the Rb Raf-1 interaction without targeting the binding partners of Rb and Raf-1, such as E2F1, prohibitm, HDACl and MEK1/2 Further, compound 3a functions by inhibiting the interaction of Raf-1 and Rb without inhibiting Raf-1 kinase activity or the kinase activity associated with cyclms D or E Also, compound 3 a inhibited cell cycle and decreased the levels of cyclm D while cdk activity was unaffected Compound 3a demonstrated Rb dependence to inhibit cell cycle progression and tumor growth in cell lines These results further confirm the specificity of 3a for targeting Rb Raf-1 Mice harboring A549 tumors responded to treatment with 3a administered by i p or oral gavage Tumor tissue displayed a decrease m proliferation, Rb phosphorylation, and angiogenesis and an increase m apoptosis Importantly, A- 549 tumors where Rb was knockdown are resistant to 3a, further suggesting that 3a inhibits tumor growth by targeting the Rb Raf-1 interaction
These results show that the mechanism of 3a mediated growth arrest is likely by targeting the Rb Raf-1 interaction Aberrant signaling mechanisms surrounding the Ras/MAPK and Rb/E2F1 pathways are commonly present in cancers The disclosed compounds, such as compound 3a, could inhibit S-phase entry in potentially 35%-90% of all of the cell lines Based on the substantial in vitro and in vivo results disclosed herein, it is believed that the disclosed compounds, in particular compound 3a, are excellent candidates for the treatment of cancer patients whose tumors harbor genetic aberrations that lead to inactivation of Rb by Raf-1
The compounds, pharmaceutical compositions, and methods of treatment descπbed in this application are believed to be effective for inhibiting cellular proliferation, particularly of cells which proliferate due to a mutation or other defect in the Rb Raf-1 regulatory pathway The disclosed compounds, pharmaceutical compositions, and methods of treatment are therefore believed to be effective for treating cancer and other proliferative disorders which can be inhibited by disrupting Rb Raf-1 binding interactions in the proliferating cells
The disclosed compounds can participate in a protem-ligand complex A protein hgand complex includes a compound and at least one protem selected from the group consisting of retinoblastoma tumor suppressor protein and serine-threonine kinase Raf-1 The complex can include a disclosed compound, retinoblastoma tumor suppressor protein, and serme-threonme kinase Raf-1
Various methods of treatment of cells and subjects are provided For example, a method of inhibiting proliferation of a cell includes contacting the cell with an effective amount of the disclosed compounds or compositions Typically, regulation of proliferation in the cell is mediated by at least one protem selected from the group consisting of retinoblastoma tumor suppressor protem and serme-threonine kinase Raf-1 For example, m various embodiments, the cells have an elevated level of Rb, Raf-1, or Rb bound to Raf-1 In some embodiment, the method includes assaying the level of Rb, Raf-1, or Rb bound to Raf- I m the cell
A method of modulating the Rb Raf-1 interaction in a proliferating cell is provided The method includes contacting the cell with an effective amount of the disclosed compounds or compositions
A method of modulating the Rb Raf-1 interaction in a proliferating cell is provided The method includes contacting the cell with a modulator of the Rb Raf-1 interaction that is suitable for oral administration In some embodiments, the modulator of the Rb Raf-1 interaction is orally administered
A method of treating or ameliorating a cell proliferation disorder is provided The method includes contacting the proliferating cells with an effective amount of the disclosed compounds or compositions Typically, regulation of cell proliferation in the disorder can be mediated by at least one protem selected from the group consisting of retinoblastoma tumor suppressor protein and seπne-threonme kinase Raf-1 The regulation of proliferation in the cells may be mediated by the interaction between retinoblastoma rumor suppressor protem and serme-threomne kinase Raf-1 The cell proliferation disorder may be cancer or a non-cancerous cell proliferation disorder The cell proliferation disorder may include angiogenesis or the cell proliferation disorder may be mediated by angiogenesis
A method of treating or ameliorating a cell proliferation disorder may also include administering the compound, or a pharmaceutically acceptable salt thereof, to a patient in need of such treatment
In various embodiments, the cell proliferation disorder is or the proliferating cells are deπved from a cancererous or a non-cancerous cell proliferation disorder Exemplary cancererous and non-cancerous cell proliferation disorders include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewmg's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, chorio carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pmealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, lymphocytic leukemia, large granular lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkm's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm's macroglobulmemia, heavy chain disease, lymphoblastic leukemia, T-cell leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cell leukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloid leukemias, myelocytic leukemia, myelogenous leukemia, neutrophilic leukemia, eosinophilic leukemia, monocytic leukemia, myelomonocytic leukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia, osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, and prostate cancer In certain embodiments, the cell proliferation disorder is osteosarcoma, promyelocyte leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, or prostate cancer
A method of inhibiting angiogenic tubule formation in a subject in need thereof includes administering to the subject an effective amount of the disclosed compounds or compositions
In some embodiments, the preceding methods of treating subjects or cells can also include coadministration of an anticancer drug or a compound that modulates angiogenic tubule formation, particularly coadministration of a compound that inhibits angiogenic tubule formation Exemplary anticancer drugs and compounds that can modulate angiogenic tubule Examples of suitable chemotherapeutic agents include any of abarehx, aldesleukin, alemtuzumab, ahtretmoin, allopunnol, altretamine, anastrozole, arsenic tπoxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabme, carboplatm, carmustine, cetuximab, chlorambucil, cisplatm, cladnbme, clofarabine, cyclophosphamide, cytarabine, dacarbazme, dactinomycin, daltepaπn sodium, dasatimb, daunorubicin, decitabine, demleukin, denileukm diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicm, erlotimb, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuπdme, fludarabme, fluorouracil, fulvestrant, gefitimb, gemcitabme, gemtuzumab ozogamicm, goserehn acetate, histrelin acetate, ibntumomab tiuxetan, ldarubicm, lfosfamide, imatmib mesylate, interferon alfa 2a, innotecan, lapatimb ditosylate, lenahdomide, letrozole, leucovoπn, leuprolide acetate, levamisole, lomustme, meclorethamine, megestrol acetate, melphalan, mercaptopunne, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabme, nofetumomab, oxaliplatin, paclitaxel, pamidronate, pamtumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatm, pipobroman, phcamycm, procarbazine, quinacπne, rasbuncase, ntuximab, sorafemb, streptozocm, sumtimb, sunitimb maleate, tamoxifen, temozolomide, temposide, testolactone, thalidomide, thioguamne, thiotepa, topotecan, toremifene, tosimmomab, trastuzumab, tretinoin, uracil mustard, valmbicin, vinblastine, vincristine, vmorelbme, voπnostat, and zoledronate A method of assessing a subject for treatment with an inhibitor of Rb Raf-1 binding interactions includes determining, in the subject or m a sample from the subject, a level of
Rb, Raf-1, or Rb bound to Raf-1, wherein treatment with an inhibitor of Rb Raf-1 binding interactions is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal
A method of identifying a subject for therapy includes the steps of providing a sample from the subject, determining a level of Rb, Raf-1, or Rb bound to Raf-1 in the sample, and identifying the subject for therapy with an inhibitor of Rb Raf-1 binding interactions when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal A kit includes an antibody specific for Rb, Raf-1, or Rb bound to Raf-1, and instructions for determining the level of Rb, Raf-1, or Rb bound to Raf-1 m a sample using the antibody specific for Rb, Raf-1, or Rb bound to Raf-1
In various embodiments, methods relating to cells can be conducted on cells in vitro or in vivo, particularly wherein the cell is in vivo in a subject The subject can be, for example, a bird, a fish, or a mammal, e g , a human
The compounds according to the invention may be administered to individuals (mammals, including animals and humans) afflicted with a cell proliferation disorder such as cancer, malignant and benign tumors, blood vessel proliferative disorders, autoimmune disorders, and fibrotic disorders The compounds are believed effective against a broad range of tumor types, including but not limited to the following ovarian cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, lung cancer, renal cancer, colorectal cancer, skm cancer, bram cancer, leukemia, including acute myeloid leukemia, chrome myeloid leukemia, acute lymphoid leukemia, and chrome lymphoid leukemia Examples of cancers include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewmg's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, lymphocytic leukemia, large granular lymphocytic leukemia, acute myelocytic leukemia, chrome leukemia, polycythemia vera, Hodgkm's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm's macroglobuhnemia, heavy chain disease, lymphoblastic leukemia, T-cell leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cell leukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloid leukemias, myelocytic leukemia, myelogenous leukemia, neutrophilic leukemia, eosinophilic leukemia, monocytic leukemia, myelomonocytic leukemia, Naegeh-type myeloid leukemia, nonlymphocytic leukemia, osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, or prostate cancer
