WO2012149102A1 - Novel compounds as wip1 inhibitors - Google Patents

Novel compounds as wip1 inhibitors Download PDF

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Publication number
WO2012149102A1
WO2012149102A1 PCT/US2012/035114 US2012035114W WO2012149102A1 WO 2012149102 A1 WO2012149102 A1 WO 2012149102A1 US 2012035114 W US2012035114 W US 2012035114W WO 2012149102 A1 WO2012149102 A1 WO 2012149102A1
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WO
WIPO (PCT)
Prior art keywords
amino
oxopropan
thiophene
methyl
substituted
Prior art date
Application number
PCT/US2012/035114
Other languages
French (fr)
Inventor
Mark A. Bobko
Michael G. Darcy
Karen A. Evans
Thomas H. Faitg
Arun Chandar KAURA
Xin Peng
Martha A. Sarpong
Mark A. Seefeld
Dai-Shi Su
Original Assignee
Glaxosmithkline Llc
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Filing date
Publication date
Application filed by Glaxosmithkline Llc filed Critical Glaxosmithkline Llc
Publication of WO2012149102A1 publication Critical patent/WO2012149102A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof

Definitions

  • This invention relates to the acylated alanine derivatives for the inhibition and/or induced destabilization of the Wipl (PPM1 D/PP2C5) phosphatase.
  • the invention relates to the use of the acylated alanine derivatives in the treatment of cancer.
  • Wild-type P53 inducible Phosphatase 1 (Wipl /PPM I D/ PP2C5) is a serine threonine phosphatase that plays a key role in the homeostatic down-regulation of various stress response and DNA repair signaling pathways.
  • Wipl is a member of the type 2C family protein phosphatase family that are dependent on Mg 2+ or Mn 2+ for their catalytic activity. As its name suggests, Wip l can be induced by various DNA damaging treatments including ionizing or UV irradiation in a P53 dependent manner (Fiscella et al. PNAS 94, 6048-6053 ( 1997)).
  • Wipl While its RNA expression is rapidly induced by various DNA damaging agents, translation of the RNA into Wipl protein is reportedly delayed by the miR- 16 micro RNA (Zhang et al., 201 0). Upon its induction, Wipl acts upon multiple effectors of the DNA damage response pathway to dephosphorylate and consequently inactivate them.
  • UNG2 Li et al., Molecular Cell 15, 621 -634 (2004)
  • MDM2 Li et al., Cancer Cell 12, 342-354 (2008)
  • M DMX Xinna et al., Journal of Biological Chemistry 281 , 24847-24862 (2009)
  • ATM Shreeram et al., Journal of Experimental Medicine 203, 2793-2799 (2006)
  • ⁇ 2 ⁇ Choa et al., Cancer Research 70, 41 12-4122 (2010)
  • p38MAPK Bulavin et al., Nature Genetics 36, 343-350 (2004)
  • Chk2 Flujimoto et al., Cell Death and Differentiation 13 , 1 1 70- 1 1 80 (2006)
  • Chk l and p53 Li et al., Genes and Development 19, 1 162- 1 1 74 ( 2005)
  • Wip l Several lines of evidence have suggested an oncogenic role for Wip l . I n cellular studies, overexpression of Wip l in E 1 A transduced rat embryonic fibroblasts was sufficient to induce transformation foci (Nannenga et al., Molecular Carcinogenesis 45, 594-604 (2006)). Conversely, murine embryonic fibroblasts homozygous for Wip l deletion showed significantly reduced tumor growth when co-transfected with the transforming oncogenes HRAS and E l A, HRAS and ErbB2, or HRAS and Myc (Bulavin et.al., Nature Genetics 36, 343-350 (2004)).
  • Wip l -/- mice also demonstrated tumor-resistance to both induced tumor models, using infection with M TV driven ErbB2 or HRAS, as well as a decreased incidence of spontaneous leukemias and sarcomas. Similarly, Wipl -/- mice were relatively resistant to ⁇ -Myc induced lymphomas (Shreeram et al., Journal of Experimental Medicine 203, 2793-2799 (2006)).
  • Wipl Overexpression of Wipl is reported to result in delay or suppression of base-excision repair by inhibition of UNG2 and P53. Similarly, its overexpression suppresses DNA double-strand-break repair by inhibition of the ATM response, inhibiting repair by both homologous recombination and non-homologous end joining.
  • Wipl /PPM I D gene locus has been reported in breast cancer (Li et al., Nature Genetics 3 1 , 133- 134.(2002)), ovarian clear cell carcinoma (Hirasawa et al., Clinical Cancer Research 9, 1995-2004 (2003)), neuroblastoma (Saito- Ohara et al., Journal of Experimental Medicine 203, 2793-2799 (2003)), and pancreatic adenocarcinoma (Loukopoulos et al., Cancer Science 98, 392-400 (2007)).
  • Wip l inhibitors describing antiproliferative activity in Wip l amplified or overexpressing cell lines (Belova et al., Cancer Biology and Therapy 4, 1 1 54- 1 1 58. (2005); Rayter et al., Oncogene 27, 1036- 1044 (2008)). Taken together, these observations suggest that Wip l a compelling target for pharmacological inhibition in the treatment of cancer.
  • This invention relates to a compound of Formula (I);
  • Ri is hydrogen, Ci-C4alkoxy, C
  • R 3 is hydrogen, C
  • This invention also relates to pharmaceutical compositions, which comprise compounds of Formula (I) and pharmaceutically acceptable carriers.
  • This invention also relates to methods of treating cancer which comprise administering an effective amount of a compound of Formula (I) to a human in need thereof.
  • This invention also relates to methods of treating cancer which comprise co-administering an compound of Formula (1) and a second compound to a human in need thereof.
  • the present invention relates to compounds of Formula (I);
  • the present invention also relates to compounds of Formula (II):
  • Ri is hydrogen, C)-C4alkoxy, Ci-C 4 alkyl, substituted Ci-C 4 alkyl, C3-C7cycloalkyl, or C3- C7heterocycloalkyl, wherein said C3-C7cycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -O(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl; R 2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C 3 alkyl, substituted Ci-C3alkyl, Ci- C 4 alkoxy, hydroxyl, amino, substituted amino, C 3 -C7cycloalkyl, cyano, ester, carboxylic acid, and C 3 -C6heterocycloalkyl;
  • R3 is hydrogen, Ci-C6alkyl, substituted C
  • R4 is cyclohexyl or cyclopi
  • X is , O or S; or a pharmaceutically acceptable salt thereof.
  • the present invention also relates to compounds of Formula (III):
  • Ri is hydrogen, Ci-C4alkoxy, C]-C4alkyl, substituted Ci-C4alkyl, C3-C 7 cycloalkyl, or C3- C7heterocycloalkyl, wherein said C3-C7cycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -0(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl;
  • R 2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, C
  • R3 is hydrogen, Ci -C6alkyl, or substituted Ci-C6alkyl
  • R4 is cyclohexyl or cyclopentyl; or a pharmaceutically acceptable salt thereof.
  • the present invention also relates to compounds according to any one of Formulas (1)- (III), wherein Ri is Ci-C 4 alkyl or substituted C
  • the present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R] is C3-C7cycloalkyl.
  • the present invention also relates to compounds according to any one of Formulas (I)- (II I), wherein R
  • the present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R 2 is phenyl, which may be substituted with one to four substituents selected from the group consisting of halogen, Ci-C3alkyl, substituted Ci-C3alkyl, Ci-C 4 alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalkyl, C3-C 7 heterocycloalkyl, cyano, ester, and carboxylic acid.
  • R 2 is phenyl, which may be substituted with one to four substituents selected from the group consisting of halogen, Ci-C3alkyl, substituted Ci-C3alkyl, Ci-C 4 alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalkyl, C3-C 7 heterocycloalkyl, cyano, ester, and carboxylic acid.
  • the present invention also relates to compounds according to any one of Formulas (I)- (III), wherein R 2 is pyridinyl, which may be substituted with one to three substituents selected from the group consisting of halo, C
  • the present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R3 is hydrogen.
  • the present invention also relates to compounds according to any one of Formulas (I)- (III), wherein R4 is cyclopentyl.
  • the present invention also relates to compounds selected from the group consisting of:
  • the salts of the present invention are pharmaceutically acceptable salts.
  • Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention.
  • Salts of the compounds of the present invention may comprise acid addition salts.
  • the salts are formed from pharmaceutically acceptable inorganic and organic acids.
  • suitable acid salts include maleic, hydrochloric, hydrobromic, sulphuric, phosphoric, nitric, perchloric, fiimic, acetic, propionic, succinic, glycolic, formic, lactic, aleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methansulfonic (mesylate), naphthalene-2-sulfonic,
  • salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, calcium edetate, camsylate, carbonate, clavulanate, citrate, dihydrochloride, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, a
  • the compound of Formula (I) or a salt thereof may exist in stereoisomeric forms (e.g., it contains one or more asymmetric carbon atoms).
  • the individual stereoisomers (enantiomers and diastereomers) and mixtures of these are included within the scope of the present invention.
  • the invention also covers the individual isomers of the compound or salt represented by Formula (I) as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • a compound or salt of Formula (I) may exist in tautomeric forms other than that shown in the formula and these are also included within the scope of the present invention. It is to be understood that the present invention includes all combinations and subsets of the particular groups defined hereinabove.
  • the scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerica!ly enriched mixtures. Also included within the scope of the invention are any wholly or partial ly deuteuriated isotopes of the compounds of Formula (1). Also included within the scope of the invention are individual isomers of the compound represented by Formula (I), as well as any wholly or partially equilibrated mixtures thereof. The present invention also includes the individual isomers of the compound or salt represented by the Formula (I) as well as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • Preferred prodrugs for compounds of the invention include: esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals.
  • the scope of the present invention also includes prodrugs of the present compounds.
  • alkyl refers to a straight or branched chain alkyl, preferably having from one to twelve carbon atoms, which may be saturated or unsaturated with multiple degrees of substitution included within the present invent ion.
  • alkyl as used herein include methyl, ethyl, propyl, isopropyl, isobutyl, n- butyl, t-butyl, isopentyl, n-pentyl, and the like, as well as substituted versions thereof.
  • substituted alkyl refers to a straight or branched chain alkyl, preferably having from one to twelve carbon atoms, which may be saturated or unsaturated with multiple degrees of substitution included within the present invention.
  • Suitable substituents are selected from the group consisting of: halogen, amino, substituted amino, urea, cyano, hydroxyl, alkoxy, alkylthio, alkylsiil fonyl, amidosulfonyl, carboxylic acid, ester, carboxamide, and aminocarbonyl.
  • cycloalkyl refers to an unsubstituted or substituted mono- or polycyclic non-aromatic saturated ring, which optionally includes an alkylene linker through which the cycloalkyl may be attached.
  • exemplary "cycloalkyl” groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, as well as unsubstituted and substituted versions thereof.
  • alkoxy refers to the group -OR a , where R a is Ci-Caalkyl or C3-7cycloalkyl as defined above.
  • substituted amino is meant -NR'R" wherein each R' and R" is independently selected from a group including hydrogen, unsubstituted Ci- Cealkyl, acyl, unsubstituted C3-C 7 cycloalkyl, wherein at least one of R' and R" is not hydrogen.
  • substituted amino includes, but are not limited to alkylamino, dialkylaminio, acylamino, and cycloalkylamino.
  • heterocycle or “heterocyclyl” or “heterocycloalkyl” refers to unsubstituted and substituted mono- or polycyclic non-aromatic ring system containing one or more heteroatoms.
  • Preferred heteroatoms include N, O, and S, including N-oxides, sulfur oxides, and dioxides.
  • the ring is three to eight- membered and is either fully saturated or has one or more degrees of nsaturation.
  • heterocyclic groups include, but are not limited to oxetanyl, tetrahydrofuranyl, pyranyl, 1 ,4-dioxanyl, 1 ,3-dioxanyl, piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, piperazinyl, pyrrolidinonyl, piperazinonyl, pyrazolidinyl, and their various tautomers, as well as unsubstituted and substituted versions thereof.
  • aryl aromatic, hydrocarbon, ring system.
  • the ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.), substituted or unsubstituted.
  • the monocyclic aryl ring is C5-C 10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system.
  • a C6 ring system i.e. a phenyl ring, is a suitable aryl group.
  • the polycyclic ring is a bicyclic aryl group, where suitable bicyclic aryl groups are C8-C 12, or C9-C 1 0.
  • a naphthyl ring, which has 10 carbon atoms, is a suitable polycyclic aryl group. Suitable substituents for aryl are described in the definition of "optionally substituted".
  • heteroaryl an aromatic ring system containing carbon(s) and at least one heteroatom.
  • Heteroaryl may be monocyclic or polycyclic, substituted or unsubstituted.
  • a monocyclic heteroaryl group may have 1 to 4 heteroatoms in the ring, while a polycyclic heteroaryl may contain 1 to 10 hetero atoms.
  • a polycyclic heteroaryl ring may contain fused, spiro or bridged ring junctions, for example, bicyclic heteroaryl is a polycyclic heteroaryl.
  • Bicyclic heteroaryl rings may contain from 8 to 12 member atoms.
  • Monocyclic heteroaryl rings may contain from 5 to 8 member atoms (carbons and heteroatoms).
  • heteroaryl groups include: benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinol ine, quinazoline, quinoxaline, thiazole, and thiophene.