Cancers may be solid tumors that may or may not be metastatic Cancers may also occur, as in leukemia, as a diffuse tissue Thus, the term "tumor cell", as provided herein, includes a cell afflicted by any one of the above identified disorders The compounds are also believed useful in the treatment of non-cancer cell proliferation disorders, that is, cell proliferation disorders which are characterized by benign indications Such disorders may also be known as "cytoprohferative" or "hyperproliferative" in that cells are made by the body at an atypically elevated rate In vanous embodiments, the non-cancerous cell proliferation disorder includes cells that have a mutation or defect in the Rb Raf-1 pathway Non-cancer cell proliferation disorders believed treatable by compounds according to the invention include, for example, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy, cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometπosis, fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis, desmoid tumors, mtimal smooth muscle cell hyperplasia, restenosis, vascular occlusion, hyperplasia m the bile duct, hyperplasia m the bronchial airways, hyperplasia m the kidneys of patients with renal interstitial fibrosis, psoriasis, Reiter's syndrome, pityriasis rubra pilaris, a hyperproliferative disorder of keratimzation, or scleroderma In vaπous embodiments, the cancer includes cells that have a mutation or defect m the Rb Raf-1 pathway In certain embodiments, the cancer is osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, or prostate cancer The methods descπbed above can be applied performed any of the compounds or embodiments thereof described in the Summary or Section II above, or their salts descπbed in Section IV above In particular, the methods can be earned out with any of the compounds whose structures are given below, particularly the 2,4-dichlorophenyl ammdmoisothiourea whose structure is provided, or with salts of such compounds as described m Section IV above
Figure imgf000060_0001
Based on the utilities described herein, the compounds disclosed or claimed herein are provided for use m medicine The compounds are also provided for use in the therapeutic methods descπbed or claimed herein, and for manufactunng a medicament for carrying out the therapeutic methods descπbed or claimed herein
X. EXAMPLES METHODS
Chemistry All reagents were purchased from commercial suppliers and used without further purification 1H NMR spectra were recorded using a Mercury 400 NMR spectrometer (Varian, Palo Alto, CA) 13C NMR spectra were recorded at 100 MHz, in some cases usmg
Distortionless Enhancement by Polanzation Transfer Solvents employed were CDCI3 or dβ-DMSO (dimethyl sulfoxide) All coupling constants are measured in Hertz (Hz) and the chemical shifts (5H and δc) are quoted in parts per million (ppm) relative to the internal standard, e g , CDCI3, dβ-DMSO, or TMS (tetramethyl silane) Atmospheric pressure ionization (API) and electrospray (ES) mass spectra and accurate mass determinations were recorded using a time of flight (TOF) mass spectrometer (Agilent 6210 LC/MS (ESI-TOF), Agilent/Hewlett Packard, Santa Clara, CA) Microwave reactions were performed in CEM 908005 model and Biotage initiator 8 machines High Performance Liquid Chromatography (HPLC) analysis was performed using a HPLC system equipped with a PU-2089 Plus quaternary gradient pump and a UV-2075 Plus UV-VIS detector (JASCO, Easton, MD), e g , using an Alltech Kromasil C- 18 column (150 x 4 6 mm, 5 μm) Infra red spectra were recorded using a FTIR-4100 spectrometer (JASCO) Melting points were determined using either a MEL-TEMP Electrothermal melting point apparatus or a Barnstead international melting point apparatus and are uncorrected Column chromatography was conducted using silica gel 63-200 mesh (Merck & Co , Whitehouse Station, NJ) Silica thin layer chromatography (TLC) was conducted on pre-coated aluminum sheets (60 F254, Merck & Co or Fisher), with observation under UV when necessary Anhydrous solvents (acetomtπle, dimethyl formamide, ethanol, isopropanol, methanol and tetrahydrofuran) were used as purchased from Aldrich HPLC grade solvents (methanol, acetontπle and water) were purchased from Burdick and Jackson for HPLC and mass analysis Cell culture and transfection. The human promyelocyte leukemia cell line U937 was cultured in RPMI (Mediatech, Hernden, VA) containing 10% fetal bovine serum (FBS, Mediatech) U2-OS, Saos-2, MCF7, PANCl and MDA-MB-231 cell lines were cultured in Dulbecco modified Eagle Medium (DMEM, Mediatech) containing 10% FBS A549 cells and A549 shRNA Rb cell lines were maintained m Ham F-12K supplemented with 10% FBS ShRNA cells lines were maintained in media containing 0 5μg/mL puromycm Hl 650, PC-9 and Aspcl cell line were cultured in RPMI (Gibco/Invitrogen, Carlsbad, CA) contaimng 10% FBS PANCl and CAPAN2 pancreatic cell lines and the A375 Melanoma cell line was grown m DMEM supplemented with 10% FBS Human aortic endothelial cells (HAECs, Clonetics, San Diego, CA) were cultured in endothelial growth medium, supplemented with 5% FBS, according to the manufacturer's instructions U251MG and U87MG glioma cell lines were maintained m DMEM supplemented with non-essential ammo acids, 5OmM β-mercaptoethanol, and 10% FBS ShRNA cell lines were made by stably transfectmg A549 cells with two different shRNA constructs that specifically target Rb obtained from a library The adenovirus (Ad) constructs Ad-green fluorescent protein (GFP) and Ad-E2F1 were obtained from W D Cress Ad-cyclin D was provided by I Cozar-Castellano
In vitro library screening assays. Enzyme Linked Immunosorbent Assay (ELISA) 96-well plates were coated with lμg/mL of a glutathione S-transferase (GST) Raf-1 (l-149aa) overnight at 4°C Subsequently the plates were blocked and GST Rb at 20μg/mL was rotated at room temperature (RT) for 30 minutes in the presence or absence of the compounds at 20 micromolar (μM) GST-Rb +/- compounds were then added to the plate and incubated for 90 minutes (mm) at 37°C The amount of Rb bound to Raf-1 was detected by Rb polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) 1 1000 incubated for 60 mm at 37°C Donkey-anti-rabbit-IgG-HRP (1 10,000) was added to the plate and incubated at 37°C for 60 minutes The color was developed with orthophenylenediamme (Sigma, St Louis, MO) and the reaction was terminated with 3 molar (M) H2SO4 Absorbance was read at 490 nanometers (nm) To determine disruption of Rb to E2F1, Phb, or HDACl the above protocol was used with the exception of coating GST Rb on the ELISA plate and adding the drugs in the presence or absence of GST E2F1, Phb, or HDACl E2F1 monoclonal antibody (1 2000) was used to detect the amount of Rb bound to E2F1 Prohibitin monoclonal antibody was used at 1 1000 to detect the amount of Rb bound to Prohϊbitm For disruption of MEK-Raf-1 binding ELISAs, Raf-1 1 microgram/milliliter (μg/mL) was coated on the plate and GST-MEK (20μg/mL) was incubated +/- the compounds for 30 minutes at room temperature Mekl polyclonal antibody was used at 1 1000 to detect the binding of Raf-1 to Mekl The IC50 concentrations for the Rb Raf-1 inhibitors were determined by plotting with Origin 7 5 software (Origin, Northampton, MA)
In vitro binding assays. Glutathione S-transferase (GST) fusion of Rb, Raf-1, E2F1, and MEKl have been previously described (Dasgupta P, Sun J, Wang S, et al MoI Cell Biol 2004,24(21) 9527-9541) First, 200 micrograms (μg) of U937 asynchronous lysates were pre-mcubated with lOμM of the indicated drugs or lμM of the Raf-1 peptide for 30 minutes at 4°C Next, 200μg of the U937 lysates were incubated with glutathione beads carrying an equal amount of the GST fusion proteins in 200μl of protein binding buffer (20 mM Tπs [pH 7 5], 50 mM KCL, 0 5 mM EDTA, 1 mM dithiothreitol, 0 5% NP-40, 3mg of bovme serum albumm/mL) at 40C for 2h (Wang S, Ghosh R, Chellappan S MoI Cell Biol 1998,18(12) 7487-7498)
Matrigel Assays. Matrigel (Collaborative Biomedical Products) was used to promote the differentiation of HAECs into capillary tube-like structures (Dasgupta P, Sun J, Wang S, et al MoI Cell Biol 2004,24(21) 9527-9541) A total of lOOμl of thawed Matπgel was added to 96-well tissue culture plates, followed by incubation at 37°C for 60 minutes to allow polymerization Subsequently, 1 X lO4 HAECs were seeded on the gels in EGM medium supplemented with 5% FBS in the presence or absence of 20μM concentrations of the indicated compounds, followed by incubation for 24 hours at 37°C Capillary tube formation assessed by using a Leica DMIL phase contrast microscope Lysate preparation, immunoprecipitation, and Western blotting. Lysates from cells treated with different agents were prepared by NP-40 lysis as descπbed earlier (Wang 1998) Tumor lysates were prepared with T-Per tissue lysis buffer (Pierce) and a Fischer PowerGen 125 dounce homogemzer Physical interaction between proteins in vivo was analyzed by immunoprecipitation- Western blot analyses with 200μg of lysate with lμg of the indicated antibody as previously descπbed (Wang 1998) Polyclonal E2F1 and Cyclin D were obtained from Santa Cruz Biotechnology Monoclonal Rb and Raf-1 were supplied by BD Transduction laboratories (San Jose, CA) Polyclonal antibodies to phospho-Rb (807,811) phospho- MEK1/2, MEK1/2, phospho-Erkl/2 and ERK1/2 were supplied by Cell Signaling (Danvers, MA) Chromatin Immunoprecipitation (ChIP) assay. A549 cells were rendered quiescent by serum starvation and re-stimulated with serum for 2h or 16h m the presence or absence of RRD 251 at 20μM Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature Subsequently, the cells were harvested and lysates were prepared Immunoprecipitations were analyzed for the presence of E2F1, Rb, Raf-1, Brgl, HPl, and HDACl by PCR as previously descnbed (Dasgupta 2004) Rabbit anti-mouse secondary antibody was used as the control for all reactions The sequences of the PCR primers used in the PCRs were as follows Cdc6 promoter (forward pπmer), 5'- GGCCTCACAG CGACTCTAAGA-3', and Cdc6 promoter (reverse pπmer),
5'-CTCGGACTCACCACAAGC-S ' TS promoter (forward pπmer), and 5'-GAC GGA GGC AGG CCA AGT G-3' TS promoter (reverse pnmer) The cdc25A and c-fos pnmers are descπbed in (Dasgupta, 2004)
In vitro kinase assay. The kinase reaction for Raf-1 was earned out with 100 nanograms (ng) of Raf-1 (Upstate Signaling, Charlottesville, VA), 0 5μg of full-length Rb protein (QED Bioscience, San Diego, CA) as the substrate, lOμM ATP, lOμCi of [γ-32P] ATP in the kinase assay buffer in the presence or absence of the drugs at 300C for 30 minutes Cyclin D and E kinase assays are descnbed in (Dasgupta 2004)
Proliferation assays. Bromodeoxyuπdme (BrdU) labeling kits were obtained from Roche Biochemicals (Indianapolis, IN) Cells were plated m poly-D-lysine coated chamber slides at a density of 10,000 cells per well and rendered quiescent by serum starvation for 24 hours Cells were then re-stimulated with serum in the presence or absence of the indicated drugs for 18h S-ρhase cells were visualized by microscopy and quantitated by counting 3 fields of 100 m quadruplicate