  • Suitable substituents for heteroaryl are described in the definition of "optionally substituted”.
  • cyano refers to the group -CN.
  • acyl refers to the group -C(0)Rb, where Rb is alkyl, cycloalkyl, or heterocyclyl, as each is defined herein.
  • the term "optionally” means that the subsequently described event(s) may or may not occur, and includes both event(s) that occur and event(s) that do not occur.
  • substituted or variations thereof denote a substitution, including multiple degrees of substitution, with one or more substitutents, preferably one, two or three. The phrase should not be interpreted as duplicative of the substitutions herein described and depicted.
  • substituent groups include acyl, alkyl, substituted alkyl, alkylsulfonyl, alkoxy, alkoxycarbonyl, cyano, halogen, haloalkyl, hydroxyl, oxo, amide, sulfamide, urea, amino, substituted amino, acylamino, phenylcarbonyl, dialkylaminosulfonamide, morpholino, sul fonamide, thiourea, carboxylic acid, ester, and nitro.
  • the invention further provides a pharmaceutical composition (also referred to as pharmaceutical formulation) comprising a compound of Formula (I) or pharmaceutically acceptable salt, thereof and one or more excipients (also referred to as carriers and/or diluents in the pharmaceutical arts).
  • a pharmaceutical composition also referred to as pharmaceutical formulation
  • the excipients are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof (i.e., the patient).
  • a process for the preparation of a pharmaceutical composition comprising mixing (or admixing) a compound of Formula (1) or salt thereof with at least one excipient .
  • compositions may be in unit dose form containing a predetermined amount of active ingredient per unit dose.
  • a unit may contain a therapeutically effective dose of the compound of Formula (I) or salt thereof or a fraction of a therapeutically effective dose such that multiple unit dosage forms might be administered at a given time to achieve the desired therapeutically effective dose.
  • Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fract ion thereof, of an active ingredient.
  • such pharmaceutical compositions may be prepared by any of the methods well-known in t he pharmacy art.
  • compositions may be adapted for administration by any appropriate route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes.
  • oral including buccal or sublingual
  • rectal nasal
  • topical including buccal, sublingual, or transdermal
  • vaginal or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes.
  • parenteral including subcutaneous, intramuscular, intravenous, or intradermal
  • compositions When adapted for oral administration, pharmaceutical compositions may be in discrete units such as tablets or capsules; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; oil-in-water l iquid emulsions or water-in-oil liquid emulsions.
  • the compound or salt thereof of the invention or the pharmaceutical composition of the invention may also be incorporated into a candy, a wafer, and/or tongue tape formulation for administration as a "quick-dissolve" medicine.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • Powders or granules are prepared by comminuting the compound to a suitable fine size and mixing with a simi larly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agents can also be present.
  • Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin or non-gelatinous sheaths.
  • Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, solid polyethylene glycol can be added to the powder mixture before the filling operation.
  • a disintegrating or sokibil izing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicine when the capsule is ingested.
  • suitable binders include starch, gelatin, natural sugars, such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, sodium alginate,
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the l ike. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets.
  • a powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, and aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt, and/or an absorption agent such as - bentonite, kaolin, or dicalcium phosphate.
  • the powder mixture can be granulated by wetting a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen.
  • the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules.
  • the granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil.
  • the lubricated mixture is then compressed into tablets.
  • the compound or salt of the present invention can also be combined with a free-flowing inert carrier and compressed into tablets directly without going through the granulating orslugging steps.
  • a clear opaque protective coating consisting of a sealing coat of shellac, a coating of sugar, or polymeric material, and a polish coating of wax can be provided.
  • Dyestuffs can be added to these coatings to distinguish different dosages.
  • Oral fluids such as solutions, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of active ingredient.
  • Syrups can be prepared by dissolving the compound or salt thereof of the invention in a suitably flavoured aqueous solution, while elixirs are prepared through the use of a non- toxic alcoholic vehicle.
  • Suspensions can be formulated by dispersing the compound or salt of the invention in a non-toxic vehicle.
  • Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additives such as peppermint oil, natural sweeteners, saccharin, or other artificial sweeteners, and the like, can also be added.
  • dosage unit formulations for oral adm inistration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as, for example, by coating or embedding particulate material in polymers, wax, or the like.
  • tablets and capsules are preferred for delivery of the pharmaceutical composition.
  • treatment includes prophylaxis and refers to alleviating the specified condition, eliminating or reducing one or more symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying the reoccurrence of the condition in a previously afflicted or diagnosed patient or subject.
  • Prophylaxis or prevention or delay of disease onset is typically accomplished by administering a drug in the same or similar manner as one would to a patient with the developed disease or condition.
  • the present invention provides a potential treatment in a mammal, especially a human, suffering from disease conditions targeted by the present compounds.
  • Such treatment comprises the step of administering a therapeutically effective amount of a compound of Formula (I) or salt thereof to said mammal, particularly a human.
  • Treatment can also comprise the step of administering a therapeutically effective amount of a pharmaceutical composition containing a compound of Formula (I) or salt thereof to said mammal, particularly a human.
  • the term "effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
  • therapeutically effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
  • therapeutically effective amounts of a compound of Formula (1), as well as salts thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition.
  • a therapeutically effective amount of a compound of Formula (I) or salt thereof may be administered as the raw chemical, it is typically presented as the active ingredient of a pharmaceutical composit ion or formulation.
  • a compound of Formula (1) or salt thereof wi ll be given for the treatment in the range of about 0.01 to 100 mg/kg body weight of recipient (patient, mammal) per day and more usually in the range of 0.1 to 10 mg/kg body weight per day.
  • Acceptable daily dosages may be from about 1 to about 1000 mg/day, and preferably from about 1 to about 1 00 mg/day.
  • This amount may be given in a single dose per day or in a number (such as two, three, four, five, or more) of sub-doses per day such that the total daily dose is the same.
  • An effective amount of a salt thereof may be determined as a proportion of the effective amount of the compound of Formula (I) per se. Similar dosages should be appropriate for treatment (including prophylaxis) of the other conditions referred herein for treatment. In general, determination of appropriate dosing can be readily arrived at by one skilled in medicine or the pharmacy art.
  • a compound of Formula (I) When a compound of Formula (I) is administered for the treatment of cancer, the term “co-administering" and derivatives thereof as used herein is meant either simultaneous administration or any manner of separate sequential administration of a LSD 1 inhibiting compound, as described herein, and a further active ingredient or ingredients, known to be useful in the treatment of cancer, including chemotherapy and radiation treatment.
  • the term further active ingredient or ingredients, as used herein includes any compound or therapeutic agent known to or that demonstrates advantageous properties when administered to a patient in need of treatment for cancer.
  • the compounds are administered in a close time proximity to each other.
  • the compounds are administered in the same dosage form, e.g. one compound may be administered topically and another compound may be administered orally.
  • any anti-neoplastic- agent that has activity versus a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention.
  • examples of such agents can be found in Cancer Principles and Pract ice f Oncology by V.T. Devita and S. Hellman (editors), 6 lh edition (February 15, 2001 ), Lippincott Wi lliams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved.
  • Typical anti-neoplastic agents useful in the present invention include, but are not limited to, anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase 11 inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and anti- folate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors.
  • anti-microtubule agents such as diterpenoids and vinca alkaloids
  • Anti-microtubule or anti-mitotic agents are phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle.
  • anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.
  • Diterpenoids which are derived from natural sources, are phase specific anti - cancer agents that operate at the G 2 /M phases of the cell cycle. It is believed that the diterpenoids stabilize the ⁇ -tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following. Examples of diterpenoids include, but are not limited to, paclitaxel and its analog docetaxel.
  • Paclitaxel 5 p,20-epoxy- l ,2a,4,7P, 10p, 13a-hexa-hydroxytax- l l -en-9-one 4, 1 0- diacetate 2-benzoate 13-ester with (2R,3 S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL®. It is a member of the taxane family of terpenes. It was first isolated in 1971 by Wani et al. J. Am. Chem, Soc, 93 :2325.
  • Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman et al., Yale Journal of Biology and Medicine, 64:583, 1991 ; McGuire et al., Ann. Intern, Med., 1 1 1 :273, 1989) and for the treatment of breast cancer (Holmes et al., J. Nat. Cancer Inst., 83 : 1 797, 1991 .) It is a potential candidate for treatment of neoplasms in the skin (Einzig et. al., Proc. Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastire et. al., Sem. Oncol., 20:56, 1 990).
  • the compound also shows potential for the treatment of polycystic kidney disease (Woo et. al., Nature, 368:750. 1994), lung cancer and malaria.
  • Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lineages, Ignoff, R.J. et. al, Cancer Chemotherapy Pocket Guic ⁇ 1998) related to the duration of dosing above a threshold concentration (50nM) (Kearns, CM. et. al., Seminars in Oncology, 3(6) p.16-23, 1 995).
  • Docetaxel (2R,3S)- N-carboxy-3-phenylisoserine,N- r/-butyl ester, 1 3-ester with 5P-20-epoxy- l ,2a,4,7P, 10p, 13ot-hexahydroxytax- l l -en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE®.
  • Docetaxel is indicated for the treatment of breast cancer.
  • Docetaxel is a semisynthetic derivative of paclitaxel q. v. , prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree. The dose limiting toxicity of docetaxel is neutropenia.
  • Vinca alkaloids are phase specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine.
  • Vinblastine vincaleukoblastine sulfate
  • VELBAN® an injectable solution.
  • Myelosuppression is the dose limiting side effect of vinblastine.
  • Vincristine vincaleukoblastine, 22-oxo-, sulfate
  • ONCOVIN® an injectable solution.
  • Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas.
  • Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosiipression and gastrointestinal mucositis effects occur.
  • Vinorelbine 3 ',4'-didehydro -4'-deoxy-C'-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate ( 1 :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELB INE®), is a semisynthetic vinca alkaloid.
  • Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose limiting side effect of vinorelbine.
  • Platinum coordination complexes are non-phase specific anti-cancer agents, which are interactive with DNA.
  • the platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand crosslinks with DNA causing adverse biological effects to the tumor.
  • Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.
  • Cisplatin cis-diamminedichloroplatinum
  • PLAT1NOL® an injectable solution.
  • Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer.
  • the primary dose limiting side effects of cisplatin are nephrotoxicity, which may be controlled by hydration and diuresis, and ototoxicity.
  • Carboplatin platinum, diammine [l , l -cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN® as an injectable solution.
  • Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma. Bone marrow suppression is the dose limiting toxicity of carboplatin.
  • Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death.
  • alkylating agents include, but are not lim ited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine.
  • Cyclophosphamide 2-[bis(2-chloroethy I)amino]tetrahydro-2H- 1 ,3,2- oxazaphosphorine 2-oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea, vomiting and leukopenia are the most common dose limiting side effects of cyclophosphamide.
  • Melphalan 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary. Bone marrow suppression is the most common dose lim iting side effect of melphalan.
  • Chlorambucil 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN® tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. Bone marrow suppression is the most common dose limiting side effect of chlorambucil.
  • Busulfan 1 ,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® TABLETS. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose limiting side effects of busulfan.
  • Carmustine, l ,3-[bis(2-chloroethyl)- l -nitrosourea is commercially available as single vials of lyophilized material as BiCNU®.
  • Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppression is the most common dose limiting side effects of carmustine.
  • dacarbazine 5-(3 ,3-dimethyl- l -triazeno)-imidazole-4-carboxamide, is commercially available as single vials of material as DTIC-Dome®.
  • dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dacarbazine.
  • Antibiotic anti-neoplastics are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death.
  • antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin; and bleomycins.
  • Dactinomycin also know as Actinomycin D, is commercially available in injectable form as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilm's tumor and rhabdomyosarcoma. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dactinomycin.
  • naphthacenedione hydrochloride is commercially available as a liposomal injectable form as DAUNOXOME® or as an injectable as CERUBIDINE®.
  • Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma. Myelosuppression is the most common dose limiting side effect of daunorubicin.
  • Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas. Myelosuppression is the most common dose limiting side effect of doxorubicin.
  • Bleomycin a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is commercially available as BLENOXANE®. Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas. Pulmonary and cutaneous toxicities are the most common dose limiting side effects of bleomycin.
  • Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.
  • Epipodophyllotoxins are phase specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death fol lows. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.
  • Etoposide 4'-demethyl-epipodophyllotoxin 9[4,6-0-( )-ethylidene-P-D- glucopyranoside] is commercially available as an injectable solution or capsules as VePESI D® and is commonly known as VP- 16.
  • Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non- small cell lung cancers. Myelosuppression is the most common side effect of etoposide. The incidence of leucopenia tends to be more severe than thrombocytopenia.
  • Teniposide 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-thenylidene-P-D- glucopyranoside], is commercially available as an injectable solution as VUMON® and is commonly known as VM-26.
  • Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia in children. Myelosuppression is the most common dose limiting side effect of teniposide. Teniposide can induce both leucopenia and thrombocytopenia.
  • Antimetabolite neoplastic agents are phase specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows.
  • Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.
  • 5-fluorouracil 5-fluoro-2,4- (1 H,3H) pyrimidinedione
  • fluorouracil is commercially available as fluorouracil.
  • Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death.
  • 5-fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Myelosuppression and mucositis are dose limiting side effects of 5- fluorouracil.
  • Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.