Soft Agar assay. Soft agar assays were done in triplicate in 12-well plates (Corning, Corning NY) First, the bottom layer of agar (0 6%) was allowed to solidify at room temperature Next the top layer of agar was (0 3%) was mixed with 5,000 cells per well and the indicated drug The drugs were added twice weekly in complete media to the agar wells Colonies were quantified by staining with MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 1 mg/mL for 1 hour at 370C Animal Studies. Nude mice (Charles River, Wilmington, MA, USA) were maintained in accordance with Institutional Animal Care and Use Committee (IACUC) procedures and guidelines A549 cells were harvested and resuspended in PBS, and then injected s c into the nght and left flanks (10 x 106 cells per flank) of 8-week old female nude mice as reported previously (Sun 99) When tumors reached about 100-200mm3, animals were dosed mtrapentoneally i p or orally by gavage with 0 ImL solution once daily Control animals received a vehicle, whereas treated animals were given compound at the indicated doses The tumor volumes were determined by measuπng the length (/) and the width (w) and calculating the volume (V= Iw2H) as described previously (Sun 99) Statistical significance between control and treated animals were evaluated using Student's West Immunohistochemistry staining.Upon termination of xenograft anti-tumor expeπments, tumors were removed and fixed in 10% neutral-buffered formalin before processing into paraffin blocks Tissue sections (5 micrometers (μm) thick) were cut from the blocks and stained with Ki-67, CD31, TUNEL, and phospho-Rb antibodies Paraffin sections were rehydrated to PBS and processed using the following protocols Sections were πnsed in dEbO, and then subjected to microwave 'antigen retrieval' for 20 minutes on 70% power, with a 1 minute cooling peπod after every 5 minutes, m 0 01 M sodium citrate, pH 6 0 (Janssen PJ, Brmkmann AO, Boersma WJ, Van der Kwast TH J Histochem Cytochem 1994,42(8) 1169-75, Shi SR, Key ME, Kalra KL J Histochem Cytochem 1991,39(6) 741-748) Sections were cooled for 20 minutes, nnsed 3 times m dH2O, twice in PBS and incubated in 5% normal goat serum for 30 minutes Sections were incubated in primary antibody for 1 hour in 5% normal goat serum, nnsed 3 times in PBS For color development the slides were treated with ABC kit (Vector Labs, Burhngame, CA) rinsed in dH2θ, and developed using DAB as chromogen After a final πnse in dH2θ, sections were lightly counterstained m hematoxylin, dehydrated, cleared and covershpped Tissue sections were stained with hematoxylin and eosin (H&E) using standard histological techniques Tissue sections were also subjected to immunostaimng for CD31 (BD Biosciences, San Diego, CA, USA) using the avidm-biotm peroxidase complex technique Mouse monoclonal antibody was used at 1 50 dilution following microwave antigen retrieval (four cycles of 5 mm each on high in 0 1 M citrate buffer) Apoptotic cells were detected using DeadEnd Coloπmetπc TUNEL system (Promega, Madison, WI)
General Synthetic Procedures for Modulators of Rb:Raf 1 interactions Reference compounds 1 and 2 were discovered by screening a library of compounds using a glutathione S-transferase-retinoblastoma/ glutathione S-transferase-Raf-1 kinase Enzyme-Linked Immunosorbent Assay screen (GST-Rb/GST-Raf-1 ELISA) Two structurally related compounds (1) and (2) were discovered that strongly inhibited the Rb Raf-1 interaction at a concentration of 20 μM (100% for 1 and 95% for 2)
Figure imgf000066_0001
NSC-35400 (1 ) NSC-35950 (2)
Benzyhsothiourea deπvatives 3, lacking substitution at the α benzyhc position, are prepared in good yields by reaction of thiourea with the appropriate benzyl hahde (Scheme 3, Table 1) (Yong 1997) When not commercially available the desired benzyl halides are obtained from the corresponding benzyl alcohols (prepared when necessary by NaBFLt reduction of the corresponding aldehyde) followed by reaction with thionyl chloride to generate the corresponding benzyl chloride The corresponding benzylisothiourea deπvatives 3 are usually obtained in good to quantitative yields
Figure imgf000066_0002
Reagents and Conditions i ethanol, 100 °C, 1-2 hours, or microwave irradiation, 100 °C, 10 minutes, 100 Watts
Scheme 3
Amidmoisothiourea compounds 10a-j and lla-b are synthesized according to Scheme 4
Figure imgf000066_0003
Reagents and Conditions i microwave 110 0C, 30-45 mm , ethanol
Scheme 4
Benzylisothiouronium deπvatives 4 beaπng an alkyl group at the benzyhc position may be prepared by the reaction of thiourea with the appropriate α-substituted benzyl halides The α-substituted benzyl halides may be prepared by addition of an alkylmagnesmm bromide to the appropπate benzaldehyde, followed by treatment of the intermediate alcohol with thionyl chloπde Substituted amidinoisothiourea compounds may be prepared by analogous methods
Figure imgf000067_0001
Reagents and Conditions i ethanol, 100 0C, 1-2 hours, or microwave irradiation, 100 0C, 10 minutes, 100 Watts, ii RCHbMgBr, tetrahydrofuran or diethyl ether, reflux, 1 hour, m Toluene, thionyl chloride, 100 0C, 2-10 hours
Scheme 5
Benzylguamdmium salts 6 may be obtained via the reaction between di-tert butoxycarbonyl thiourea and the appropnate benzylamme, (Yong 1997) followed by deprotection of the corresponding di-tert-butoxycarbonyl guamdme product with tm(rV) chloπde (Miel 1997) or tnfluoroacetic acid, (Guisado 2002)
Figure imgf000067_0002
Reagents and Conditions i Mukaiyama's reagent (l-methyl-2-chloropyridmium iodide), tπethylamme, dimethylformamide, room temperature, 20 minutes, ii CF3CO2H, dichloromethane, room temperature, overnight or SnCU, ethyl acetate, room temperature, overnight
Scheme 6
Typical Reaction Conditions for Synthesis of Compounds 3, 10 and 11.
A microwave reaction tube (2 mL) is charged with a mixture of ethanol (0 5-1 mL), the appropnate benzyl chloπde (1-2 mmol) and thiourea or guanylthiourea (1 molar eq ) The tube is capped and heated in a microwave reactor (Biotage Initiator I) at 110-1200C for 30-45 minutes The reactions are momtored by thm layer chromatography (ethyl acetate hexane, 1 4, v v) After the reaction is complete, the reaction mixture was concentrated under vacuum and the residue is washed with hexane The solid product is filtered and dried under high vacuum to give the product Typical Reaction Conditions for Synthesis of Compounds 3.
A lO milliliter (mL) microwave reaction tube is charged with the benzyl halide (1 0 milhmole, mmol) and thiourea (76 mg, 1 0 mmol) in ethanol (1 5 mL) The tube is capped and irradiated in the microwave reactor (single-mode CEM Discover™ system, CEM,
Matthews, NC) at 100 0C for 15 minutes The solid is filtered and solid washed with cold ethanol The solid product is dπed under high vacuum to give the product
The following compounds were prepared by the foregoing methods Example 1. (2,4-Dichlorophenyl)methyl Isothiourea Hydrochloride (3a).
Figure imgf000068_0001
White solid, mp 222-223 0C, 1H NMR (400 MHz, d6-DMSO) δ 4 58 (s, 2H), 7 47 (dd, J = 8 0 and 2 0 Hz, IH), 7 63 (d, /= 8 0 Hz, IH), 7 70 (d, J = 2 0 Hz, IH), 9 31 (br s, 2H), 9 39 (br s, 2H), 13C NMR (100 MHz, d6-DMSO) δ 32 6, 128 5, 130 0, 132 5, 133 3, 134 5, 135 1, 169 4, MS (ESI) m/z 235 0 (100%, [M + H]+), HRMS calcd for C8H9Cl2N2S 234 9858, observed 234 9854, HPLC analysis (Alltech Cl 8) 90% methanol, 10% acetomtnle, flow rate 0 5 mL/min fø 3 26 mm 90% acetomtnle, 10% water, flow rate 0 75 mL/min rR 2 05 min 100% methanol, flow rate 0 5 mL/mm fe 3 05 mm Example 2. (4-Chloro-2-nitrophenyl)methyl Isothiourea Hydrochloride (3u).
Figure imgf000068_0002
White solid, 44% 1H NMR (400 MHz, OUSO-d6) δ 4 72 (s, 2H), 7 75 (d, J = 8 4 Hz, IH), 7 90 (dd, J = 8 4, 2 2 Hz, IH), 8 22 (d, J = 2 2 Hz, IH), 9 22 (bs, 4H), HRMS calcd for C8H8ClN3O2S (M-Cl)+ 246 00985, found 246 01283 Example 3. 2-Chloro-4-fluorophenyl)methyI isothiourea Hydrochloride (3v).
Figure imgf000069_0001
White solid, 100%. 1H NMR (400 MHz, OMSO-d6) δ 4.57 (s), 7.28 (td, J = 8.4, 2.4 Hz, IH), 7.55 (dd, J = 8.6, 2.4 Hz, IH), 7.66 (dd, J = 8.4, 6.2 Hz, IH), 9.29 (bs, 4H); HRMS calcd. for C8H8ClFN2S (M-Cl)+ 219.01535, found 219.01549. Example 4. — (2,4-Difluorophenyl)methyl isothiourea Hydrochloride (3w).
Figure imgf000069_0002
White solid, 100%. 1H NMR (400 MHz, DMSO^) δ 4.55 (s, 2H), 7.14 (t, J = 8.1 Hz, IH), 7.34 (t, J= 9.8 Hz, IH), 7.60 (q, J= 7.9 Hz, IH), 9.30 (bs, 2H) 9.37 (bs, 2H); HRMS calcd. for C8H8ClN3O2S (M-
Example 5. (2-Chloro-4-fluorophenyl)methyl Amidinoisothiourea Hydrochloride (10a).
Figure imgf000069_0003
White solid, 75%; m.p. 154-156 0C; 1H NMR (400 MHz, DMSO-^) δ 4.28 (s, 2H), 7.20 (td, J= 8.5, 2.6 Hz, IH), 7.48 (dd, J= 8.8, 2.6 Hz, IH), 7.57 (dd, J =8.7, 6.24 Hz, IH), 8.00 (bs, 4H), 8.10(s, 2H); HRMS calcd. for C9HnClFN4S (M-Cl)+ 261.03715, found 261.03737.
Example 6. (2,4-Difluorophenyl)methyl Amidinoisothiourea Hydrochloride (10b).
Figure imgf000070_0001
White solid, 78%; m.p. 144-146 0C; 1H NMR (400 MHz, OUSO-d6) δ 4.22 (s, 2H), 7.06 (td, J = 8.6, 2.3 Hz, IH), 7.25 (td, J = 9.8, 2.4 Hz, IH), 7.51 (td, J = 8.6, 6.2 Hz, IH), 7.98 (bs, 4H), 8.09 (s, 2H); HRMS calcd. for C9H11F2N4S (M-Cl)+ 245.06670, found 245.06731.
Example 7. (2,4-Dichlorophenyl)methyI Amidinoisothiourea Hydrochloride (10c).
Figure imgf000070_0002
Whitfe solid, 74%; m.p. 139-142 °C 1H NMR (400 MHz, CD3OD) δ 4.34 (s, 2H), 7.30 (dd, J = 8.3, 2.1 Hz, IH), 7.47 (d, J = 2.1 Hz, IH), 7.50 (d, J = 8.3 Hz, IH), HRMS calcd. for C9H1 !Cl2N4S (M-Cl)+ 277.00760, found 277.00741.
Example 8. (2-Nitro-4-chlorophenyl)methyl Amidinoisothiourea Hydrochloride (1Od).
Figure imgf000070_0003
Off-white solid, 28%; m.p. 183-185 0C 1H NMR (400 MHz, CD3OD) δ 4.52 (s, 2H), 7.66- 7.72 (m, 2H), 8.06 (s, IH); HRMS calcd. for C9Hi0ClN5O2S (M-Cl)+ 288.03165, found 288.03168.
Example 9. (4-CyanophenyI)methyI Amidinoisothiourea Hydrochloride (1Oe).
Figure imgf000071_0001
White solid, 42% 1H NMR (400 MHz DMS(W15) δ 8 05 (bs, 4H), 7 78 (d, 2H, J = 8 2 Hz), 7 71 (bs, IH), 7 55 (d, 2H, J = S 1 Hz), 4 27 (s, 2H), HRMS calcd for Ci0H12N5S (M-Cl)+ 234 08079, found 23408155 Example 10. (2,5-Dichlorophenyl)methyl Amidinoisothiourea Hydrochloride (1Of). H
Figure imgf000071_0002
White solid, 47% 1H NMR (400 MHz DMSO-<4) δ 8 10 (bs, 6H), 7 56 (d, IH, J= 2 5 Hz), 7 50 (d, IH, 7 = 8 6 Hz), 7 40 (dd, IH, J = 2 6, 8 6 Hz), 427 (s, 2H), HRMS calcd for C9HnCl2N4S (M-Cl)+ 277 00760, found 277 00839
Example 11. (2-Chloro-6-fluorophenyl)methyl Amidinoisothiourea Hydrochloride
(1Og).
Figure imgf000071_0003
White solid, 57% 1H NMR (400 MHz DMSO-<4) δ 8 08 (bs, 4H), 7 85 (bs, 2H), 7 43-7 35 (m, 2H)1 7 28-723 (m, IH), 4 33 (s, 2H), HRMS calcd for C9Hi1ClFN4S (M-Cl)+ 261 03807, found 261 03801
Example 12. (6-Chlorobenzo[d] [l,3dioxoI-5-yl)methyI Amidinoisothiourea Hydrochloride (1Oh).
Figure imgf000072_0001
White solid, 67% 1H NMR (400 MHz, CD3OD) δ 6 97 (s, IH), 6 90 (s, IH), 5 98 (s, 2H), 4 28 (s, 2H), HRMS calcd for CIOHI2C1FN402S (M-Cl)+ 287 03640, found 287 04802 Example 13. (4-Chloro-3-fluorophenyl)methyl Amidinoisothiourea Hydrochloride (1Oi).
Figure imgf000072_0002
White solid, 45% 1H NMR (400 MHz DMSO-*) δ 8 07-7 89 (m, 6H), 7 51 (t, IH, J = 8 1 Hz), 7 39 (dd, IH, J = 1 8, 10 4 Hz), 7 22 (dd, IH, / = 1 8, 8 3 Hz), 4 21 (s, 2H), HRMS calcd for C9HnClN4S (M-Cl)+ 261 03715, found 261 03706
Example 14. (2,6-Difluorophenyl)methyl Amidinoisothiourea Hydrochloride (1Oj).
Figure imgf000072_0003
White solid, 53% 1H NMR (400 MHz, DMSO-rftf) δ 8 06 (s, 4H), 7 82 (s, 2H), 7 46-7 38 (m, IH), 7 12 (t, 2H, / = 8 1 Hz), 4 24 (s, 2H), HRMS calcd for C9Hi1F2NO2S (M-Cl)+ 245 06670, found 245 06687
Example 15. (2-Naphthyl)methyI Amidinoisothiourea hydrochloride (lla).
Figure imgf000073_0001
White solid, 80%, m p 175-177 0C 1H NMR (400 MHz, OMSO-d6) δ 4 39 (s, 2H), 7 49-7 52 (m, 3H), 7 85-7 89 (m, 4H), 8 08 (bs, IH), 9 37(bs, 3H, disappeared on D2O shake), HRMS calcd for C13Hi4BrN4S (M-Cl)+ 259 10119, found 259 10063 Example 16. 2-(l-Bromonaphthyl)methyI amidinoisothiourea hydrochloride (lib).
Figure imgf000073_0002