  • Cytarabine 4-amino- l-P-D-arabinofuranosyl-2 ( 1 H)-pyrimidinone, is commercial ly available as CYTOSAR-U® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include 5-azacytidine and 2',2 '-difluorodeoxycytidine (gemcitabine). Cytarabine induces leucopenia, thrombocytopenia, and mucositis.
  • Mercaptopurine l ,7-dihydro-6H-purine-6-thione monohydrate
  • PUR1NETHOL® is commercially available as PUR1NETHOL®.
  • Mercaptopurine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Myelosuppression and gastrointestinal mucositis are expected side effects of mercaptopurine at high doses.
  • a useful mercaptopurine analog is azathioprine.
  • Thioguanine 2-amino- l ,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®.
  • Thioguanine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism.
  • Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia.
  • Myelosuppression including leucopenia, thrombocytopen ia, and anemia, is the most common dose limiting side effect of thioguanine administration. However, gastrointestinal side effects occur and can be dose limiting.
  • Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.
  • Gemcitabine 2'-deoxy-2 ', 2'-difluorocytidine monohydrochloride ( ⁇ -isomer), is commercially available as GEMZAR®.
  • Gemcitabine exhibits cell phase specificity at S- phase and by blocking progression of cells through the G l /S boundary.
  • Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.
  • Myelosuppression including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of gemcitabine adm inistration.
  • Methotrexate N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino] benzoyl]-L- glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate.
  • Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder.
  • Myelosuppression (leucopenia, thrombocytopenia, and anemia) and mucositis are expected side effect of methotrexate administration.
  • Camptothecins including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors. Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to irinotecan, topotecan, and the various optical forms of 7-(4-methylpiperazino-methylene)- 1 0, l l -ethylenedioxy-20-camptothecin described below.
  • Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I - DNA complex. It is bel ieved that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I : DNA : irintecan or SN-38 ternary complex with replication enzymes. Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum.
  • the dose limiting side effects of irinotecan HC1 are myelosuppression, including neutropenia, and GI effects, including diarrhea.
  • Topotecan HC1 (S)- 10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy- l H- pyrano[3 ',4',6,7] indolizino[l ,2-b]quinoline-3, 14-(4H, 12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN®.
  • Topotecan is a derivative of camptothecin which binds to the topoisomerase I - DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule.
  • Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.
  • the dose limiting side effect of topotecan HCI is myelosuppression, primarily neutropenia.
  • camptothecin derivative of formula A following, currently under development, including the racemic mixture (R,S) form as well as the R and S enantiomers:
  • Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer.
  • hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children ; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrms such as megestrol acetate useful in the treatment of hormone dependent breast cancer and endometrial carcinoma; estrogens, androgens, and anti-androgens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a-reducta
  • GnRH gonadotropin-releasing hormone
  • LH leutinizing hormone
  • FSH follicle stimulating hormone
  • Signal transduction pathway inhibitors are those inhibitors, which block or inhibit a chemical process which evokes an intracellular change. As used herein this change is cell proliferation or differentiation.
  • Signal tranduction inhibitors useful in the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3domain blockers, serine/threonine kinases, phosphotidy! inositol-3 kinases, myoinositol signaling, and Ras oncogenes.
  • protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth.
  • protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.
  • Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are generally termed growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, i.e. aberrant kinase growth factor receptor activity, for example by over- expression or mutation, has been shown to result in uncontrol led cell growth. Accordingly, the aberrant activity of such kinases has been linked to malignant tissue growth. Consequently, inhibitors of such kinases could provide cancer treatment methods.
  • Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2, erbB4, vascu lar endothelial growth factor receptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (TIE-2), insulin growth factor -I (IGFI) receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FG F) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptors, and the RET protooncogene.
  • EGFr epidermal growth factor receptor
  • PDGFr platelet derived growth factor receptor
  • erbB2 erbB4
  • VEGFr vascu lar endothelial growth factor receptor
  • TIE-2 vascu lar endothelial growth
  • inhibitors of growth receptors include ligand antagonists, antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides.
  • Growth factor receptors and agents that inhibit growth factor receptor function are described, for instance, in Kath, John C, Exp. Opin. Ther. Patents (2000) 1 0(6): 803-818; Shawver et al DDT Vol 2, No. 2 February 1997; and Lofts, F. J. et al, "Growth factor receptors as targets", New Molecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr, David, CRC press 1994, London.
  • Non-receptor tyrosine kinases which are not growth factor receptor kinases are termed nonreceptor tyrosine kinases.
  • Non-receptor tyrosine kinases useful in the present invention include cSi c, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl.
  • Such non- receptor kinases and agents which inhibit non-receptor tyrosine kinase function are described in Sinh, S.
  • SH2/SH3 domain blockers are agents that disrupt SH2 or SJ-I3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunil, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP.
  • SH2/SH3 domains as targets for anti-cancer drugs are discussed in Smithgall, T.E. ( 1995), Journal o f Pharmacological and Toxicological Methods. 34(3) 125-32.
  • Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracel lular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta). IkB kinase family (IKKa, I Kb), PKB family kinases, AKT kinase family members, and TGF beta receptor kinases.
  • MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracel lular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including
  • Serine/Threonine kinases and inhibitors thereof are described in Yamamoto, T., Taya, S., Kaibuchi, K., ( 1 999), Journal of B iochemistry. 126 (5) 799-803; Brodt, P, Samani, A., and Navab, R. (2000), B iochemical Pharmacology, 60. 1 101 - 1 107; Massague, J., Weis-Garcia, F. ( 1 996) Cancer Surveys. 27:41 -64; Philip, P.A., and Harris, A.L. (1995), Cancer Treatment and Research. 78: 3-27, Lackey, K.
  • Myo-inositol signaling inhibitors such as phospholipase C blockers and Myoinositol analogues.
  • signal inhibitors are described in Powis, G., and Kozikowski A., ( 1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press 1994, London.
  • Ras Oncogene Another group of signal transduction pathway inhibitors are inhibitors of Ras Oncogene.
  • Such inhibitors include inhibitors of farnesyltransfei ase, geranyl-geranyl transferase, and CAAX proteases as well as anti-sense oligonucleotides, ribozymes and immunotherapy.
  • Such inhibitors have been shown to block ras activation in cells containing wild type mutant ras , thereby acting as antiproliferation agents.
  • Ras oncogene inhibition is discussed in Scharovsky, O.G., Rozados, V.R., Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4) 292-8; Ashby, M.N. ( 1 998), Current Opinion in Lipidology. 9 (2) 99 - 102; and BioChim. B iophys. Acta, (19899) 1423(3): 1 9-30.
  • antibody antagonists to receptor kinase ligand binding may also serve as signal transduction inhibitors.
  • This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand binding domain of receptor tyrosine kinases.
  • Imclone C225 EGFR specific antibody see Green, M.C. et al, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat.
  • Herceptin ® erbB2 antibody see Tyrosine Kinase Signalling in Breast cancerrerbB Family Receptor Tyrosine Kniases, Breast cancer Res., 2000, 2(3), 1 76- 1 83
  • 2CB VEGFR2 specific antibody see Brekken, R.A. et al, Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice, Cancer Res. (2000) 60, 51 1 7-5124).
  • Non-receptor kinase angiogenesis inhibitors may also find use in the present invention.
  • Inhibitors of angiogenesis related VEGFR and ⁇ 2 are discussed above in regard to signal transduction inhibitors (both receptors are receptor tyrosine kinases).
  • Angiogenesis in general is linked to erbB2/EGFR signaling since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression.
  • an erbB2/EGFR inhibitor with an inhibitor of angiogenesis makes sense.
  • non-receptor tyrosine kinase inhibitors may be used in combination with the EGFR/erbB2 inhibitors of the present invention.
  • anti-VEGF antibodies which do not recognize VEGFR (the receptor tyrosine kinase), but bind to the ligand; small molecule inhibitors of integrin (alpha v betas) that will inhibit angiogenesis; endostatin and angiostatin (non-RTK) may also prove useful in combination with the disclosed erb family inhibitors.
  • VEGFR the receptor tyrosine kinase
  • small molecule inhibitors of integrin alpha v betas
  • endostatin and angiostatin non-RTK
  • Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I).
  • immunologic strategies to generate an immune response against erbB2 or EGFR. These strategies are generally in the realm of tumor vaccinations.
  • the efficacy of immunologic approaches may be greatly enhanced through combined inhibition of erbB2/EGFR signaling pathways using a small molecule inhibitor. Discussion of the immunologic/tumor vaccine approach against erbB2/EGFR are found in Reilly RT et al. (2000), Cancer Res. 60: 3569-3576; and Chen Y, Hu D, Eling DJ, Robbins J, and Kipps TJ. (1998), Cancer Res. 58 : 1 965- 1971 .
  • Agents used in proapoptotic regimens may also be used in the combination of the present invention.
  • Members of the Bcl-2 family of proteins block apoptosis. Upregulation of bcl-2 has therefore been linked to chemoresistance.
  • EGF epidermal growth factor
  • Cell cycle signalling inhibitors inhibit molecules involved in the control of the cell cycle.
  • a family of protein kinases called cyclin dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle.
  • CDKs cyclin dependent kinases
  • Several inhibitors of cell cycle signalling are under development. For instance, examples of cyclin dependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for the same are described in, for instance, Rosania et al, Exp. Opin. Ther. Patents (2000) 1 0(2):21 5-230.
  • p21 WAF 1 /CIP 1 has been described as a potent and universal inhibitor of cyclin-dependent kinases (Cdks) (Ball et al., Progress in Cell Cycle Res., 3: 125 ( 1997)).
  • Modulators of the Retinoid Acid Receptor have been used to treat leukemias.
  • the pathology of the leukemia is associated with the abnormal accumulation of immature progenitor cells that are sensitive to retinoc acid therapy.
  • APL acute promyelocytic leukemia
  • RAR retinoic acid receptor
  • PA4L promyelocytic leukemia
  • Tretinoin acts on PML-RAR to lift this block, causing the immature promyelocytes to differentiate to normal mature blood cells thus decreasing promyelocytes and promoting a population of terminally differentiated cells with a restricted lifespan.
  • Talazorole is an experimental drug in the same class as Tretinoin.
  • Cancer specific changes are often associated with silencing of tumor suppressor genes via histone modifications and modi fications to DNA including DNA hypermethylation.
  • Epigenetic pharmaceuticals control regulatory regions associated with tumor suppressor genes by causing conformational changes in histones and removing repressive
  • epigenetic agents include histone deacetylase inhibitors and DNA methylation inhibitors.
  • Histone deacetylase inhibitors are a class of compounds that interfere with the function of histone deacetylases. Inhibitors of histone deacetylases have been shown to be useful in the treatment of cutaneous T-cell lymphoma. They are being investigated in the clinic for multiple other tumor types. Examples of HDAC inhibitors approved for use are Vorinostat and Romidepsin. .These compounds are thought to inhibit the activity of HDACs and result in the accumulation of acetylation to histones promoting gene expression.
  • Azacitidine (INN) or 5-azacytidine, sold under the trade name Vidaza, is a chemical analogue of cytidine, a nucleoside present in DNA and RNA.
  • Azacit idine and its deoxy derivative, decitabine also known as 5-aza-2'deoxycytidine
  • Azacitidine acts as a false substrate and potent inhibitor of DNA methyltransferases leading to reduction of DNA methylation.
  • DNA methyltransferases incorporate azacitidine into DNA during replication and into RNA during transcription in the cell.
  • Inhibition of DNA methylation occurs through the formation of stable complexes between the molecule and DNA methyltransferases, thereby saturating cell methylation machinery. This results in a loss of DNA methylation and can affect the way cell regulation proteins, such as transcriptional machinery, are able to associate with the DNA.
  • HDAC inhibitors examples include:
  • Vorinostat including pharmaceutically acceptable salts thereof. Marks et al., Nature Biotechnology 25, 84 to 90 (2007); Stenger, Community Oncology 4, 384-386 (2007).
  • Vorinostat has the following chemical structure and name:
  • Romidepsin including pharmaceutically acceptable salts thereof. Vinodhkumar et al., Biomedicine & Pharmacotherapy 62 (2008) 85-93. Romidepsin, has the following chemical structure and name:
  • Panobinostat including pharmaceutically acceptable salts thereof.
  • Panobinostat has the following chemical structure and name:
  • Valproic acid has the following chemical structure and name:
  • Mocetinostat (MGCDOl 03), including pharmaceutically acceptable salts thereof. Balasubramanian et al., Cancer Letters 280: 21 1 -221 (2009). Mocetinostat, has the following chemical structure and name:
  • HDAC inhibitors are included in Bertrand European Journal of Medicinal Chemistry 45, (2010) 2095-21 16, particularly the compounds of table 3 therein as indicated below.
  • proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins, like the p53 protein.
  • proteasome inhibitors are marketed or are being studied in the treatment of cancer.
  • Suitable proteasome inhibitors for use in combination herein include: 1. Bortezomib (Velcade®), including pharmaceutically acceptable salts thereof. Adams J, Kauffman M (2004), Cancer Invest 22 (2): 304-1 1 .
  • Bortezomib has the following chemical structure and name.
  • Disulfiram including pharmaceutically acceptable salts thereof. Bouma et al. ( 1998). J. Antimicrob. Chemother. 42 (6): 817-20. Disulfiram has the following chemical structure and name.
  • Epigallocatechin gallate has the following chemical structure and name.