White solid, 62%, m p 160-162 0C 1H NMR (400 MHz, DMSO-4) δ 4 53 (s, 2H), 7 61(m, 2H), 7 67-7 71 (m, IH), 7 93 (bs, IH, disappeared on D2O shake), 7 94 (d, J = 8 5 Hz, IH), 7 98 (d, J= 8 1 Hz, IH), 8 13 (bs, 3H, disappeared on D2O shake), 8 20 (d, J= 8 5 Hz, IH), HRMS calcd for Ci3Hi4BrN4S (M-Cl)+ 337 01171, found 337 01251
Rb:Raf-l Binding Inhibition Activity For the Example Compounds
The compounds were screened for Rb Raf-1 binding inhibitory properties using a
GST-Rb/GST-Raf-1 ELISA assay The results are reported as inhibition of Rb Raf-1 binding at a concentration of 10 or 20 micromolar (μM, Tables 1-4) The compounds can be futher evaluated by generating a dose response for the most active compounds - those that inhibit the interaction by 80% or greater at 20 μM to generate an ICso value
The most active compounds tended to possess a monosustituted or disubstituted benzene ring, bearing at least one hahde in either one or both of the positions ortho, meta, or para to the carbon bound to the isothiouronmm group
Table 1. Structures, yields of compounds 3a-z, and inhibition of Rb Raf-1 binding
Figure imgf000074_0001
Figure imgf000074_0003
+ signifies 25-50% inhibition at 10 μM, ++ signifies 50-100% inhibition at 10 μM Table 2. Structures of compounds 10a-d, yields, and inhibition of Rb Raf-1 binding
Figure imgf000074_0002
Figure imgf000074_0004
+ signifies 25-50% inhibition at 10 μM, ** signifies 50-100% inhibition at 20 μM
Table 3. Structures of compounds lla-b, yields, and inhibition of Rb Raf-1 binding
Figure imgf000075_0001
11a-b
Figure imgf000075_0002
+ signifies 25-50% inhibition at 10 μM, -H- signifies 50-100% inhibition at 10 μM
Example 17: Modulators of Rb:Raf 1 interactions Disrupt Rb:Raf-l In Intact Cells. U937 cells were serum starved serum starved for 48 hours and subsequently serum stimulated for 2 hours in the presence or absence of 20 μM of the compounds Compounds 10b and 10c significantly inhibited the binding of Raf-1 to Rb, as seen by lmmunoprecipitation-Western blot analysis (FIG. IA) Raf-1 peptide conjugated to penetratm was used as a positive control Thus it appears that these two compounds were capable of disrupting the Rb Raf-1 interaction
Example 18: Compounds 10b & 10c Inhibited Epithelial Lung Cancer Cells.
Compounds 10b and 10c inhibited the proliferation of epithelial lung cancer cells To investigate whether compounds 10b and 10c require a functional Rb to inhibit tumor cell proliferation, A549 cells (human epithelial lung carcinoma) were stably transfected with two different shRNA constructs (sh6 and sh8) to knock down Rb expression (FIGs. IB and 1C) A549 cells stably expressing the Rb shRNAs had significantly less Rb protein compared to parental A549 cells Compounds 10b and 10c were very effective at inhibiting S-phase entry m parental A549 cells but had little or no effect on cells stably expressing sh6 and sh8, which lacked Rb This result confirms that compounds 10b and 10c arrest the proliferation of epithelial lung cancer cells in a Rb dependent manner
Example 19: Dose-Dependent Inhibition of Cancer Cells by 3w, 10a, 10b and 10c.
Compounds 3w, 10a, 10b and 10c inhibited the proliferation of epithelial lung cancer cells in a dose-dependent manner Similar to the preceding example, A549 cells (human epithelial lung carcinoma) were contacted with compounds 3w, 10a, 10b and 10c (FIG. ID) A BrdU incorporation assay at compound concentrations of 5, 10, 20, 30 and 50 μM shows dose-dependent inhibition of wild-type A549 cells by compounds 3w, 10a, 10b and 10c This result confirms that compounds 3w, 10a, 10b and 10c arrest the proliferation of epithelial lung cancer cells Example 20: Modulators of Rb:Raf 1 interactions Disrupt Angiogenesis.
An experiment was performed to determine whether angiogenic tubule formation could be inhibited by compounds 10b and 10c Human aortic endothelial cells (HAECs) were grown in matngel in the presence or absence of 20, 50 and 100 μM of 10b or 10c, or 100 μM of compound 3a It was found that while angiogenic tubules formed in control (no drug) wells, compounds 10b and 10c significantly inhibited angiogenic tubule formation in a dose dependent fashion, and showed inhibition comparable to that of compound 3a at 100 μM (FIG IE)
Example 21: Modulators of Rb:Raf 1 interactions 3a & 9a Significantly Inhibited Human Tumor Line in vivo. Experiments were performed to assess whether compounds 10b and 10c could inhibit human tumor growth in vivo using a nude mice xenograft model Athymic nude mice were implanted with IXlO7 A549 cells bilaterally and the tumors were allowed to reach 200mm3 in size before treatment began FIG IF shows that tumors from vehicle treated mice grew to an average size of over 1200 mm3. In contrast, tumors treated with compounds 10b and 10c at at 150 mg/kg were substantially inhibited
Example 22: Compound 10c Inhibited 7 Disparate Human Cancer Cell Lines
Compound 10c inhibited the proliferation of a wide range of cancer cells at 20 μM as shown in FIG. IG In a BrdU incorporation assay, compound 10c was contacted with a range of cancer cells including PANC-I (human pancreatic carcinoma, epithelial-hke), CAPAN-2 (human pancreatic ductal adenocarcinoma), Mel-5 (human malignant melanoma), MCF-7 (human breast adenocarcinoma), LNCAP (androgen-sensitive human prostate adenocarcinoma), A549 (human epithelial lung carcinoma), and PC-3 (human prostate adenocarcinoma), and compared to Rb-deficient cancer cells (A549 cells stably transfected with two different shRNA constructs (sh6 and sh8) to knock down Rb expression, and the Rb-deficient prostate cancer cell line DU145) This result confirms that compound 10c arrests the proliferation of a wide variety of cancer cells in a Rb dependent manner
Example 23: Compounds 3a, 10b and 10c Reduce the Viability of U937 Myeloid Cells
U937 myeloid cells were incubated in the absence of compound (control), or with compounds 3a, 10b, or 10c at lOμM, 20μM, or 50μM for 24 hours Cell viability was assessed by an MTT assay, a colorometπc assay which measures the number of cells by measuring the activity of enzymes that reduce 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazohum bromide The results are shown m Figure 2 A dose-dependent reduction m cell number was seen with each of the compounds, demonstrating that they reduce cell viability significantly
Example 24: Compounds 3a, 10b and 10c Reduce the Viability of Ramos Burkitt's Lymphoma Cells
Ramos cells (Burkitt's Lymphoma) were incubated in the absence of compound (control), or with compounds 3a, 10b, or 10c at lOμM, 20μM, or 50μM for 24 hours Cell viability was assessed by an MTT assay, a colorometπc assay which measures the number of cells by measuπng the activity of enzymes that reduce 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazohum bromide The results are shown m Figure 3 A dose-dependent reduction m cell number was seen with each of the compounds, demonstrating that they reduce cell viability significantly Example 25. Evidence that Inhibition of Cell Proliferation By Compounds of the Invention is Mediated by Raf-1
A549 cells lacking Raf-1 (sh-13B) were generated by stably transfectmg a shRNA to Raf-1 Control cells were generated by stably transfectmg A549 cells with a control shRNA The cells were incubated m the presence or absence of ccompounds 3a, 10b and 10c (20μM) and S-phase entry was assessed by measuring BrdU incorporation The results are shown in Figure 4 Relative to controls incubated in the absence of compound, proliferation of the cells with control shRNA (having Raf-1) was inhibited by each of the compounds In contrast, proliferation of the cells lacking Raf-1 (the cells transfected with Raf-mhibitory shRNA) was not inhibited by the compound This experiment provides evidence that inhibition of cell proliferation by compounds of the invention is mediated by Raf-1 as well as Rb and Raf-1
Example 26. Evidence that the Rb-E2F Pathway Regulates the Expression of Matrix Metalloproteinase (MMP) Genes Figure 9A hows a schematic of the promoters showing the E2F binding site on the genes for MMP2, MMP9 and MMP14 Using A549 cells transfected with an shRNA to inhibit expression of E2F1, QRT-PCR expeπments were perfomed to measure the expression of matrix metalloprotemases, MMP2, MMP9 and MMP 14 The results are shown in Figure 5 and show that when A549 cells are depleted of E2F1, the expression of MMP9 and MMP 14 is reduced This expeπment provides evidence that the Rb-E2F pathway can regulate the expression of matrix metalloprotemases (MMPs)
Example 27. Immunoprecipitation Assays Showing that Rb and E2F1 Associate with MMP Promoters
Figure 10 shows the results of chromatin immunoprecipitation assays showing the binding of E2F1 as well as the association of Rb with the promoters of matrix proteases Experiments were performed with respect to MMP9 (Figure 6A), MMP2 (Figure 6B), MMP14 (Figure 6C), and MMP15 (Figure 6D) This is as assay used to assess the binding of proteins to promoters in living cells These results provide evidence that E2F1 is associated with these promoters in the cells, regulating their expression Example 28. Evidence that Compounds of the Invention Inhibit Expression of Matrix Metalloprotemases MMP9, MMP14 and MMP15.
A quantitative real-time PCR experiment was performed to measure the effect of compounds 3a, 10b and 10c on the expression of MMP2, MMP9, MMP14 and MMP15 m MDAMB231 cells (breast cancer) The cells were incubated either in the absence of compound or in the presence of compound (50μM) for 24 hours The results are shown in Figures 7A (MMP2), 7B (MMP9), 7C (MMP 14) and 7D (MMP 15) Expression of MMP9, MMP14 and MMP15 was inhibited by each of the compounds These results provide evidence that the compounds of the invention are effective in controlling the expression of genes that are involved in metastasis Example 29. Evidence that Rb and E2F Associate with and Induce FLTl and KDR Promoters.
The data shown in Figure 12 promoters for VEGF receptors, FLTl and KDR, have
E2F binding sites, sshown chematically m Figure 8A Figures 8B-D show the results of chromatin immunoprecipitation assay performed using pπmary endothelial cells human aortic endothelial cells HAEC (Figure 8B), human umbilical cord vein endothelial cell
(HUVEC) (Figure 8C) and human microvascular endothelial cells from the lung (HMEC-L)
(Figure 8D) Treatment of the primary endothelial cells (human aortic endothelial cells, human umbilical cord vein endothelial cells or human microvascular endothelial cells from the lung) with VEGF induced the binding of E2F1 to the FLTl and KDR promoters This provides evidence that these promoters can be regulated by the Rb-E2F pathway and could possibly be targeted by the Rb-Raf-1 disruptors
The data shown m Figure 9 demonstrates transient transfection of E2F1 induces FLTl and KDR promoters and that Rb can repress these promoters The transfection assays were performed in both A549 and HUVEC cells
Example 30. Evidence that Compounds of the Invention Inhibit The Expression of FLTl and KDR.
A quantitative real-time PCR expeπment was performed to measure the effect of compounds 3a, 10b and 10c on the expression of FLTl and KDR in human aortic endothelial cells The cells were incubated either in the absence of compound or in the presence of compound (50μM) for 18 hours The results are shown in Figure 10 Each of the compounds inhibits expression of both FLT and KDR These results provide evidence that the compounds of the invention inhibit the expression of FLT and KDR
REFERENCES The entire teachings of each document cited herein, including each of the following, are incorporated by reference
Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh DA Role of Raf in vascular protection from distinct apoptotic stimuli Science 2003, 301(5629), 94-96
Arkin MR, Wells JA Small-molecule inhibitors of protein protem interactions progressing towards the dream Nat Rev DrugDiscov 2004, 3(4), 301-317 Bagchi S, Weinmann R, Raychaudhuπ P The retinoblastoma protein copuπfies with E2F-I, an El A-regulated inhibitor of the transcription factor E2F Cell 1991, 65(6) 1073-82
Beyersbergen RL, Bernards R Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins Biochim Biophys Acta 1996,1287(2-3) 103-20 Chau BN, Wang JY Coordinated regulation of life and death by RB Nat Rev Cancer
2003,3(2) 130-8
Chellappan S, Kraus VB, Kroger B, et al Adenovirus ElA, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product Proc Natl Acad Sa USA 1992,89 4549-53 Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevms JR The E2F transcription factor is a cellular target for the RB protem Cell 1991,65(6) 1053-61
Chittenden T, M LD1 Jr KWG RB associates with an E2F-hke, sequence-specific DNA-bmding protem Cold Spring Harb Symp Quant BwI 1991,56 187-95
Classon M, Dyson N plO7 and pl30 versatile proteins with interesting pockets Exp Cell Res 2001,264(1) 135-47
Classon M, Salama S, Gorka C, Mulloy R, Braun P, Harlow E Combinatorial roles for pRB, plO7, andpl30 in E2F-mediated cell cycle control Proc Natl Acad Sa USA 2000,97(20) 10820-5
Cobrmik D Pocket proteins and cell cycle control Oncogene 2005, 24(17), 2796-2809
Dasgupta P, Betts V, Rastogi S, et al Direct binding of apoptosis signal regulating kinase 1 to retinoblastoma protem novel links between apoptotic signaling and cell cycle machinery J Biol Chem 2004, 279(37), 38762-38769
Dasgupta P, Betts V, Rastogi S, et al Direct binding of apoptosis signal-regulating kinase 1 to retinoblastoma protem novel links between apoptotic signaling and cell cycle machinery J Biol Chem 2004,279(37) 38762-9 Dasgupta P, Chellappan S Nicotine-mediated cell proliferation and angiogenesis New twists to an old story Cell Cycle 2006, in press
Dasgupta, P, Rastogi, S, Pillai, S, Ordonez-Ercan, D, Morns, M et al Nicotine mduces cell proliferation by beta-arrestm-mediated activation of Src and Rb Raf-1 pathways J Clin Invest 2006, 116, 2208-2217 Dasgupta, P, Sun, JZ, Wang, S, Fusaro, G, Betts, V et al Disruption of the Rb Raf-1 interaction inhibits rumor growth and angiogenesis MoI Cell Biol 2004, 24, 9527-9541 de Brum A, Maiti B, Jakoi L, Timmers C, Buerki R, Leone G Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation J BwI Chem 2003,278(43) 42041-9
DeGregon J, Leone G, Miron A, Jakoi L, Nevms JR Distinct roles for E2F proteins in cell growth control and apoptosis Proc Natl Acad Sci USA 1997,94(14) 7245-50
DeGregon J, Leone G, Ohtani K, Miron A, Nevms JR E2F-1 accumulation bypasses a Gl arrest resulting from the inhibition of Gl cycltn-dependent kinase activity Genes Dev 1995,9(23) 2873-87
Derossi D, Chassamg G, Prochiantz A Trojan peptides the penetratin system for intracellular delivery Trends Cell Biol 1998,8(2) 84-7 Derossi, D, Joliot, AH, Chassamg, G, Prochiantz, A The 3rd Helix of the Antennapedia
Homeodomam Translocates through Biological-Membranes J Biol Chem 1994, 269, 10444-10450
Di Stefano L, Jensen MR, Helm K E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes Embo /2003,22(23) 6289-98
Dyson N, Gmda P, McCaIl C, E H Adenovirus ElA makes two distinct contacts with the retinoblastoma protein J Virol 1992,66(7) 4606-11
Dyson N, Howley PM, Munger K, Harlow E The human papilloma virus-16 E7 oncoprotein is able to bind to the retmoblastoma gene product Science 1989,243(4893) 934-7
Guisado, O, Martinez, S, Pastor, J A novel, facile methodology for the synthesis of N,N '-bis(tert-butoxycarbonyl)-protected guanidines using polymer-supported carbodiimide Tetrahedron Lett 2002, 43, 7105-7109
Hakem R, Mak TW Animal models of tumor-suppressor genes Ann Rev Genet 2001,35 209-41
Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through Gl CeK 1999, 98(6), 859-869 Harbour, JW, Dean, DC Rb function in cell-cycle regulation and apoptosis Nat Cell BwI 2000,
2, E65-E67
Harbour, JW, Dean, DC The Rb/E2F pathway expanding roles and emerging paradigms Genes Dev 2000, 14, 2393-2409
Hood JD, Cheresh DA Targeted delivery of mutant Raf kinase to neovessels causes tumor regression Cold Spring Harbor Symp on Quant Biol 2002, 67, 285-291
Ishida S, Huang E, Zuzan H, et al Role for E2F m control of both DNA replication and mitotic functions as revealed from DNA microarray analysis MoI Cell Biol 2001,21(14) 4684-99 Johnson DG, Schneider Broussard R Role of E2F in cell cycle control and cancer Front Biosci 1998,3 d447-8
Johnson DG, Schwarz, J K , Cress, W D and Nevins, J R Expression of transcription factor E2F1 induces quiescent cells to enter S phase Nature 1993,365 349-52 Kasid, U Raf-1 protein kinase, signal transduction, and targeted intervention of radiation response Experimental Biology and Medicine 2001, 226, 624-625
Kato J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D dependent kinase CDK4 Genes Dev 1993,7 331-42 Knudsen ES, Wang JY Differential regulation of retinoblastoma protein function by specific Cdk phosphorylation sites J Biol Chem 1996, 271(14), 8313-8320
Knudsen ES, Wang JY Dual mechanisms for the inhibition of E2F binding to RB by cyclin- dependent kinase-mediated RB phosphorylation MoI Cell 2?ω/ 1997, 17(10), 5771-5783
Lam EW, La TN DP and E2F proteins coordinating transcription with cell cycle progression Curr Opm Cell Biol 1994,6(6) 859-66
Lee JO, Russo AA, Pavletich NP Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7 Nature 1998,391(6670) 859-65
Macleod K Tumor suppressor genes Curr Opin Genet Dev 2000,10(1) 81-93
Miel, H, Rault, S Total deprotection of N,N'-bis(tert-butoxycarbonyl)guanidmes using SnCW Tetrahedron Lett 1997, 38, 7865 7866
Morns EJ, Dyson NJ Retinoblastoma protein partners Adv Cancer Res 2001,82 1-54
Muller H, Bracken AP, Vernell R, et al E2Fs regulate the expression of genes involved m differentiation, development, proliferation, and apoptosis Genes Dev 2001, 15(3) 267-85
Muller H, Helm K The E2F transcription factors key regulators of cell proliferation Biochim Bwphys Acta 2000,1470(1) Ml M12
Nevins JR Cell cycle targets of the DNA tumor viruses Curr Opm Genet Devel 1994, 4(1), 130-134
Nevms JR Disruption of cell cycle control by viral oncoproteins Biochemical Society Transactions 1993,21(4) 935-8 Nevins JR E2F a link between the Rb tumor suppressor protein and viral oncoproteins Science
1992, 258(5081), 424 429
Nevins, JR The Rb/E2F pathway and cancer Hum MoI Genet 2001, 10, 699-703
Norbury C, Nurse P Animal cell cycles and their control Ann Rev Biochem 1992,61 441-70 Paggi, MG, Baldi, A, Bonetto, F, Giordano, A Retinoblastoma protein family in cell cycle and cancer A review / Cell Biochem 1996, 62, 418-430
Pπves C, Hall PA The p53 pathway J Pathol 1999,187(1) 112-26
Reddy GP Cell cycle regulatory events in G1->S transition of mammalian cells / Cell Biochem 1994, 54(4), 379-386
Reed SI Control of the Gl/S transition Cancer Surveys 1997, 29, 7 23
Ren B, Cam H, Takahashi Y, et al E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints Genes Dev 2002,16(2) 245-56
Rim, BI Sorafenib Expert Opinion on Pharmacotherapy 2006, 7, 453-461 Rudrn, CM, HolmLund, J, Fleming, GF, Mam, S, Stadler, WM et al Phase I trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1 , administered by 24-hour weekly infusion to patients with advanced cancer Clin Cancer Res 2001, 7, 1214-1220
Sager R Tumor suppressor genes in the cell cycle Curr Opin Cell Biol 1992,4155-160
Sager R Tumor suppressor genes The puzzle and the promise Science 1989,246 1406 12 Sherr CJ, McCormick F The RB and p53 pathways in cancer Cancer Cell 2002,2(2) 103-12
Sherr CJ, Roberts JM Inhibitors of mammalian Gl cyclin-dependent kinases Genes Dev 1995,9(10) 1149-63
Sherr CJ Cell cycle control and cancer Harvey Lect 2000,96 73-92
Sherr CJ Mammalian Gl cyclins and cell cycle progression Proc Assoc Am Physicians 1995,107(2) 181-6
Sherr CJ The ms and outs of RB Coupling gene expression to the cell cycle clock Trends Cell
Figure imgf000083_0001
15-18
Sherr CJ The ms and outs of RB Coupling gene expression to the cell cycle clock Trends Cell BwI 1994,4 15-8 Slansky JE, Farnham PJ Introduction to the E2F family protein structure and gene regulation
Curr Top Microbiol Immunol 1996,208(1) 1-30
Stiegler P, Kasten M, Giordano A The RB family of cell cycle regulatory factors J Cell Biochem Suppl 1998,31 30-6
Takahashi Y, Rayman JB, Dynlacht BD Analysis of promoter binding by the E2F and pRb families in vivo distinct E2F proteins mediate activation and repression Genes Dev 2000,14 804-16
Taya Y, Yasuda H, Kamyo M, et al In vitro phosphorylation of the tumor suppressor gene RBprotem by mitosis-specific histone Hl kinase Biochem Biophys Res Commun 1989,164(1) 580-6 Taya Y RB kinases and RB-bmdmg proteins new points of view Trends Biochem Sa X997,22(l) 14-7
Taylor WR, Stark GR Regulation of the G2/M transition by p53 Oncogene 2001,20(I5) 1803- I5 Tonini T, Hillson C, Claudio PP Interview with the retinoblastoma family members do they help each other? J Cell Physiol 2002, 192(2) 138-50
Trraiarchi JM, Lees JA Transcription Sibling rivalry in the E2F family Nat Rev MoI Cell Biol 2002,3(1) 11-20
Wang S, Ghosh R, Chellappan S Raf-1 physically interacts with Rb and regulates its function A link between mitogemc signaling and cell cycle regulation MoI Cell Biol 1998,18(12) 7487-98
Wang S, Nath N, Minden A, Chellappan S Regulation of Rb and E2F by signal transduction cascades divergent effects of JNKl and ρ38 kinases Embo J 1999, 18(6) 1559 70
Wang, S, Ghosh, RN, Chellappan, SP Raf-1 physically interacts with Rb and regulates its function a link between mitogemc signaling and cell cycle regulation MoI Cell Biol 1998, 18, 7487-7498
Weinberg RA E2F and cell proliferation a world turned upside down CeW 1996,85(4) 457-9
Weinberg, RA The Retinoblastoma Protem and Cell-Cycle Control Cell 1995, 81, 323-330
Welch PJ, Wang JY Disruption of retmoblastoma protein function by coexpression of its C pocket fragment Genes Dev 1995,9(1) 31-46 Welch PJ, Wang JYJ A C terminal protem-bindmg domain m the retmoblastoma protein regulates nuclear c-Abl tyrosine in the cell cycle Cell 1993,75 779-90
Wilhelm S, Carter C, Lynch M, et al Discovery and development of sorafenib a multikinase inhibitor for treating cancer Nat Rev DrugDiscov 2006, 5(10), 8358-8344
Wilhelm, SM, Carter, C, Tang, LY, Wilkie, D, McNabola, A et al BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis Cancer Res 2004, 64, 7099-7109
Ym H, Hamilton AD Strategies for targeting protem protem interactions with synthetic agents Angew Chem Int Ed Engl 2005, 44(27), 4130-4163
Yong, YF, Kowalski, JA, Lipton, MA Facile and efficient guanylation of amines using thioureas and Mukaiyama's reagent J Org Chem 1997, 62, 1540-1542