  • Salinosporamide A including pharmaceutically acceptable salts thereof. Feling et at., (2003), Angew. Chem. Int. Ed. Engl. 42 (3): 355-7.
  • Salinosporamide A has the following chemical structure and name.
  • Carfilzomib including pharmaceutically acceptable salts thereof. uhn DJ, et al, Blood, 2007, 1 1 0:3281 -3290.
  • Carfilzomib has the following chemical structure and name.
  • Hsp70s and Hsp90s are a families of ubiquitously expressed heat shock proteins. Hsp70s and Hsp90s are over expressed certain cancer types. Several Hsp70s and Hsp90s inhibitors are being studied in the treatment of cancer. Suitable Hsp70s and Hsp90s inhibitors for use in combination herein include:
  • 17-AAG(Geldanamycin) has the following chemical structure and name.
  • Radicicol has the following chemical structure and name.
  • Inhibitors of cancer metabolism Many tumor cells show a markedly different metabolism from that of normal tissues. For example, the rate of glycolysis, the metabol process that converts glucose to pyruvate, is increased, and the pyruvate generated is reduced to lactate, rather than being further oxidized in the mitochondria via the tricarboxylic acid (TCA) cycle. This effect is often seen even under aerobic conditions and is known as the Warburg Effect. Lactate dehydrogenase A (LDH-A), an isoform of lactate dehydrogenase expressed in muscle cells, plays a pivotal role in tumor cell metabolism by performing the reduction of pyruvate to lactate, which can then be exported out of the cell. The enzyme has been shown to be upregulated in many tumor types. The alteration of glucose metabolism described in the Warburg effect is critical for growth and proliferation of cancer cells and knocking down LDH-A using RNA-i has been shown to lead to a reduction in cell proliferation and tumor growth in xenograft models.
  • LDH-A lactate de
  • FAS fatty acid synthase
  • Inhibitors of cancer metabolism including inhibitors of LDH-A and inhibitors of fatty acid biosynthesis (or FAS inhibitors), are suitable for use in combination with the compounds of this invention.
  • the cancer treatment method of the claimed invention includes the co-administration a compound of Formula (I) and/or a pharmaceutically acceptable salt, hydrate, solvate or pro-drug thereof and at least one anti-neoplastic agent, such as one selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors,
  • topoisomerase I inhibitors include hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, FAS inhibitors, HDAC inhibitors, LDH-A inhibitors and cell cycle signaling inhibitors.
  • I. a Protection step, for example: CbzCI, NaOH, water ;
  • I.b, I.d Amidification step, for example: EDC, HOAt, DIEA, DMF ;
  • l.c Deprotection step, for example: 3 ⁇ 4 Pd/C, EtOAc, EtOH.
  • protecting groups are available to one skilled in the art and can be used here as long as they do not interfere with the processes listed herein. Methods for the protection of amines are described in standard reference volumes, such as Greene "Protective Groups in Organic Synthesis" (published by Wiley-Interscience).
  • amino group of Aminoacid 1 can be protected by, but not exclusively, tert- butyl carbamate (Boc), (9H-fluoren-9-yl)methyl (Fmoc) or benzyl carbamate (Cbz).
  • Typical conditions include (Boc ⁇ O, FmocCI or CbzCI, with or without a base like NaOH, NaHC0 3 or triethylamine in an organic solvent like THF, ACN, DM F, EtOAc or even in water.
  • Protected amino-acid 2 can be transformed into the amide 3 by typical amide coupling conditions.
  • a variety of amide coupling reagents such as EDC, HOBt, HOAt, HATU, PyBrop, etc. are commercially available and can be used here.
  • Amide coupling reactions are generally run in solvents such as DC or DMF, utilizing an organic base like ET3N or (i-Pr) 2 NEt. The amino group of 3 was deprotected to afford 4.
  • I I. a Reductive animation step, for example: 1 ) MeOH, 2) NaCNBl-b ; l l.b : Alkylation step for example: Etl, NaH, DMF ; II.c: Hydrolysis step, for example: I N LiOH, THF.
  • Reductive amination of 5 with an arylamine can afford the carboxylic acid 6, which can eventually be alkylated to give 7. Subsequent saponi fication can give the carboxylic acid 8.
  • acid for example AcOH
  • a dehydrating agent for example molecular sieves
  • the reactions are often conducted in methanol or a chlorinated solvent like DCM or 1 ,2-dichloroethane, using a reduction agent like NaB Hi, NaCNB3 ⁇ 4, NaBH(OAc)3 or even LAH.
  • the alkylation reaction can be typically conducted with an alkylated agent such as an alkyl halide (EtI for example), a strong base l ike NaH in a polar solvent l ike DMF.
  • an alkylated agent such as an alkyl halide (EtI for example), a strong base l ike NaH in a polar solvent l ike DMF.
  • the hydrolysis/saponification step can be conducted with a base like LiOH or NaOH in water, or in acidic conditions like ( IN HC1 for example).
  • I II .a Reduction step, for example: NaBH 4 , MeOH ; Ill.b : Esterification step for example: MeOH, H2SO4 ; lll.c: Mitsunobu reaction step, for example : ArOH, DEAD, Ph 3 P, TITF ; I ll.d: Bromination step, for example: CBr «i, DCM ; l ll.e: Alkylation step, for example: ArOH, CS2CO3, ACN, DMF. Reduction of the formyl group of compound 5 using for example NaBPLi can afford compound 9.
  • Such reduction could also be conducted with other reducing agents like lithium aluminium hydride, diisobutylaluminium hydride, sodium borohydride, L- selectride, diborane, diazene or aluminum hydride.
  • Sodium cyanoborohydride, 9-BBN- pyridine and tribut ltin hydride are also known to be selective for aldehydes.
  • Hydrogenation using platinum or ruthenium as catalysts could also be an option.
  • the esterification of 9 into 10 could typically be conducted in methanol as solvent in the presence of catalytic sulfuric acid.
  • Alcohol H) may be converted to bromo derivative 11 using CBr , hydrobromic acid or phosphorus tribromide (PBr3).
  • PBr3 phosphorus tribromide
  • This transformation can also take place using radical conditions in water instead of an organic solvent and the bromine is obtained by oxidation of hydrobromic acid with hydrogen peroxide.
  • An incandescent light bulb is then sufficient for bromine radical generation.
  • Final ly compound 12 can be obtained from a phenol or hydroxy-pyridine by M itsunobu reaction with 10 or alkylation with 11. Mitsunobu reactions are well known to those skilled in the art of organic synthesis. Methods and reaction conditions for such transformations are discussed in Synthesis 1981 , 1 -28.
  • Alkylation conditions include but are not limited to using an inorganic base (
  • y,z N or CH or C-alkyl
  • Reduction of the nitro group can be achieved by a wide variety of methods known to the one skilled in the art, including using SnC12 with HC1 in EtOH, or metal Fe in AcOH, or hydrazine or even hydrogenation using a catalyst such as palladium on carbon.
  • reaction mixture was washed with water three times.
  • the combined aqueous layers were extracted with DCM (2x25mL).
  • the combined organic layers were washed with brine, dried over sodium sulfate, filtered, concentrated, and vacuum pumped to a white solid ( 1 .14 g, >90% pure by LC-MS) that was used directly in the next reaction without further purification.
  • Triethylamine (0.394mL, 2.83mmol), 1 - hydroxybenzotriazole (144mg, 0.942mmol) and finally l -ethyl-3-(3- dimethylaminopropyl)carbodiimide ( 1 81 mg, 0.942mmol) were added and the contents allowed to stir at room temperature for 3 days. The reaction was then poured into 250mL of water and extracted (3 x 1 50mL) with ethyl acetate. The organic fractions were pooled, washed (2 x 200mL) with water and (2 x 200mL) with brine, dried over sodium sul fate, filtered and evaporated to dryness.
  • Ci3 ⁇ 4)-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpiOpanoic acid (l .Og, 3.89mmol), O-methylhydroxylamine hydrochloride (1.558g, 4.66mmol) and 1 - ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.894g, 4.66mmol) in DMF (6.1 mL) was added in one portion 1 -hydroxy-7-azabenzotriazole (0.635g, 4.66mmol).
  • Reaction 'continued to stir overnight in order to push to completion, which was confirmed by LCMS, LC-MS(ES) m/z 3 1 5.4 [M+H]+.
  • Reaction mixture was transferred to a separatory funnel, then washed with water, saturated aqueous NaHC03 solution, brine, dried over sodium sulfate, filtered, concentrated and vacuum pumped overnight to afford a yellow oil.
  • the product was redissolved in DCM and purified thru a silica plug using 100% EtOAc to remove baseline impurities. The filtrate was
  • the reaction was diluted with DCM and a saturated aqueous NaHC03 solution then transferred to a separatory funnel. It was observed that much of the desired product had gummed up and was stuck to the walls of the separatory funnel.
  • the upper aqueous layer was neutralized by addition of 2N HCI. 35mL of 1PA were then added to the 350mL of DCM already in the funnel. This made the brown gum dissolve and gave two homogeneous layers.
  • the layers were separated and the aqueous layer was extracted with 50mL more of 10% 1PA:DCM.
  • the combined IPA/DCM layers were dried over Na2S04, filtered, and concentrated to give a tan foam (2.44g).
  • Wipl(A2-K420) Recombinant Wipl protein (Wipl(A2-K420)) was expressed in Sf9 cells, lysed in 0.5% CHAPS, purified over 1.) Nickel Column, 2.) Superdex 200 pool3, then 3.) MonoQ p2 , and finally stored at -20°C in storage buffer (50mM Tris-HCl (pH 8.0), 20% Glycerol, 0.2M aCl,lmM TCEP, ImM CHAPS, 0.25mM Imidazole). Assay reaction was carried out in reaction buffer containing 50mM Tris pH7.5, 30mM MgCl 2 , 0.05mg/mL BSA, 0.05% CHAPS ,and ImM DTT. Fluorescein diphosphate (FDP, tetiaammonium salt; Invitrogen) solution was prepared as l OniM stock made in 50mM Tris (pH7.5), and stored at -20°C.
  • FDP Fluorescein diphosphate
  • Recombinant full-length Wip l protein (FL-Wip l ) was expressed in Sf9 cells, lysed in 0.5% CHAPS, purified over 1 .) NiNTA column, then 2.)Superdex 200 pool4, and finally stored at -20°C in storage buffer (50mM Tris-HCl (pH 8.0), 20% Glycerol, 0.1 5M NaCl, 0. 1 M EGTA, l mM TCEP).
  • Recombinant GST-tagged full-length p38 was expressed in Sf9 cells, lysed, purified over 1.) NiNTA column, 2.)Superdex 200 pool 3. Recovered protein was then treated with recombinant M K6 (U.
  • compounds were serially-diluted from a maximum final concentration of 50 ⁇ , 3-fold drops for 1 1 points to a minimum final concentration of 0.85nM, and 0.1 ⁇ of compounds in DMSO (or neat DMSO for vehicle control) were added to Non-binding 384 well plate (Corning).
  • a final concentration of 15nM FL-Wip l was added to the assay plate in assay buffer (50mM Tri ( pH 7.5), 30mM MgCl 2 , 0.1 mM EGTA, O. l mg/mL BSA, 1 mM CHAPS, 1 mM DTT) for a 1 5min preincubation.
  • MultiLabel Reader Perkin Elmer. Readout in Relative Fluorescent units (FUs) were compared with vehicle ( 1 % DMSO) treated control wells. Curves were analyzed using ActivityBase and XLfit, and XC50 results expressed as pIC50 values.
  • a p53-Luciferase reporter BacMam construct was made that contains a p53 response element ((CCTGGACTTGCCTGGCCTG) 15) upstream of a minimal promoter derived from HSV TK and the firefly Luciferase open reading frame.
  • the construct was cloned into a BacMam expression vector, BacMam virus was cultured from DH 1 Obac cells, and titer was determined.
  • MX-1 breast carcinoma cell line was obtained from the NCI and cultured as recommended in RPMI+10% fetal bovine serum (FBS).
  • MX- 1 cells were trypsinized and viable cells were counted and diluted in OptiMEM (Invitrogen)+ 5% FBS to achieve 10,000 cells per 48uL (2.1 x 10 5 cells/mL).
  • OptiMEM Invitrogen
  • FBS FBS
  • MX- 1 cells were then seeded 48uL/well into 384 well black polystyrene cell culture cr oplates (Greiner) and placed in tissue culture incubator at 37°C and 5% C0 2 for 24 hours. The following day, compounds were serially-diluted by 3-fold drops for 10 point curves.
  • Cell lines used for the cellular outgrowth assay included MX- 1 breast carcinoma line (NCI), A549 lung carcinoma (ATCC), Molt3 T-Cell leukemia (ATCC), and HN5 head and neck carcinoma cell line (ICR-UK). All cel l lines were cultured according to
  • Cell lines were split in duplicate T-75 flasks two to three days prior to assay set-up in ratios which yielded70-80% confluence at time o f harvest for plate seeding. Cells were trypsinized, viable cell concentrations were determined using a using ViCell XR (Beckman Coulter), and cells were resuspended to yield 2,000 cells per ml. Into 96 well black polystyrene cell culture microplates (Nalgene) 96 ds cell suspension was added and seeded cells were allowed to adhere overnight in 37°C + 5% C02 incubator.