Claims

WHAT IS CLAIMED IS:
1 A compound according to formula (I)
Figure imgf000085_0001
or a salt thereof, wherein
Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl,
Y is optionally substituted methylene,
X1 is -O-, -S-, or optionally substituted -NH-,
X3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene,
X2 is S or optionally substituted NH,
X4 is S or optionally substituted NH, or X2 and X4 are both N and are linked together through an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl ring,
X is an optionally substituted — NH2 or 3-7 membered heteroaryl or heterocyclyl ring, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R8, -OC(O)Ra, -C(O)ORa, -SRa, -C(S)Ra, -OC(S)R", -C(S)ORa, -C(O)SRa, -C(S)SRa, -S(O)Ra, -SO2Ra, -SO3R", -OSO2R3, -OSO3Ra, -PO2RaRb, -OPO2RaRb, -PO3RaRb, -0P03RaRb, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2Rc, -C(O)NRaSO2Rc, -C(O)NR3CN, -S02N(RaRb), -NR3SO2R", -NR0C(O)R3, -NRcC(0)0Ra, -NRcC(0)N(RaRb), -C(NRc)-N(RaRb),
-NRd-C(NRc)-N(RaRb), -NR3N(R3R"), -CRc=CR3Rb, -C≡CRa, =O, =S, =CRaRb, =NRa, =NORa, or =NNRa, or two optionally substitutable carbons are linked with C1.3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -ORa, -C(O)Ra, -C(O)Ra-aryl, -OC(O)R3, -C(O)ORa, -SRa, -S(O)Ra, -SO2R3, -SO3R3, -N(RaRb), -C(O)N(RaRb), -C(O)NRaNRbSO2R°, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R3, -NR0C(O)OR3, -NR°C(0)N(RaRb), or oxygen to form an N-oxide, and is optionally protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a countenon, and wherein each of R3, Rb, R° and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or m any occurrence of -N(RaRb), R3 and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group with the proviso that when X1 is NH, X2 is NH, X3 is NH, X4 is NH, X5 is NH2, and Y is CH2, then πng A is other than 2-tπfiuoromethylphenyl, 3-methoxyphenyl, 3- mtrophenyl, 3-trifluoromethylphenyl, 3-vinylphenyl, 4-t-butylphenyl, 4-chlorophenyl, 4- fluorophenyl, 4-methoxyphenyl, 4-methylphenyl, 4-nitrophenyl, 4-tnfluoromethylphenyl, 4-vmylphenyl, 3,4-dichlorophenyl, 3,5-ditπfiuoromethylphenyl, and 2-hydroxy-5- mtrophenyl A compound according to claim 1, or a salt thereof, wherein Group A is substituted phenyl or optionally substituted naphthyl or pyndyl A compound according to claim 1 or 2, or a salt thereof, wherein in Group A, an unsubstituted πng atom is adjacent to the πng atom attached to Y A compound according to any one of claims 1, 2, or 3, or a salt thereof, wherem Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, C] 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic A compound according to any one of claims 1, 2, or 3, or a salt thereof, wherein Y is C(O), or methylene optionally substituted with hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, or Ci 6 alkyl substituted with aryl A compound according to any one of claims 1, 2, or 3, or a salt thereof, wherein Y is methylene optionally substituted with hydroxyl, Ci e alkyl, Ci β alkoxy, or Ci β alkyl substituted with aryl
A compound according to any one of claims 1, 2, or 3, or a salt thereof, wherein Y is methylene optionally substituted with Ci 3 alkyl
A compound according to any one of claims 1, 2, or 3, or a salt thereof, wherein Y is methylene
A compound according to any one of claims 1 to 8, or a salt thereof, wherein the compound is represented by the following structural formula (Ia)
Figure imgf000087_0001
or a salt thereof, wherein
RL is hydrogen, hydroxyl, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, Ci-6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic,
R2 is hydrogen, hydroxyl, C1.6 alkyl, Ci_6 alkoxy, C1.6 haloalkyl, Ci^ haloalkoxy, Ci-6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic,
R3 is hydrogen, hydroxyl, Ci-6 alkyl, Ci_e alkoxy, Ci-6 haloalkyl, C1.6 haloalkoxy, Cue alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic,
R4 is hydrogen, hydroxyl, C1.6 alkyl, Q.6 alkoxy, Ci g haloalkyl, Ci β haloalkoxy, Ci-6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, and
R5 is hydrogen, hydroxyl, Ci 5 alkyl, Ci $ alkoxy, Ci 6 haloalkyl, Ci s haloalkoxy, Ci-6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic A compound according to claim 9, or a salt thereof, wherein
R1 is hydrogen, hydroxyl,
Figure imgf000087_0002
alkyl, Ci_6 alkoxy, Ci_6 haloalkyl, Q.s haloalkoxy, or Ci-6 alkyl substituted with aryl, R2 is hydrogen, hydroxyl, Ci β alkyl, Ci 6 alkoxy, Ci β haloalkyl, Ci 6 haloalkoxy, or Ci 6 alkyl substituted with aryl,
R3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci-g alkoxy, Ci β haloalkyl, Ci g haloalkoxy, or Ci_5 alkyl substituted with aryl,
R4 is hydrogen, hydroxyl, Ci 6 alkyl, Ci s alkoxy, Ci e haloalkyl, Ci 6 haloalkoxy, or Ci β alkyl substituted with aryl, and
R5 is hydrogen, hydroxyl, Ci s alkyl, Ci β alkoxy, Ci β haloalkyl, Ci β haloalkoxy, or Ci 6 alkyl substituted with aryl A compound according to claim 9, or a salt thereof, wherein
R1 is hydrogen, hydroxyl, Ci e alkyl, Ci β alkoxy, or Ci ^ alkyl substituted with aryl,
R2 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci β alkyl substituted with aryl,
R3 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci_6 alkyl substituted with aryl,
R4 is hydrogen, hydroxyl, Ci β alkyl, Ci & alkoxy, or Ci_6 alkyl substituted with aryl, and
R5 is hydrogen, hydroxyl, Ci β alkyl, Ci_6 alkoxy, or Ci_6 alkyl substituted with aryl A compound according to claim 9, or a salt thereof, wherein
R1 is hydrogen or C1.3 alkyl, R2 is hydrogen or Cj 3 alkyl, R3 is hydrogen or C1 3 alkyl, R4 is hydrogen or C1.3 alkyl, and R5 is hydrogen or Ci_3 alkyl A compound according to claim 9, or a salt thereof, wherein
R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R4 is hydrogen, and R5 is hydrogen A compound according to any one of claims 9 to 13, or a salt thereof, wherein A is substituted phenyl A compound according to claim 14, or a salt thereof, wherein
Y is methylene, R1 is hydrogen, R2 is hydrogen, R is hydrogen,
R4 is hydrogen, and R5 is hydrogen A compound according to any one of claims 9 to 13, or a salt thereof, wherein A is optionally substituted naphthyl A compound according to claims 16, or a salt thereof, wherein
Y is methylene, R1 is hydrogen, R2 is hydrogen, R3 is hydrogen,
R4 is hydrogen, and R5 is hydrogen A compound according to claim 16 or 17, or a salt thereof, wherein A is optionally substituted 1 -naphthyl A compound according to claim 16 or 17, or a salt thereof, wherein A is optionally substituted 2-naphthyl A compound according to any one of claims 1 to 19, or a salt thereof, wherein one or more substitutable carbons in Group A is substituted with a substitutent selected from -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R3, -OC(O)Ra, -C(O)OR3, -SR3, -SO2R3, -SO3R", -OSO2R3, -OSO3Ra, -N(R3R"), -C(0)N(RaRb), -C(0)NRaNRbS02Rc, -C(O)NRaSO2Rc, -SO2N(RaRb), -NRaS02Rb, -NRcC(0)Ra, and -NR0C(O)OR3, or two substitutable carbons are linked with Ci 3 alkylenedioxy
A compound according to any one of claims 1 to 19, or a salt thereof, wherein one, two or three substitutable carbons m Group A are substituted with a substituent independently selected from -F, -Cl, -Br, -I, -CN, -NO2, d.6 alkyl, Ci.6 alkoxy, -CF3, and Ci 6 haloalkoxy, or two substitutable carbons are linked with Ci_2 alkylenedioxy
A compound according to claim 21 wherein Group A is phenyl, wherein one, two or three substitutable carbons of the phenyl are substituted with a substituent independently selected from -F, -Cl, -Br, -I, -CN, -NO2, C1 6 alkyl, C1 6 alkoxy, -CF3, and C1^ haloalkoxy, or two substitutable carbons are linked with C1^ alkylenedioxy
A compound according to claim 22, or a salt thereof, wherein the compound is selected from the following compounds
Figure imgf000090_0001
and salts thereof A compound according to any one of claims 1 to 15 or 20 to 22, or a salt thereof, wherein Group A is phenyl unsubstituted at its 6-position A compound according to any one of claims 1 to 15, or 20 to 22, or a salt thereof, wherein Group A is 2,4-substituted phenyl A compound according to any one of claims 1 to 15 or 20 to 22, or a salt thereof, wherein Group A is phenyl monosubstituted at its 2, 3, or 4 positions or independently disubstituted at its 2,3, 2,4, 2,5 or 3,4 positions with -F, -Cl, -Br, -NO2, Ci 6 alkyl, or -CF3 A compound according to any one of claims 1 to 15 or 20 to 22, or a salt thereof, wherein Group A is phenyl independently disubstituted at its 2,3, 2,4, 3,4, or 2,5 positions with -NO2, -Cl, -F or -CF3 A compound according to any one of claims 1 to 15 or 20 to 22, or a salt thereof, wherein Group A is phenyl monosubstituted at its 2, 3, or 4 position with -NO2, -Cl or -F A compound according to any one of claims 1 to 15 or 20 to 22, or a salt thereof, wherein Group A is phenyl independently disubstituted at its 2,4 positions with -NO2, -Cl or -F A compound according to claim 29, or a salt thereof, wherein the compound is selected from the following compounds
Figure imgf000091_0001
and salts thereof A compound according to claim 29, or a salt thereof, wherein the compound is the following compound, H2
Figure imgf000091_0002
or a salt thereof A compound according to any one of claims 1 to 13 or 16 to 19, or a salt thereof, wherein Group A is unsubstituted 2- naphthyl or 1 -substituted 2- naphthyl A compound according to any one of claims 1 to 13 or 16 to 19, or a salt thereof, wherein Group A is naphthyl optionally substituted with one or more of -F, -Cl, -Br, -NO2, Ci 6 alkyl, or -CF3 A compound according to any one of claims 1 to 13 or 16 to 19, or a salt thereof, wherein Group A is naphthyl optionally monosubstituted with -F, -Cl, -Br, -NO2, or -CF3 A compound according to any one of claims 1 to 13 or 16 to 19, or a salt thereof, wherein Group A is naphthyl optionally monosubstituted with -F, -Cl, or -Br A compound according to claim 34, or a salt thereof, wherein the compound is selected from the following compounds
Figure imgf000092_0001
and salts thereof A compound according to formula (II)
Figure imgf000092_0002
or a salt thereof, wherein
Y is optionally substituted methylene,
X1 is -O-, -S-, or optionally substituted -NH-,
X2 is S or optionally substituted NH, and
R6 and R7 are independently -F, -Cl, -Br, -I, -NO2, -CN, -CF3, or C1-C6 alkoxy, provided that R6 and R7 are not both -Cl and R6 and R7 are not both -CF3, with the further proviso that when Y is -CH2-, X1 is S and X2 is NH, then R6 and R7 are not both -F, R6 and R7 are not both -Br, R6 and R7 are not both -I, R6 and R7 are not both -NO2, and R6 and R7 are not both -CH3, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)Ra, -OC(O)R3, -C(O)OR3, -SRa, -C(S)Ra, -OC(S)Ra, -C(S)OR3, -C(O)SR3, -C(S)SR3, -S(O)R3, -SO2R3, -SO3Ra, -OSO2R3, -OSO3R3, -P02RaRb, -OPO2R3R11, -PO3R3Rb, -OPO3RaRb, -N(RaRb), -C(O)N(RaRb), -C(0)NRaNRbS02Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R", -NR0C(O)OR0, -NR°C(0)N(RaRb), -C(NR°)-N(R3Rb),
-NRd-C(NR°)-N(RaRb), -NR3N(R3R5), -CRc-CR3Rb, -C≡CR3, =O, =S, =CRaRb, =NRa, =N0Ra, or =NNRa, or two optionally substitutable carbons are linked with Ci 3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -0Ra, -C(O)R3, -C(0)R3-aryl, -OC(O)Ra, -C(0)0Ra, -SR3, -S(O)R3, -SO2R3, -SO3R3, -N(RaRb), -C(0)N(RaRb), -C(0)NRaNRbS02Rc, -C(O)NR3SO2R0, -C(O)NR3CN, -SO2N(RaRb), -NR3SO2Rb, -NR0C(O)R3, -NR0C(O)OR3, -NR°C(O)N(RaRb), or oxygen to form an N-oxide, and optionally is protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable counteπon, and wherein each of Ra, Rb, R° and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or in any occurrence of -N(R3Rb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group A compound according to claim 37, or a salt thereof, wherein R6 and R7 are not both F, R6 and R7 are not both -Br, R6 and R7 are not both -I, R6 and R7 are not both -NO2, and R6 and R7 are not both -CH3 A compound according to claim 37 or 38, or a salt thereof, wherein Y is C(O), C(S), or methylene optionally substituted with hydroxyl, Ci β alkyl, Ci β alkoxy, Ci β haloalkyl, Ci e haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic A compound according to claim 37 or 38, or a salt thereof, wherein Y is methylene optionally substituted with hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci β alkyl substituted with aryl A compound according to claim 37 or 38, or a salt thereof, wherein Y is methylene optionally substituted with Ci 3 alkyl A compound according to claim 37 or 38, or a salt thereof, wherein Y is methylene A compound according to any one of claims 37 to 42, or a salt thereof, wherein the compound is represented by the following structural formula
Figure imgf000094_0001