  • Exemplified compounds of the present invention were tested according to the above assays and were found to be inhibitors of WIP l .
  • the plCso values ranged from about 5.3-8.5.
  • the IC50 values of the present compounds range from about 3 to 5000 nM.
  • the IC50 values of the more active compounds range from about 3 to 100 nM.
  • the most active compounds are under 10 nM.
  • the plC50 of compounds of Examples 1 -3 and 6-9 ranges from 7.5 to 8.0.
  • the pIC50 of compounds of Examples 14, 1 7, 4 ranges from 8.0-8.5.

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Abstract

This invention relates to the use of acylated alanine derivatives for the modulation, notably the inhibition of the activity of WIP1. Suitably, the present invention relates to the use of acylated alanine derivatives in the treatment of cancer.

Description

NOVEL COMPOUNDS AS W1P1 INHIBITORS
This application claims the benefit of US Provisional Application No. 61 /480454 filed 29 April 201 1 , which is incorporated herein in its entirety.
FIELD OF THE INVENTION This invention relates to the acylated alanine derivatives for the inhibition and/or induced destabilization of the Wipl (PPM1 D/PP2C5) phosphatase. The invention relates to the use of the acylated alanine derivatives in the treatment of cancer.
BACKGROUND OF THE INVENTION Wild-type P53 inducible Phosphatase 1 (Wipl /PPM I D/ PP2C5) is a serine threonine phosphatase that plays a key role in the homeostatic down-regulation of various stress response and DNA repair signaling pathways. Wipl is a member of the type 2C family protein phosphatase family that are dependent on Mg2+ or Mn2+ for their catalytic activity. As its name suggests, Wip l can be induced by various DNA damaging treatments including ionizing or UV irradiation in a P53 dependent manner (Fiscella et al. PNAS 94, 6048-6053 ( 1997)). While its RNA expression is rapidly induced by various DNA damaging agents, translation of the RNA into Wipl protein is reportedly delayed by the miR- 16 micro RNA (Zhang et al., 201 0). Upon its induction, Wipl acts upon multiple effectors of the DNA damage response pathway to dephosphorylate and consequently inactivate them. Among its reported substrates are UNG2 (Lu et al., Molecular Cell 15, 621 -634 (2004)), MDM2 (Lu et al., Cancer Cell 12, 342-354 (2008)), M DMX (Xinna et al., Journal of Biological Chemistry 281 , 24847-24862 (2009)), ATM (Shreeram et al., Journal of Experimental Medicine 203, 2793-2799 (2006)), γΗ2ΑΧ (Cha et al., Cancer Research 70, 41 12-4122 (2010)) , p38MAPK (Bulavin et al., Nature Genetics 36, 343-350 (2004)), Chk2 (Fujimoto et al., Cell Death and Differentiation 13 , 1 1 70- 1 1 80 (2006)), Chk l and p53(Lu et al., Genes and Development 19, 1 162- 1 1 74 ( 2005)).
Several lines of evidence have suggested an oncogenic role for Wip l . I n cellular studies, overexpression of Wip l in E 1 A transduced rat embryonic fibroblasts was sufficient to induce transformation foci (Nannenga et al., Molecular Carcinogenesis 45, 594-604 (2006)). Conversely, murine embryonic fibroblasts homozygous for Wip l deletion showed significantly reduced tumor growth when co-transfected with the transforming oncogenes HRAS and E l A, HRAS and ErbB2, or HRAS and Myc (Bulavin et.al., Nature Genetics 36, 343-350 (2004)). Wip l -/- mice also demonstrated tumor-resistance to both induced tumor models, using infection with M TV driven ErbB2 or HRAS, as well as a decreased incidence of spontaneous leukemias and sarcomas. Similarly, Wipl -/- mice were relatively resistant to Εμ-Myc induced lymphomas (Shreeram et al., Journal of Experimental Medicine 203, 2793-2799 (2006)).
Overexpression of Wipl is reported to result in delay or suppression of base-excision repair by inhibition of UNG2 and P53. Similarly, its overexpression suppresses DNA double-strand-break repair by inhibition of the ATM response, inhibiting repair by both homologous recombination and non-homologous end joining. Conversely, silencing or knockout of Wip l has been reported to elevate the activity of multiple stress response pathways, resulting in elevated levels of phospho-p38 , phospho-p53(S l 5), p53 response genes p21/Waf, p l 6INK4A, and ARF (Bulavin et al., Nature Genetics 36, 343-350 (2004)), and elevated γΗ2ΑΧ and DNA damage-associated nuclear foci (Moon et al., Journal of Biological Chemistry 285, 12935- 12947 (2010)).
Amplified or overexpressed Wip l has also been proposed to promote tumorigenesis in multiple cancers by suppressing the regulatory activity of a host of its tumor suppressor substrates. Amplification of the Wipl /PPM I D gene locus on 17q23 has been reported in breast cancer (Li et al., Nature Genetics 3 1 , 133- 134.(2002)), ovarian clear cell carcinoma (Hirasawa et al., Clinical Cancer Research 9, 1995-2004 (2003)), neuroblastoma (Saito- Ohara et al., Journal of Experimental Medicine 203, 2793-2799 (2003)), and pancreatic adenocarcinoma (Loukopoulos et al., Cancer Science 98, 392-400 (2007)). Moreover, elevated expression of Wip l is reported in mediilloblastomas (Castellino et al., Journal of N euro-Oncology 86, 245-256 (2008)) and gastric carcinomas (Fuku et al., Pathology International 57, 566-571 (2007)). In several of these tumors, gene ampli fication has been confirmed to correlate with elevated protein expression.
Inhibition of Wip l by small molecule inhibitors or by RNA interference has been shown to have antiproliferative effects in tumor cells. In cell culture studies, silencing of Wip l by RNA interference results in elevated phosphorylation and activation of multiple tumor suppressors including p53, Chk2 and γΗ2ΑΧ, and results in tumor cell growth inhibition.(Fujimoto et al., Cell Death and Differentiation 13, 1 1 70- 1 1 80 (2006); Lu et al., Cancer Cell 12, 342-354 (2007)). Several groups have also reportedly identified Wip l inhibitors, describing antiproliferative activity in Wip l amplified or overexpressing cell lines (Belova et al., Cancer Biology and Therapy 4, 1 1 54- 1 1 58. (2005); Rayter et al., Oncogene 27, 1036- 1044 (2008)). Taken together, these observations suggest that Wip l a compelling target for pharmacological inhibition in the treatment of cancer.
SUMMARY OF THE INVENTION
This invention relates to a compound of Formula (I);
Figure imgf000004_0001
(I)
wherein
Ri is hydrogen, Ci-C4alkoxy, C|-C4alkyl, substituted Ci-C4alkyl, C3-C7cycloalkyl, or C3- C7heterocycloalkyl, wherein said C3-C7cycloalkyl may be substituted with one to three substiluents selected from the group consisting of hydroxyl and -0(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl; R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C3alkyl, substituted Ci-C3alkyl, Ci- C alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalk l, cyano, ester, carboxylic acid, and C3-C6heterocycloalkyl;
R3 is hydrogen, C|-C6alkyl, substituted d-Cealkyl, or R3 is absent when X is O or S; R4 is cyclohexyl or cyclopentyl; X is N, O or S; or a pharmaceutically acceptable salt thereof. This invention also relates to pharmaceutical compositions, which comprise compounds of Formula (I) and pharmaceutically acceptable carriers.
This invention also relates to methods of treating cancer which comprise administering an effective amount of a compound of Formula (I) to a human in need thereof. This invention also relates to methods of treating cancer which comprise co-administering an compound of Formula (1) and a second compound to a human in need thereof.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds of Formula (I);
The present invention also relates to compounds of Formula (II):
Figure imgf000005_0001
(Π);
wherein
Ri is hydrogen, C)-C4alkoxy, Ci-C4alkyl, substituted Ci-C4alkyl, C3-C7cycloalkyl, or C3- C7heterocycloalkyl, wherein said C3-C7cycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -O(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl; R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C3alkyl, substituted Ci-C3alkyl, Ci- C4alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalkyl, cyano, ester, carboxylic acid, and C3-C6heterocycloalkyl;
R3 is hydrogen, Ci-C6alkyl, substituted C|-C6alkyl, or R3 is absent when X is O or S; R4 is cyclohexyl or cyclopi
X is , O or S; or a pharmaceutically acceptable salt thereof.
The present invention also relates to compounds of Formula (III):
Figure imgf000006_0001
(i n);
wherein
Ri is hydrogen, Ci-C4alkoxy, C]-C4alkyl, substituted Ci-C4alkyl, C3-C7cycloalkyl, or C3- C7heterocycloalkyl, wherein said C3-C7cycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -0(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl;
R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, C|-C3alkyl, substituted Ci-C3alkyl, C|- C alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalkyl, cyano, ester, carboxylic acid, and C3-C6heterocycloalkyl;
R3 is hydrogen, Ci -C6alkyl, or substituted Ci-C6alkyl;
R4 is cyclohexyl or cyclopentyl; or a pharmaceutically acceptable salt thereof.
The present invention also relates to compounds according to any one of Formulas (1)- (III), wherein Ri is Ci-C4alkyl or substituted C|-C4alkyl.
The present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R] is C3-C7cycloalkyl.
The present invention also relates to compounds according to any one of Formulas (I)- (II I), wherein R| is cyclopropyl or cyclobiityl, wherein said cyclopropyl and cyclobutyl may be substituted with hydroxyl or-0(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci- Cealkyl.
The present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R2 is phenyl, which may be substituted with one to four substituents selected from the group consisting of halogen, Ci-C3alkyl, substituted Ci-C3alkyl, Ci-C4alkoxy, hydroxyl, amino, substituted amino, C3-C7cycloalkyl, C3-C7heterocycloalkyl, cyano, ester, and carboxylic acid.
The present invention also relates to compounds according to any one of Formulas (I)- (III), wherein R2 is pyridinyl, which may be substituted with one to three substituents selected from the group consisting of halo, C|-C3alkyl, and substituted Ci-C3alkyl.
The present invention also relates to compounds according to any one of Formulas (1)- (III), wherein R3 is hydrogen.
The present invention also relates to compounds according to any one of Formulas (I)- (III), wherein R4 is cyclopentyl. The present invention also relates to compounds selected from the group consisting of:
(S-5-{[(5-chloropyridin-3-yl)amino]methyl}-N-{3-cyclopentyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; f'S)-5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}-N-{3-cyclopentyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; CS 5-{[(4-chIoropyridin-2-yl)amino]methyl}-N-{3-cyclopentyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; ¾)-5-{[(5-chloro-pyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l-(oxetan-3-ylamino)- l-oxopropan-2-yl]thiophene-2-carboxamide;
(S-5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l-(oxetan-3- ylamino)-l-oxopropan-2-yl]thiophene-2-carboxamide; fS^-5-{[(4-chloro-pyridin-2-yl)amino]methyl}-N-[3-cyclopentyl-l-(oxetan-3-ylamino)- l-oxopropan-2-yl]thiophene-2-carboxamide; f¾)-N-(l-amino-3-cyclopentyl-l-oxopropan-2-yl)-5-{[(5-chloropyridin-3- yl)amino]methyl}thiophene-2-carboxamide;
(^-5-{[(5-chloropyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l-(methylamino)-l- oxopropan-2-yl]thiophene-2-carboxamide; (^-5-{[(5-chloropyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l-(dimet ylarnino)-l- oxopropan-2-yl]thiophene-2-carboxamide;
(¾)-5-{[(5-chloropyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l-(methoxyamino)-l- oxopropan-2-yl]thiophene-2-carboxamide;
(¾j-5-{[(5-chloropyridin-3-yl)amino]methyl}-N-{3-cyclopent l-l-[(3- hydiOxypropyl)amino]-l-oxopropan-2-yl}thiopliene-2-carboxamide;
(¾)-5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyI}-N-{3-cyclopenty]-l-[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(¾)-5-{[(4-chloropyridin-2-yl)amino]methyl}-N-{3-cyclopent l-l-[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; fS 5-{[(5-chloropyridin-3-yl)amino]methyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(¾ -5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(¾j-5-{[(4-chloropyridin-2-yl)amino]rnethyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(^-5-{[(4-chloropyridin-2-yl)amino]met yl}-N-[3-cyclohexyl-l-(met ylamino)-l- oxopropan-2-yl]thiophene-2-carboxamide;
(¾)-5-{[(5-chloropyridin-3-yI)oxy]methyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; fS-5-{[(5-chloro-2-methylpyridin-3-oxy]methyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide; ^-5-{[(5-chloropyridin-3-yl)oxy]methyl}-N-[3-cyclopentyl-l-(oxetan-3-ylamino)-l- oxopropan-2-yl]thiophene-2-carboxamide;
(^-5-{[(5-chloropyridin-3-yl)oxy]methyI}-N-[3-cyclopentyl-l-(oxetan-3-ylamino)-l- oxopropan-2-yl]thiophene-2-carboxamide; (^-5-{[(3-chlorophenyl)amino]methyl}-N-{3-cyclohexyl-l-[(2-methoxyethyl)am l-oxopropan-2-yl}thiophene-2-carboxamide;
5-{[(5-chIoro-2-methylpyridin-3-yl)amino]methyl}-N^
3-hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}^
3-hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(^-5-{[(5-chloro-2-rnethylpyridin-3-yl)amino]methyl}-N-[3-cyclopentyl-l- (cycIopropy!amino)-l-oxopropan-2-yl]thiophene-2-carboxamide;
^5,5r 3-[^-2-(5-{[(5-chloro-2-methylpyridin -yl)amino]methyl}thiophene-2- carboxamido)-3-cyclopentylpropanamido]cyclobutyl acetate; and (S (7S,3S-3-[^-2-(5-{[(5-chloro-2-me
carboxarnido)-3-cyclopentylpropanamido]cyclobiityl 2,6-diaminohexanoate;
This invention also relates to compounds exemplified in the Experimental section. Typically, but not absolutely, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention. Salts of the compounds of the present invention may comprise acid addition salts. In general, the salts are formed from pharmaceutically acceptable inorganic and organic acids. More specific examples of suitable acid salts include maleic, hydrochloric, hydrobromic, sulphuric, phosphoric, nitric, perchloric, fiimic, acetic, propionic, succinic, glycolic, formic, lactic, aleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methansulfonic (mesylate), naphthalene-2-sulfonic,
benzenesulfonic, hydroxynaphthoic, hydroiodic, malic, teroic, tannic, and the like.