or a salt thereof, wherein R8 is hydrogen, hydroxyl, Ci s alkyl, Ci (, alkoxy, Ci β haloalkyl, Ci s haloalkoxy, Ci ^ alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic A compound according to claim 43, or a salt thereof, wherein R8 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, or Ci 6 alkyl substituted with aryl A compound according to claim 43, or a salt thereof, wherein R8 is hydrogen or Ci 3 alkyl A compound according to claim 43, or a salt thereof, wherein R8 is hydrogen A compound according to any one of claims 43 to 46, or a salt thereof, wherein R6 and R7 are independently -F, -Cl, -Br, -NO2, or -CF3 A compound according to claim 47, or a salt thereof, wherein the compound is selected from the group consisting of
Figure imgf000095_0001
and salts thereof A pharmaceutical composition composing a compound of any one of claims 1-48, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable earner A method of treating or ameliorating a cell proliferation disorder, comprising administering to a subject in need of such treatment an effective amount of a compound according to formula (I)
Figure imgf000095_0002
or a pharmaceutically acceptable salt thereof, wherein
Group A is substituted phenyl, optionally substituted 6-membered heteroaryl, or optionally substituted fused bicyclic 9-10 membered aryl or heteroaryl,
Y is optionally substituted methylene,
X1 is -O-, -S-, or optionally substituted -NH-,
X3 is -O-, -S-, optionally substituted -NH- or optionally substituted methylene,
X2 is S or optionally substituted NH,
X4 is S or optionally substituted NH, or X2 and X4 are both N and are linked together through an optionally substituted alkyl, alkenyl, heteroalkyl, or heteroalkenyl linking group, thereby forming an optionally substituted 5-7 membered heteroaryl or heterocyclyl πng,
X5 is an optionally substituted -NH2 or 3-7 membered heteroaryl or heterocyclyl πng, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(O)R", -OC(O)Ra, -C(O)ORa, -SRa, -C(S)Ra, -OC(S)R", -C(S)OR8, -C(O)SR", -C(S)SR", -S(O)Ra, -SO2R8, -SO3R", -OSO2R3, -OSO3R", -PO2R"Rb, -OPO2R"Rb, -PO3RaRb, -OPO3R"Rb, -N(RaRb), -C(0)N(RaRb), -C(0)NRaNRbS02Rc, -C(O)NR1SO2R0, -C(O)NRaCN, -S02N(RaRb), -NRaS02Rb, -NRcC(0)Ra, -NR0C(O)OR", -NR°C(O)N(RaRb), -C(NRc)-N(RaRb),
-NRd-C(NRc)-N(RaRb), -NRaN(RaRb), -CR°=CRaRb, -C≡CRa, =O, =S, =CR"Rb, =NRa, =NORa, or =NNR", or two optionally substitutable carbons are linked with Ci 3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -0Ra, -C(O)R", -C(O)R"-aryl, -OC(O)R", -C(O)OR", -SRa, -S(O)Ra, -SO2R3, -SO3Ra, -N(R"Rb), -C(O)N(RaRb), -C(O)NR"NRbSO2R°, -C(O)NR8SO2R0, -C(O)NR2CN, -S02N(RaRb), -NRaSO2Rb, -NR0C(O)R3, -NR0C(O)OR", -NR°C(O)N(RaRb), or oxygen to form an N-oxide, and is optionally protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a counteπon, and wherein each of Ra, Rb, Rc and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cyclo aliphatic, or in any occurrence of -N(R"Rb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group A a method according to claim 50, wherein the compound is represented by the following structural formula (Ia)
Figure imgf000096_0001
or a pharmaceutically acceptable salt thereof, wherein
R1 is hydrogen, hydroxyl, C1-5 alkyl, C1 6 alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloaliphatic, R2 is hydrogen, hydroxyl, Ci β alkyl, Ci g alkoxy, Ci 6 haloalkyl, Ci 6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic,
R3 is hydrogen, hydroxyl, Ci β alkyl, Ci β alkoxy, Ci 6 haloalkyl, Ci β haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic,
R4 is hydrogen, hydroxyl, Ci g alkyl, Ci g alkoxy, Ci 5 haloalkyl, Ci 5 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, and
R5 is hydrogen, hydroxyl, Ci 6 alkyl, Ci 6 alkoxy, Ci β haloalkyl, Ci 6 haloalkoxy, Ci 6 alkyl substituted with aryl, aryl, heteroaryl, heterocyclyl, or cycloahphatic A method according to claim 51 wherein
Y is CH2, R1 is hydrogen, R2 is hydrogen, R3 is hydrogen, R is hydrogen, and R5 is hydrogen A method according to any one of claims 50 to 52, wherein A is substituted phenyl A method of treating or ameliorating a cell proliferation disorder, comprising administering to a subject in need of such treatment an effective amount of a compound according to formula (II)
Figure imgf000097_0001
or a pharmaceutically acceptable salt thereof, wherein Y is optionally substituted methylene, X1 is -O-, -S-, or optionally substituted -NH-, X2 is S or optionally substituted NH, and R6 and R7 are independently -F, -Cl, -Br, -I, -NO2, -CN, -CF3, or C1-C6 alkoxy, provided that R6 and R7 are not both -Cl and R6 and R7 are not both -CF3, wherein each optionally substitutable carbon is optionally substituted with -F, -Cl, -Br, -I, -CN, -NO2, -Ra, -ORa, -C(0)Ra, -OC(O)R", -C(O)ORa, -SRa, -C(S)Ra, -OC(S)R3, -C(S)ORa, -C(O)SRa, -C(S)SR", -S(O)Ra, -SO2Ra, -SO3R3, -OSO2R", -OSO3R3, -PO2RaRb, -OPO2R"Rb, -PO3RaRb, -OPO3RaRb, -N(RaRb), -C(0)N(RaRb), -C(O)NR"NRbSO2Rc, -C(O)NRaSO2Rc, -C(0)NRaCN, -SO2N(RaRb), -NRaSO2Rb, -NR0C(O)R3, -NR0C(O)OR3, -NR°C(0)N(RaRb), -C(NRc)-N(RaRb),
-NRd-C(NR°)-N(RaRb), -NRaN(R3Rb), -CR°=CRaRb, -C≡CR3, =O, =S, "CRaRb, =NRa, =N0R3, or =NNRa, or two optionally substitutable carbons are linked with Ci 3 alkylenedioxy, each optionally substitutable nitrogen is optionally substituted with -CN, -NO2, -Ra, -0Ra, -C(O)R3, -C(O)Ra-aryl, -OC(O)R3, -C(O)ORa, -SRa, -S(O)R", -SO2R3, -SO3R", -N(RaRb), -C(0)N(RaRb), -C(O)NR3NRbSO2R°, -C(O)NR3SO2R0, -C(O)NR1CN, -SO2N(RaRb), -NR3SO2R", -NR0C(O)R3, -NR0C(O)OR3, -NR°C(O)N(RaRb), or oxygen to form an N-oxide, and optionally is protonated or quaternary substituted with a nitrogen substituent, thereby carrying a positive charge which is balanced by a pharmaceutically acceptable counteπon, and wherein each of Ra, Rb, Rc and Rd is independently -H, alkyl, haloalkyl, aralkyl, aryl, heteroaryl, heterocyclyl, or cycloahphatic, or in any occurrence of -N(RaRb), Ra and Rb taken together with the nitrogen to which they are attached optionally form an optionally substituted heterocyclic group A method according to claim 53 wherein Y is CH2, X1 is S and X2 is NH A method of treating or ameliorating a cell proliferation disorder, comprising administering to a subject in need of such treatement an effective amount of a compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof A method according to any one of claims 50 to 56, wherein the cell proliferation disorder is a cancer A method according to claim 57 wherein the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcmoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pmealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, lymphocytic leukemia, large granular lymphocytic leukemia, acute myelocytic leukemia, chrome leukemia, polycythemia vera, Hodgkm's lymphoma, non-Hodgkm's lymphoma, multiple myeloma, Waldenstrobm's macroglobulmemia, heavy chain disease, lymphoblastic leukemia, T-cell leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cell leukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloid leukemias, myelocytic leukemia, myelogenous leukemia, neutrophilic leukemia, eosinophilic leukemia, monocytic leukemia, myelomonocytic leukemia, Naegeli-type myeloid leukemia, nonlymphocytic leukemia, osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, and prostate cancer A method according claim 57, wherein the cancer is selected from the group consisting of osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, and prostate cancer A method according claims 58 or 59, further comprising administering an anticancer drug to the subject A method according to any one of claims 50 to 56, wherein the cell proliferation disorder is angiogenesis or the cell proliferation disorder is mediated by angiogenesis A method according to any one of claims 50 to 61, wherein the proliferating cells of the cell proliferation disorder have an elevated level of Rb, Raf-1, or Rb bound to Raf-1 A method according to any one of claims 50 to 62, wherein the regulation of proliferation in the proliferating cells of the cell proliferation disorder is mediated by at least one protein selected from the group consisting of retinoblastoma tumor suppressor protein and seπne- threonine kinase Raf-1 A method of inhibiting proliferation of a cell, comprising contacting the cell with an effective amount of a compound according to any one of claims 1 to 48, or a salt thereof A method according to claim 64, wherein the regulation of proliferation in the cell is mediated by at least one protein selected from the group consisting of retinoblastoma tumor suppressor protein and serine-threonine kinase Raf-1 A method of modulating Rb Raf-1 binding m a proliferating cell, composing contacting the cell with an effective amount of a compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof A method according to any one of claims 64 to 66, wherein the cell is in a subject A method according to claim 50, wherein the compound is orally administered A method of treating or ameliorating a cell proliferation disorder, comprising contacting proliferating cells with an effective amount of a compound according to any one of claims 1 to 48, or a salt thereof A method according to claim 69, wherein the regulation of proliferation in the cells is mediated by at least one protein selected from the group consisting of retinoblastoma tumor suppressor protein and seπne-threomne kinase Raf-1 A method according to any one of claims 69 or 70, wherein the regulation of proliferation in the cells is mediated by the interaction between retinoblastoma tumor suppressor protein and serme-threonine kinase Raf-1 A method according to claim 69 to 71, wherein the cells have an elevated level of Rb, Raf-1 , or Rb bound to Raf-1 A method according to any one of claims 69 to 72, wherein the cell proliferation disorder is a cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, hposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choπocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pmealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, lymphocytic leukemia, large granular lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrobm's macroglobulmemia, heavy chain disease, lymphoblastic leukemia, T-cell leukemia, T-lymphocytic leukemia, T-lymphoblastic leukemia, B cell leukemia, B-lymphocytic leukemia, mixed cell leukemias, myeloid leukemias, myelocytic leukemia, myelogenous leukemia, neutrophilic leukemia, eosinophilic leukemia, monocytic leukemia, myelomonocytic leukemia, Naegeh-type myeloid leukemia, nonlymphocytic leukemia, osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, and prostate cancer
74 A method according to any one of claims 69 to 72, wherein the cell proliferation disorder is a cancer is selected from the group consisting of osteosarcoma, promyelocytic leukemia, non-small cell lung cancer, epithelial lung carcinoma, pancreatic carcinoma, pancreatic ductal adenocarcinoma, glioblastoma, metastatic breast cancer, melanoma, and prostate cancer
75 A method according to any one of claims 69 to 72, wherein the cell proliferation disorder is angiogenesis or a cell proliferation disorder mediated by angiogenesis
76 A method according to any one of claims 64 to 75, wherein the cells have an elevated level of Rb, Raf-1, or Rb bound to Raf-1
77 A method according to claim 64 to 76, further comprising assaying the level of Rb, Raf- 1, or Rb bound to Raf-1 in a cell
78 A method of assessing a subject for treatment with an inhibitor of Rb Raf-1 binding interactions, compπsing determining, in the subject or in a sample from the subject, a level of Rb, Raf-1, or Rb bound to Raf-1, wherein treatment with an inhibitor of Rb Raf-1 binding interactions is indicated when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal, wherein the inhibitor of RB Raf-1 binding interactions is a compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof
79 A method of identifying a subject for therapy, comprising obtaining a sample from the subject, determining a level of Rb, Raf-1, or Rb bound to Raf-1 in the sample, and identifying the subject for therapy with an inhibitor of Rb Raf-1 binding interactions when the level of Rb, Raf-1, or Rb bound to Raf-1 is elevated compared to normal, wherein the inhibitor of Rb Raf-1 binding interactions is a compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof
80 A compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, for use m medicine
81 A compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, for use m a method of treating or ameliorating a cell proliferation disorder
82 Use of compound according to any one of claims 1 to 48, or a pharmaceutically acceptable salt thereof, m the manufacture of a medicament for treating or ameliorating a cell proliferation disorder
PCT/US2009/055509 2008-08-29 2009-08-31 Inhibition of cell proliferation WO2010025448A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9328708P 2008-08-29 2008-08-29
US61/093,287 2008-08-29