Other representative salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, calcium edetate, camsylate, carbonate, clavulanate, citrate, dihydrochloride, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.
The compound of Formula (I) or a salt thereof may exist in stereoisomeric forms (e.g., it contains one or more asymmetric carbon atoms). The individual stereoisomers (enantiomers and diastereomers) and mixtures of these are included within the scope of the present invention. The invention also covers the individual isomers of the compound or salt represented by Formula (I) as mixtures with isomers thereof in which one or more chiral centers are inverted. Likewise, it is understood that a compound or salt of Formula (I) may exist in tautomeric forms other than that shown in the formula and these are also included within the scope of the present invention. It is to be understood that the present invention includes all combinations and subsets of the particular groups defined hereinabove. The scope of the present invention includes mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerica!ly enriched mixtures. Also included within the scope of the invention are any wholly or partial ly deuteuriated isotopes of the compounds of Formula (1). Also included within the scope of the invention are individual isomers of the compound represented by Formula (I), as well as any wholly or partially equilibrated mixtures thereof. The present invention also includes the individual isomers of the compound or salt represented by the Formula (I) as well as mixtures with isomers thereof in which one or more chiral centers are inverted.
It will be appreciated by those skilled in the art that certain protected derivatives of compounds of formula (1), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". Further, certain compounds of the invention may act as prodrugs of other compounds of the invention. All protected derivatives and prodrugs of compounds of the invention are included within the scope of the invention. It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as "pro- moieties" may be placed on appropriate functionalities when such functionalities are present within compounds of the invention. Preferred prodrugs for compounds of the invention include: esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals. The scope of the present invention also includes prodrugs of the present compounds.
DEFINITIONS
Terms are used within their accepted meanings. The following definitions are meant to clarify, but not limit, the terms defined.
As used herein, the term "alkyl" (or "alkylene") refers to a straight or branched chain alkyl, preferably having from one to twelve carbon atoms, which may be saturated or unsaturated with multiple degrees of substitution included within the present invent ion. Examples of "alkyl" as used herein include methyl, ethyl, propyl, isopropyl, isobutyl, n- butyl, t-butyl, isopentyl, n-pentyl, and the like, as well as substituted versions thereof.
As used herein, the term "substituted alkyl" (or "alkylene") refers to a straight or branched chain alkyl, preferably having from one to twelve carbon atoms, which may be saturated or unsaturated with multiple degrees of substitution included within the present invention. Suitable substituents are selected from the group consisting of: halogen, amino, substituted amino, urea, cyano, hydroxyl, alkoxy, alkylthio, alkylsiil fonyl, amidosulfonyl, carboxylic acid, ester, carboxamide, and aminocarbonyl.
As used herein, the term "cycloalkyl" refers to an unsubstituted or substituted mono- or polycyclic non-aromatic saturated ring, which optionally includes an alkylene linker through which the cycloalkyl may be attached. Exemplary "cycloalkyl" groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, as well as unsubstituted and substituted versions thereof.
As used herein, the term "alkoxy" refers to the group -ORa, where Ra is Ci-Caalkyl or C3-7cycloalkyl as defined above. As used herein, the term "substituted amino" is meant -NR'R" wherein each R' and R" is independently selected from a group including hydrogen, unsubstituted Ci- Cealkyl, acyl, unsubstituted C3-C7cycloalkyl, wherein at least one of R' and R" is not hydrogen. Examples of substituted amino includes, but are not limited to alkylamino, dialkylaminio, acylamino, and cycloalkylamino. As used herein, the term "heterocycle" or "heterocyclyl" or "heterocycloalkyl" refers to unsubstituted and substituted mono- or polycyclic non-aromatic ring system containing one or more heteroatoms. Preferred heteroatoms include N, O, and S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to eight- membered and is either fully saturated or has one or more degrees of nsaturation.
Multiple degrees of substitution, preferably one, two or three, are included within the present definition. Examples of "heterocyclic" groups include, but are not limited to oxetanyl, tetrahydrofuranyl, pyranyl, 1 ,4-dioxanyl, 1 ,3-dioxanyl, piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, piperazinyl, pyrrolidinonyl, piperazinonyl, pyrazolidinyl, and their various tautomers, as well as unsubstituted and substituted versions thereof. As used herein, the term "aryl", unless otherwise defined, is meant aromatic, hydrocarbon, ring system. The ring system may be monocyclic or fused polycyclic (e.g., bicyclic, tricyclic, etc.), substituted or unsubstituted. In various embodiments, the monocyclic aryl ring is C5-C 10, or C5-C7, or C5-C6, where these carbon numbers refer to the number of carbon atoms that form the ring system. A C6 ring system, i.e. a phenyl ring, is a suitable aryl group. In various embodiments, the polycyclic ring is a bicyclic aryl group, where suitable bicyclic aryl groups are C8-C 12, or C9-C 1 0. A naphthyl ring, which has 10 carbon atoms, is a suitable polycyclic aryl group. Suitable substituents for aryl are described in the definition of "optionally substituted".
As used herein, the term "heteroaryl", unless otherwise defined, is meant an aromatic ring system containing carbon(s) and at least one heteroatom. Heteroaryl may be monocyclic or polycyclic, substituted or unsubstituted. A monocyclic heteroaryl group may have 1 to 4 heteroatoms in the ring, while a polycyclic heteroaryl may contain 1 to 10 hetero atoms. A polycyclic heteroaryl ring may contain fused, spiro or bridged ring junctions, for example, bicyclic heteroaryl is a polycyclic heteroaryl. Bicyclic heteroaryl rings may contain from 8 to 12 member atoms. Monocyclic heteroaryl rings may contain from 5 to 8 member atoms (carbons and heteroatoms). Exemplary heteroaryl groups include: benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinol ine, quinazoline, quinoxaline, thiazole, and thiophene. Suitable substituents for heteroaryl are described in the definition of "optionally substituted".
As used herein, the term "cyano" refers to the group -CN.
As used herein, the term "acyl" refers to the group -C(0)Rb, where Rb is alkyl, cycloalkyl, or heterocyclyl, as each is defined herein.
As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and includes both event(s) that occur and event(s) that do not occur.
As used herein, unless otherwise defined, the phrase "substituted" or variations thereof denote a substitution, including multiple degrees of substitution, with one or more substitutents, preferably one, two or three. The phrase should not be interpreted as duplicative of the substitutions herein described and depicted. Exemplary substituent groups include acyl, alkyl, substituted alkyl, alkylsulfonyl, alkoxy, alkoxycarbonyl, cyano, halogen, haloalkyl, hydroxyl, oxo, amide, sulfamide, urea, amino, substituted amino, acylamino, phenylcarbonyl, dialkylaminosulfonamide, morpholino, sul fonamide, thiourea, carboxylic acid, ester, and nitro. The invention further provides a pharmaceutical composition (also referred to as pharmaceutical formulation) comprising a compound of Formula (I) or pharmaceutically acceptable salt, thereof and one or more excipients (also referred to as carriers and/or diluents in the pharmaceutical arts). The excipients are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof (i.e., the patient). In accordance with another aspect of the invention there is provided a process for the preparation of a pharmaceutical composition comprising mixing (or admixing) a compound of Formula (1) or salt thereof with at least one excipient .
PHARMACEUTICAL COMPOSITIONS
Pharmaceutical compositions may be in unit dose form containing a predetermined amount of active ingredient per unit dose. Such a unit may contain a therapeutically effective dose of the compound of Formula (I) or salt thereof or a fraction of a therapeutically effective dose such that multiple unit dosage forms might be administered at a given time to achieve the desired therapeutically effective dose. Preferred unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fract ion thereof, of an active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well-known in t he pharmacy art.
Pharmaceutical compositions may be adapted for administration by any appropriate route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes. Such compositions may be prepared by any method known in the art of pharmacy, for example, by bringing into association the active ingredient with the excipient(s).
When adapted for oral administration, pharmaceutical compositions may be in discrete units such as tablets or capsules; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; oil-in-water l iquid emulsions or water-in-oil liquid emulsions. The compound or salt thereof of the invention or the pharmaceutical composition of the invention may also be incorporated into a candy, a wafer, and/or tongue tape formulation for administration as a "quick-dissolve" medicine.
For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Powders or granules are prepared by comminuting the compound to a suitable fine size and mixing with a simi larly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing, and coloring agents can also be present. Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin or non-gelatinous sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate, solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or sokibil izing agent such as agar-agar, calcium carbonate, or sodium carbonate can also be added to improve the availability of the medicine when the capsule is ingested.
Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars, such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum, and the l ike. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant, and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, and aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt, and/or an absorption agent such as - bentonite, kaolin, or dicalcium phosphate. The powder mixture can be granulated by wetting a binder such as syrup, starch paste, acadia mucilage, or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc, or mineral oil. The lubricated mixture is then compressed into tablets. The compound or salt of the present invention can also be combined with a free-flowing inert carrier and compressed into tablets directly without going through the granulating orslugging steps. A clear opaque protective coating consisting of a sealing coat of shellac, a coating of sugar, or polymeric material, and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different dosages. Oral fluids such as solutions, syrups, and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of active ingredient.
Syrups can be prepared by dissolving the compound or salt thereof of the invention in a suitably flavoured aqueous solution, while elixirs are prepared through the use of a non- toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound or salt of the invention in a non-toxic vehicle. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additives such as peppermint oil, natural sweeteners, saccharin, or other artificial sweeteners, and the like, can also be added. Where appropriate, dosage unit formulations for oral adm inistration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as, for example, by coating or embedding particulate material in polymers, wax, or the like.
In the present invention, tablets and capsules are preferred for delivery of the pharmaceutical composition.
As used herein, the term "treatment" includes prophylaxis and refers to alleviating the specified condition, eliminating or reducing one or more symptoms of the condition, slowing or eliminating the progression of the condition, and preventing or delaying the reoccurrence of the condition in a previously afflicted or diagnosed patient or subject. Prophylaxis (or prevention or delay of disease onset) is typically accomplished by administering a drug in the same or similar manner as one would to a patient with the developed disease or condition.
The present invention provides a potential treatment in a mammal, especially a human, suffering from disease conditions targeted by the present compounds. Such treatment comprises the step of administering a therapeutically effective amount of a compound of Formula (I) or salt thereof to said mammal, particularly a human. Treatment can also comprise the step of administering a therapeutically effective amount of a pharmaceutical composition containing a compound of Formula (I) or salt thereof to said mammal, particularly a human. As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician.
The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For use in therapy, therapeutically effective amounts of a compound of Formula (1), as well as salts thereof, may be administered as the raw chemical. Additionally, the active ingredient may be presented as a pharmaceutical composition.
While it is possible that, for use in therapy, a therapeutically effective amount of a compound of Formula (I) or salt thereof may be administered as the raw chemical, it is typically presented as the active ingredient of a pharmaceutical composit ion or formulation.
The precise therapeutically effective amount of a compound or salt thereof of the invention will depend on a number of factors, including, but not limited to, the age and weight of the subject (patient) being treated, the precise disorder requiring treatment and its severity, the nature of the pharmaceutical formulation/composition, and route of administration, and will ultimately be at the discretion of the attending physician or veterinarian. Typically, a compound of Formula (1) or salt thereof wi ll be given for the treatment in the range of about 0.01 to 100 mg/kg body weight of recipient (patient, mammal) per day and more usually in the range of 0.1 to 10 mg/kg body weight per day. Acceptable daily dosages may be from about 1 to about 1000 mg/day, and preferably from about 1 to about 1 00 mg/day. This amount may be given in a single dose per day or in a number (such as two, three, four, five, or more) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt thereof may be determined as a proportion of the effective amount of the compound of Formula (I) per se. Similar dosages should be appropriate for treatment (including prophylaxis) of the other conditions referred herein for treatment. In general, determination of appropriate dosing can be readily arrived at by one skilled in medicine or the pharmacy art. COMBINATIONS
When a compound of Formula (I) is administered for the treatment of cancer, the term "co-administering" and derivatives thereof as used herein is meant either simultaneous administration or any manner of separate sequential administration of a LSD 1 inhibiting compound, as described herein, and a further active ingredient or ingredients, known to be useful in the treatment of cancer, including chemotherapy and radiation treatment. The term further active ingredient or ingredients, as used herein, includes any compound or therapeutic agent known to or that demonstrates advantageous properties when administered to a patient in need of treatment for cancer. Preferably, if the administration is not simultaneous, the compounds are administered in a close time proximity to each other. Furthermore, it does not matter i f the compounds are administered in the same dosage form, e.g. one compound may be administered topically and another compound may be administered orally.
Typically, any anti-neoplastic- agent that has activity versus a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Pract ice f Oncology by V.T. Devita and S. Hellman (editors), 6lh edition (February 15, 2001 ), Lippincott Wi lliams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Typical anti-neoplastic agents useful in the present invention include, but are not limited to, anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase 11 inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and anti- folate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors. Examples of a further active ingredient or ingredients for use in combination or coadministered with the present LSD 1 inhibiting compounds are chemotherapeutic agents. Anti-microtubule or anti-mitotic agents are phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.
Diterpenoids, which are derived from natural sources, are phase specific anti - cancer agents that operate at the G2/M phases of the cell cycle. It is believed that the diterpenoids stabilize the β-tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following. Examples of diterpenoids include, but are not limited to, paclitaxel and its analog docetaxel. Paclitaxel, 5 p,20-epoxy- l ,2a,4,7P, 10p, 13a-hexa-hydroxytax- l l -en-9-one 4, 1 0- diacetate 2-benzoate 13-ester with (2R,3 S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL®. It is a member of the taxane family of terpenes. It was first isolated in 1971 by Wani et al. J. Am. Chem, Soc, 93 :2325. 1971 ), who characterized its structure by chemical and X-ray crystallographic methods. One mechanism for its activity relates to paclitaxel's capacity to bind tubulin, thereby inhibiting cancer cell growth. Schiff et al., Proc. Natl, Acad, Sci. USA, 77: 1 561 - 1 565 ( 1980); Schiff et al., Nature, 277:665-667 ( 1979); Kumar, J. Biol, Chem, 256: 1 0435- 10441 (1981 ). For a review of synthesis and anticancer activity of some paclitaxel derivatives see: D. G. I. Kingston et al., Studies in Organic Chemistry vol. 26, entitled "New trends in Natural Products Chemistry 1986", Attaur-Rahman, P.W. Le Quesne, Eds. (Elsevier, Amsterdam, 1986) pp 219-235.
Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman et al., Yale Journal of Biology and Medicine, 64:583, 1991 ; McGuire et al., Ann. Intern, Med., 1 1 1 :273, 1989) and for the treatment of breast cancer (Holmes et al., J. Nat. Cancer Inst., 83 : 1 797, 1991 .) It is a potential candidate for treatment of neoplasms in the skin (Einzig et. al., Proc. Am. Soc. Clin. Oncol., 20:46) and head and neck carcinomas (Forastire et. al., Sem. Oncol., 20:56, 1 990). The compound also shows potential for the treatment of polycystic kidney disease (Woo et. al., Nature, 368:750. 1994), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lineages, Ignoff, R.J. et. al, Cancer Chemotherapy Pocket Guic^ 1998) related to the duration of dosing above a threshold concentration (50nM) (Kearns, CM. et. al., Seminars in Oncology, 3(6) p.16-23, 1 995).
Docetaxel, (2R,3S)- N-carboxy-3-phenylisoserine,N- r/-butyl ester, 1 3-ester with 5P-20-epoxy- l ,2a,4,7P, 10p, 13ot-hexahydroxytax- l l -en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE®. Docetaxel is indicated for the treatment of breast cancer. Docetaxel is a semisynthetic derivative of paclitaxel q. v. , prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree. The dose limiting toxicity of docetaxel is neutropenia.
Vinca alkaloids are phase specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine.
Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN® as an injectable solution. Although, it has possible indication as a second line therapy of various solid tumors, it is primarily indicated in the treatment of testicular cancer and various lymphomas including Hodgkin's Disease; and lymphocytic and histiocytic lymphomas. Myelosuppression is the dose limiting side effect of vinblastine.
Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commercially available as ONCOVIN® as an injectable solution. Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non-Hodgkin's malignant lymphomas. Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosiipression and gastrointestinal mucositis effects occur.
Vinorelbine, 3 ',4'-didehydro -4'-deoxy-C'-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate ( 1 :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELB INE®), is a semisynthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose limiting side effect of vinorelbine.
Platinum coordination complexes are non-phase specific anti-cancer agents, which are interactive with DNA. The platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand crosslinks with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.
Cisplatin, cis-diamminedichloroplatinum, is commercially available as PLAT1NOL® as an injectable solution. Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. The primary dose limiting side effects of cisplatin are nephrotoxicity, which may be controlled by hydration and diuresis, and ototoxicity.
Carboplatin, platinum, diammine [l , l -cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN® as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma. Bone marrow suppression is the dose limiting toxicity of carboplatin.
Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death. Examples of alkylating agents include, but are not lim ited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine.
Cyclophosphamide, 2-[bis(2-chloroethy I)amino]tetrahydro-2H- 1 ,3,2- oxazaphosphorine 2-oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN®. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea, vomiting and leukopenia are the most common dose limiting side effects of cyclophosphamide.
Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN®. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary. Bone marrow suppression is the most common dose lim iting side effect of melphalan.
Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN® tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. Bone marrow suppression is the most common dose limiting side effect of chlorambucil.
Busulfan, 1 ,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® TABLETS. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose limiting side effects of busulfan.
Carmustine, l ,3-[bis(2-chloroethyl)- l -nitrosourea, is commercially available as single vials of lyophilized material as BiCNU®. Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppression is the most common dose limiting side effects of carmustine.
Dacarbazine, 5-(3 ,3-dimethyl- l -triazeno)-imidazole-4-carboxamide, is commercially available as single vials of material as DTIC-Dome®. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dacarbazine.
Antibiotic anti-neoplastics are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death. Examples of antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin; and bleomycins.
Dactinomycin, also know as Actinomycin D, is commercially available in injectable form as COSMEGEN®. Dactinomycin is indicated for the treatment of Wilm's tumor and rhabdomyosarcoma. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dactinomycin.
Daunorubicin, (8S-cis-)-8-acetyl- 10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- hexopyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8, 1 1 -trihydroxy-1 -methoxy-5, 12
naphthacenedione hydrochloride, is commercially available as a liposomal injectable form as DAUNOXOME® or as an injectable as CERUBIDINE®. Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma. Myelosuppression is the most common dose limiting side effect of daunorubicin.
Doxorubicin, (8S, 10S)- 10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- hexopyranosyl)oxy]-8-glycoloyl, 7,8,9, 10-tetrahydro-6,8, l 1 -trihydroxy- 1 -methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX® or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas. Myelosuppression is the most common dose limiting side effect of doxorubicin.
Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is commercially available as BLENOXANE®. Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas. Pulmonary and cutaneous toxicities are the most common dose limiting side effects of bleomycin.
Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.
Epipodophyllotoxins are phase specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death fol lows. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.
Etoposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-( )-ethylidene-P-D- glucopyranoside], is commercially available as an injectable solution or capsules as VePESI D® and is commonly known as VP- 16. Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and non- small cell lung cancers. Myelosuppression is the most common side effect of etoposide. The incidence of leucopenia tends to be more severe than thrombocytopenia.
Teniposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-thenylidene-P-D- glucopyranoside], is commercially available as an injectable solution as VUMON® and is commonly known as VM-26. Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia in children. Myelosuppression is the most common dose limiting side effect of teniposide. Teniposide can induce both leucopenia and thrombocytopenia. Antimetabolite neoplastic agents are phase specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows. Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.
5-fluorouracil, 5-fluoro-2,4- (1 H,3H) pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death. 5-fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Myelosuppression and mucositis are dose limiting side effects of 5- fluorouracil. Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate.
Cytarabine, 4-amino- l-P-D-arabinofuranosyl-2 ( 1 H)-pyrimidinone, is commercial ly available as CYTOSAR-U® and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include 5-azacytidine and 2',2 '-difluorodeoxycytidine (gemcitabine). Cytarabine induces leucopenia, thrombocytopenia, and mucositis.
Mercaptopurine, l ,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PUR1NETHOL®. Mercaptopurine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Myelosuppression and gastrointestinal mucositis are expected side effects of mercaptopurine at high doses. A useful mercaptopurine analog is azathioprine.
Thioguanine, 2-amino- l ,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID®. Thioguanine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Myelosuppression, including leucopenia, thrombocytopen ia, and anemia, is the most common dose limiting side effect of thioguanine administration. However, gastrointestinal side effects occur and can be dose limiting. Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine.
Gemcitabine, 2'-deoxy-2 ', 2'-difluorocytidine monohydrochloride (β-isomer), is commercially available as GEMZAR®. Gemcitabine exhibits cell phase specificity at S- phase and by blocking progression of cells through the G l /S boundary. Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer. Myelosuppression, including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of gemcitabine adm inistration.
Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl]methylamino] benzoyl]-L- glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate. Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder. Myelosuppression (leucopenia, thrombocytopenia, and anemia) and mucositis are expected side effect of methotrexate administration.
Camptothecins, including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors. Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to irinotecan, topotecan, and the various optical forms of 7-(4-methylpiperazino-methylene)- 1 0, l l -ethylenedioxy-20-camptothecin described below.
Irinotecan HC1, (4S)-4, 1 l -diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]- ] H-pyrano[3 ',4', 6,7]indolizino[ l ,2-b]quinoline-3, 14(411, 1 2H)-dione hydrochloride, is commercially available as the injectable solution CAMPTOSAR®.
Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I - DNA complex. It is bel ieved that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I : DNA : irintecan or SN-38 ternary complex with replication enzymes. Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum. The dose limiting side effects of irinotecan HC1 are myelosuppression, including neutropenia, and GI effects, including diarrhea.
Topotecan HC1, (S)- 10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy- l H- pyrano[3 ',4',6,7] indolizino[l ,2-b]quinoline-3, 14-(4H, 12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN®. Topotecan is a derivative of camptothecin which binds to the topoisomerase I - DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule. Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer. The dose limiting side effect of topotecan HCI is myelosuppression, primarily neutropenia. Also of interest, is the camptothecin derivative of formula A following, currently under development, including the racemic mixture (R,S) form as well as the R and S enantiomers:
Figure imgf000027_0001
known by the chemical name "7-(4-methylpiperazino-methylene)- 10, l 1 -ethylenedioxy- 20(R,S)-camptothecin (racemic mixture) or "7-(4-methylpiperazino-methylene)- 1 0, l 1 - ethylenedioxy-20(R)-camptothecin (R enantiomer) or "7-(4-methylpiperazino-methylene)- 1 0, 1 l -ethylenedioxy-20(S)-camptothecin (S enantiomer). Such compound as well as related compounds are described, including methods of making, in U.S. Patent Nos. 6,063,923; 5,342,947; 5,559,235; 5,491 ,237 and pending U.S. patent Application No. 08/977,217 filed November 24, 1997.
Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer. Examples of hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children ; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrms such as megestrol acetate useful in the treatment of hormone dependent breast cancer and endometrial carcinoma; estrogens, androgens, and anti-androgens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a-reductases such as finasteride and dutasteride, useful in the treatment of prostatic carcinoma and benign prostatic hypertrophy; anti- estrogens such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, as well as selective estrogen receptor modulators (SERMS) such those described in U.S. Patent Nos. 5,681 ,835, 5,877,219, and 6,207,716, useful in the treatment of hormone dependent breast carcinoma and other susceptible cancers; and gonadotropin-releasing hormone (GnRH) and analogues thereof which stimulate the release of leutinizing hormone (LH) and/or follicle stimulating hormone (FSH) for the treatment prostatic carcinoma, for instance, LHRH agonists and antagagonists such as goserelin acetate and luprolide.
Signal transduction pathway inhibitors are those inhibitors, which block or inhibit a chemical process which evokes an intracellular change. As used herein this change is cell proliferation or differentiation. Signal tranduction inhibitors useful in the present invention include inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3domain blockers, serine/threonine kinases, phosphotidy! inositol-3 kinases, myoinositol signaling, and Ras oncogenes.
Several protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.
Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are generally termed growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, i.e. aberrant kinase growth factor receptor activity, for example by over- expression or mutation, has been shown to result in uncontrol led cell growth. Accordingly, the aberrant activity of such kinases has been linked to malignant tissue growth. Consequently, inhibitors of such kinases could provide cancer treatment methods. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2, erbB4, vascu lar endothelial growth factor receptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (TIE-2), insulin growth factor -I (IGFI) receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FG F) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptors, and the RET protooncogene. Several inhibitors of growth receptors are under development and include ligand antagonists, antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides. Growth factor receptors and agents that inhibit growth factor receptor function are described, for instance, in Kath, John C, Exp. Opin. Ther. Patents (2000) 1 0(6): 803-818; Shawver et al DDT Vol 2, No. 2 February 1997; and Lofts, F. J. et al, "Growth factor receptors as targets", New Molecular Targets for Cancer Chemotherapy, ed. Workman, Paul and Kerr, David, CRC press 1994, London.
Tyrosine kinases, which are not growth factor receptor kinases are termed nonreceptor tyrosine kinases. Non-receptor tyrosine kinases useful in the present invention, which are targets or potential targets of anti-cancer drugs, include cSi c, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Such non- receptor kinases and agents which inhibit non-receptor tyrosine kinase function are described in Sinh, S. and Corey, S.J., ( 1999) Journal of Hematotherapy and Stem Cell Research 8 (5): 465 - 80; and Bolen, J.B., Brugge, J.S., ( 1 997) Annual review of Immunology. 15 : 371 -404.