Publications (2)

Publication Number Publication Date
WO2010025448A2 true WO2010025448A2 (en) 2010-03-04
WO2010025448A3 WO2010025448A3 (en) 2010-07-15

Family

ID=41662061

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/055509 WO2010025448A2 (en) 2008-08-29 2009-08-31 Inhibition of cell proliferation

Country Status (2)

Country Link
US (1) US20100184851A1 (en)
WO (1) WO2010025448A2 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB960084A (en) * 1961-01-13 1964-06-10 Us Vitamin Pharm Corp Haloaralkyl biguanides
US3996232A (en) * 1970-11-23 1976-12-07 William H. Rorer, Inc. 1,5-Disubstituted biguanides
DE19641692A1 (en) * 1996-10-10 1998-04-23 Bayer Ag Substituted 2,4-diamino-1,3,5-triazines
WO2006122546A1 (en) * 2005-05-18 2006-11-23 Forschungsverbund Berlin E.V. Non-peptidic inhibitors of akap/pka interaction
WO2007062222A2 (en) * 2005-11-22 2007-05-31 University Of South Florida Inhibition of cell proliferation
WO2008026757A1 (en) * 2006-08-30 2008-03-06 Otsuka Pharmaceutical Co., Ltd. Process for producing 1-(3,4-dichlorobenzyl)-5-octylbiguanide or a salt thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL266751A (en) * 1960-07-21
US3258325A (en) * 1962-06-01 1966-06-28 Pennsalt Chemicals Corp Herbicidal method
DE19601303A1 (en) * 1996-01-16 1997-07-17 Boehringer Ingelheim Kg Novel benzoylguanidine derivatives, process for their preparation and their use in the manufacture of medicaments
US6093728A (en) * 1997-09-26 2000-07-25 Asta Medica Aktiengesellschaft Methods of modulating serine/threonine protein kinase function with azabenzimidazole-based compounds
EP1057033B1 (en) * 1998-02-18 2003-04-23 Theryte Limited Treating cancer
US7855228B2 (en) * 2005-02-10 2010-12-21 The Board Of Trustees Of The Leland Stamford Junior University Antibiotics targeting MreB

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB960084A (en) * 1961-01-13 1964-06-10 Us Vitamin Pharm Corp Haloaralkyl biguanides
US3996232A (en) * 1970-11-23 1976-12-07 William H. Rorer, Inc. 1,5-Disubstituted biguanides
DE19641692A1 (en) * 1996-10-10 1998-04-23 Bayer Ag Substituted 2,4-diamino-1,3,5-triazines
WO2006122546A1 (en) * 2005-05-18 2006-11-23 Forschungsverbund Berlin E.V. Non-peptidic inhibitors of akap/pka interaction
WO2007062222A2 (en) * 2005-11-22 2007-05-31 University Of South Florida Inhibition of cell proliferation
WO2008026757A1 (en) * 2006-08-30 2008-03-06 Otsuka Pharmaceutical Co., Ltd. Process for producing 1-(3,4-dichlorobenzyl)-5-octylbiguanide or a salt thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUMMERFIELD, RACHAEL L. ET AL: "A 2.13 .ANG. Structure of E. coli Dihydrofolate Reductase Bound to a Novel Competitive Inhibitor Reveals a New Binding Surface Involving the M20 Loop Region" JOURNAL OF MEDICINAL CHEMISTRY , 49(24), 6977-6986 CODEN: JMCMAR; ISSN: 0022-2623, 11 April 2006 (2006-04-11), XP002569042 *

Also Published As

Publication number Publication date
US20100184851A1 (en) 2010-07-22
WO2010025448A3 (en) 2010-07-15

Similar Documents

Publication Publication Date Title
US9656953B2 (en) Inhibition of cell proliferation
JP2022043073A (en) Bivalent bromodomain inhibitors and uses thereof
CN104822672B (en) The purposes of therapeutic compound and composition as well as PKM2 conditioning agents
RU2488584C2 (en) Novel imidazolidine compounds as androgen receptor modulators
ES2824801T3 (en) Halogen-substituted heterocyclic compound salt
US20160151370A1 (en) Substituted Benzylpyrazoles
JP2016519674A (en) 3-Acetylamino-1- (phenyl-heteroaryl-aminocarbonyl or phenyl-heteroaryl-carbonylamino) benzene derivatives for treating or preventing hyperproliferative disorders
RU2504542C2 (en) Hydroxylated pyrimidylcyclopentanes as protein kinase (akt) inhibitors
KR20130121818A (en) Substituted 2-hydroxy-4-(2-(phenylsulfonamido)acetamido)benzoic acid analogs as inhibitors of stat proteins
JP5539225B2 (en) Hydroxylated pyrimidylcyclopentane as an AKT protein kinase inhibitor
CN105315224A (en) Cyclopentylacrylamide derivative
CN103237791A (en) Compositions and methods for the treatment of pathological condition(s) related to GPR35 and/or GPR35-HERG complex
US20170334863A1 (en) Cdc42 INHIBITOR AND USES THEREOF
FI118589B (en) Pyrimidine or triazine carboxylic acid derivatives for use as medicaments
ES2417488T3 (en) Triazole compounds suitable for treating disorders that respond to dopamine D3 receptor modulation
Akhmadiev et al. Synthesis, crystal structure and docking studies as potential anti-inflammatory agents of novel antipyrine sulfanyl derivatives
JP2020072706A (en) Pyridinone compounds and uses thereof
WO2010025448A2 (en) Inhibition of cell proliferation
EP3568128B1 (en) Hsp90b n-terminal isoform-selective inhibitors
US11000512B2 (en) Use of DDX3 inhibitors as antiproliferative agents
WO2018133756A1 (en) Targeted autophagy agonist and application thereof in treatment of neurodegenerative diseases
US20240150301A1 (en) Methods of modulating androgen receptor condensates
TW201536285A (en) (+)-5-(3,4-difluorphenyl)-5-[(3-methyl-2-oxopyridine-1(2H)-yl)methyl] imidazolidine-2,4-dione and pharmaceuticals containing thes same
EP3351544A1 (en) Benzene disulfonamide for the treatment of cancer
WO2021202137A1 (en) Therapeutic uses of inhibitors of the rna-binding protein hur

Legal Events

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

Ref document number: 09792102

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09792102

Country of ref document: EP

Kind code of ref document: A2