SH2/SH3 domain blockers are agents that disrupt SH2 or SJ-I3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunil, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussed in Smithgall, T.E. ( 1995), Journal o f Pharmacological and Toxicological Methods. 34(3) 125-32.
Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracel lular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta). IkB kinase family (IKKa, I Kb), PKB family kinases, AKT kinase family members, and TGF beta receptor kinases. Such Serine/Threonine kinases and inhibitors thereof are described in Yamamoto, T., Taya, S., Kaibuchi, K., ( 1 999), Journal of B iochemistry. 126 (5) 799-803; Brodt, P, Samani, A., and Navab, R. (2000), B iochemical Pharmacology, 60. 1 101 - 1 107; Massague, J., Weis-Garcia, F. ( 1 996) Cancer Surveys. 27:41 -64; Philip, P.A., and Harris, A.L. (1995), Cancer Treatment and Research. 78: 3-27, Lackey, K. et al Bioorganic and Medicinal Chemistry Letters, ( 1 0), 2000, 223-226; U.S. Patent No. 6,268,391 ; and Martinez-Iacaci, L., et al, Int. J. Cancer (2000), 88( 1 ), 44-52. Inhibitors of Phosphotidyl inosito!-3 Kinase family members including blockers of P13-kinase, ATM, DNA-PK, and Ku are also useful in the present invention. Such kinases are discussed in Abraham, R.T. (1996), Current Opinion in Immunology. 8 (3) 412-8; Canman, C.E., Lim, D.S. ( 1998), Oncogene 1 7 (25) 3301-3308; Jackson, S.P. ( 1 997), International Journal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H. et al, Cancer res, (2000) 60(6), 1541 - 1545.
Also useful in the present invention are Myo-inositol signaling inhibitors such as phospholipase C blockers and Myoinositol analogues. Such signal inhibitors are described in Powis, G., and Kozikowski A., ( 1994) New Molecular Targets for Cancer Chemotherapy ed., Paul Workman and David Kerr, CRC press 1994, London.
Another group of signal transduction pathway inhibitors are inhibitors of Ras Oncogene. Such inhibitors include inhibitors of farnesyltransfei ase, geranyl-geranyl transferase, and CAAX proteases as well as anti-sense oligonucleotides, ribozymes and immunotherapy. Such inhibitors have been shown to block ras activation in cells containing wild type mutant ras , thereby acting as antiproliferation agents. Ras oncogene inhibition is discussed in Scharovsky, O.G., Rozados, V.R., Gervasoni, S. I. Matar, P. (2000), Journal of Biomedical Science. 7(4) 292-8; Ashby, M.N. ( 1 998), Current Opinion in Lipidology. 9 (2) 99 - 102; and BioChim. B iophys. Acta, (19899) 1423(3): 1 9-30.
As mentioned above, antibody antagonists to receptor kinase ligand binding may also serve as signal transduction inhibitors. This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand binding domain of receptor tyrosine kinases. For example Imclone C225 EGFR specific antibody (see Green, M.C. et al, Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev., (2000), 26(4), 269-286); Herceptin ® erbB2 antibody (see Tyrosine Kinase Signalling in Breast cancerrerbB Family Receptor Tyrosine Kniases, Breast cancer Res., 2000, 2(3), 1 76- 1 83); and 2CB VEGFR2 specific antibody (see Brekken, R.A. et al, Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice, Cancer Res. (2000) 60, 51 1 7-5124).
Non-receptor kinase angiogenesis inhibitors may also find use in the present invention. Inhibitors of angiogenesis related VEGFR and ΤΓΕ2 are discussed above in regard to signal transduction inhibitors (both receptors are receptor tyrosine kinases). Angiogenesis in general is linked to erbB2/EGFR signaling since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression. Thus, the combination of an erbB2/EGFR inhibitor with an inhibitor of angiogenesis makes sense. Accordingly, non-receptor tyrosine kinase inhibitors may be used in combination with the EGFR/erbB2 inhibitors of the present invention. For example, anti-VEGF antibodies, which do not recognize VEGFR (the receptor tyrosine kinase), but bind to the ligand; small molecule inhibitors of integrin (alphav betas) that will inhibit angiogenesis; endostatin and angiostatin (non-RTK) may also prove useful in combination with the disclosed erb family inhibitors. (See Bruns CJ et al (2000), Cancer Res., 60: 2926-2935; Schreiber AB, Winkler ME, and Derynck R. (1986), Science, 232: 1250- 1 253 ; Yen L et al. (2000), Oncogene 19: 3460-3469).
Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I). There are a number of immunologic strategies to generate an immune response against erbB2 or EGFR. These strategies are generally in the realm of tumor vaccinations. The efficacy of immunologic approaches may be greatly enhanced through combined inhibition of erbB2/EGFR signaling pathways using a small molecule inhibitor. Discussion of the immunologic/tumor vaccine approach against erbB2/EGFR are found in Reilly RT et al. (2000), Cancer Res. 60: 3569-3576; and Chen Y, Hu D, Eling DJ, Robbins J, and Kipps TJ. (1998), Cancer Res. 58 : 1 965- 1971 . Agents used in proapoptotic regimens (e.g., bcl-2 antisense oligonucleotides) may also be used in the combination of the present invention. Members of the Bcl-2 family of proteins block apoptosis. Upregulation of bcl-2 has therefore been linked to chemoresistance. Studies have shown that the epidermal growth factor (EGF) stimulates anti-apoptotic members of the bcl-2 family (i.e., mcl- 1 ). Therefore, strategies designed to downregulate the expression of bcl-2 in tumors have demonstrated cl inical benefit and are now in Phase 11/111 trials, namely Genta's G3 139 bcl-2 antisense oligonucleotide. Such proapoptotic strategies using the antisense oligonucleotide strategy for bcl-2 are discussed in Water JS et al. (2000), J. Clin. Oncol. 1 8 : 1 812- 1 823 ; and Kitada S et al. (1994), Antisense Res. Dev. 4: 71 -79.
Cell cycle signalling inhibitors inhibit molecules involved in the control of the cell cycle. A family of protein kinases called cyclin dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle. Several inhibitors of cell cycle signalling are under development. For instance, examples of cyclin dependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for the same are described in, for instance, Rosania et al, Exp. Opin. Ther. Patents (2000) 1 0(2):21 5-230. Further, p21 WAF 1 /CIP 1 has been described as a potent and universal inhibitor of cyclin- dependent kinases (Cdks) (Ball et al., Progress in Cell Cycle Res., 3: 125 ( 1997)).
Compounds that are known to induce expression of p21 WAF l /CIP l have been implicated in the suppression of cell proliferation and as having tumor suppressing activity (Richon et al., Proc. Nat Acad. Sci. U.S.A. 97(18): 10014- 1001 9 (2000)), and are included as cell cycle signaling inhibitors.
Modulators of the Retinoid Acid Receptor have been used to treat leukemias. The pathology of the leukemia is associated with the abnormal accumulation of immature progenitor cells that are sensitive to retinoc acid therapy. The majority of cases of acute promyelocytic leukemia (APL), also called acute myeloid leukemia subtype M3, involve a chromosomal translocation of chromosomes 15 and 17 that causes genet ic fusion of the retinoic acid receptor (RAR) gene to the promyelocytic leukemia (PA4L) gene. This fusion PML-RAR protein is responsible for preventing immature myeloid cells from
differentiating into more mature cells. This block in differentiation is and subsequent accumulation of less differentiated cells is thought to cause leukemia. ATRA, Tretinoin, acts on PML-RAR to lift this block, causing the immature promyelocytes to differentiate to normal mature blood cells thus decreasing promyelocytes and promoting a population of terminally differentiated cells with a restricted lifespan. Talazorole is an experimental drug in the same class as Tretinoin.
Epigenetic alterations have been implicated in virtually all types of human cancers.
Cancer specific changes are often associated with silencing of tumor suppressor genes via histone modifications and modi fications to DNA including DNA hypermethylation.
Epigenetic pharmaceuticals control regulatory regions associated with tumor suppressor genes by causing conformational changes in histones and removing repressive
modifications to DNA. These changes directly affect the formation and progression of cancer. Examples of epigenetic agents include histone deacetylase inhibitors and DNA methylation inhibitors.
Histone deacetylase inhibitors (HDAC inhibitors, HDI) are a class of compounds that interfere with the function of histone deacetylases. Inhibitors of histone deacetylases have been shown to be useful in the treatment of cutaneous T-cell lymphoma. They are being investigated in the clinic for multiple other tumor types. Examples of HDAC inhibitors approved for use are Vorinostat and Romidepsin. .These compounds are thought to inhibit the activity of HDACs and result in the accumulation of acetylation to histones promoting gene expression.
Azacitidine (INN) or 5-azacytidine, sold under the trade name Vidaza, is a chemical analogue of cytidine, a nucleoside present in DNA and RNA. Azacit idine and its deoxy derivative, decitabine (also known as 5-aza-2'deoxycytidine), are used in the treatment of myelodysplasia syndrome and are currently under study for other tumor indications. Azacitidine acts as a false substrate and potent inhibitor of DNA methyltransferases leading to reduction of DNA methylation. DNA methyltransferases incorporate azacitidine into DNA during replication and into RNA during transcription in the cell. Inhibition of DNA methylation occurs through the formation of stable complexes between the molecule and DNA methyltransferases, thereby saturating cell methylation machinery. This results in a loss of DNA methylation and can affect the way cell regulation proteins, such as transcriptional machinery, are able to associate with the DNA.
Examples of such HDAC inhibitors include:
1 . Vorinostat, including pharmaceutically acceptable salts thereof. Marks et al., Nature Biotechnology 25, 84 to 90 (2007); Stenger, Community Oncology 4, 384-386 (2007).
Vorinostat has the following chemical structure and name:
Figure imgf000033_0001
N-hydroxy-N-phenyl-octanediamide 2. Romidepsin, including pharmaceutically acceptable salts thereof. Vinodhkumar et al., Biomedicine & Pharmacotherapy 62 (2008) 85-93. Romidepsin, has the following chemical structure and name:
Figure imgf000034_0001
(1 S.4S.7Z, 1 OS, 16E,21 R)-7-ethylidene-4,21 -di(propan-2-y l)-2-oxa- 12, 13-dithia-5,8,20,23- tetrazabicyclo[8.7.6]tricos- 16-ene-3,6,9, 1 9,22-pentone
3. Panobinostat, including pharmaceutically acceptable salts thereof. Drugs of the Future 32(4): 315-322 (2007).
Panobinostat, has the following chemical structure and name:
Figure imgf000034_0002
(2£)-N-hydroxy-3-[4-({[2-(2-methyl- l H-indol-3- yl)ethyl]amino} methyl)phenyl]acrylamide 4. Valproic acid, including pharmaceutically acceptable salts thereof. Gottlicher, et al., EMBO J. 20(24): 6969-6978 (2001).
Valproic acid, has the following chemical structure and name:
CH3— CH2— CH2
CH—
CH3— CH2— CH2 /' "OH
2-propylpentanoic acid
5. Mocetinostat (MGCDOl 03), including pharmaceutically acceptable salts thereof. Balasubramanian et al., Cancer Letters 280: 21 1 -221 (2009). Mocetinostat, has the following chemical structure and name:
Figure imgf000035_0001
N-(2-Aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide
Further examples of such HDAC inhibitors are included in Bertrand European Journal of Medicinal Chemistry 45, (2010) 2095-21 16, particularly the compounds of table 3 therein as indicated below.
Figure imgf000036_0001
Proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins, like the p53 protein. Several proteasome inhibitors are marketed or are being studied in the treatment of cancer. Suitable proteasome inhibitors for use in combination herein include: 1. Bortezomib (Velcade®), including pharmaceutically acceptable salts thereof. Adams J, Kauffman M (2004), Cancer Invest 22 (2): 304-1 1 .
Bortezomib has the following chemical structure and name.
Figure imgf000037_0001
[( l R)-3-methyl- l -({(2S)-3-phenyl-2-[(pyrazm-2- ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid
2. Disulfiram, including pharmaceutically acceptable salts thereof. Bouma et al. ( 1998). J. Antimicrob. Chemother. 42 (6): 817-20. Disulfiram has the following chemical structure and name.
Figure imgf000037_0002
1 , 1 ', 1 ", 1 "'-[disulfanediylbis(carbonothioy lnitrilo)]tetraethane
3. Epigallocatechin gallate (EGCG), including pharmaceutically acceptable salts thereof. Williamson et al., (December 2006), The Journal of Allergy and Clinical Immunology 118 (6): 1369-74.
Epigallocatechin gallate has the following chemical structure and name.
Figure imgf000038_0001
[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3)4,5- trihydroxybenzoate
4. Salinosporamide A, including pharmaceutically acceptable salts thereof. Feling et at., (2003), Angew. Chem. Int. Ed. Engl. 42 (3): 355-7.
Salinosporamide A has the following chemical structure and name.
Figure imgf000038_0002
(4R,5 S)-4-(2-chloroethyl)- l -(( l S)-cyclohex-2-enyI(hydroxy)methyl) -5-methyl-6-oxa-2- azabicyclo3.2.0heptane-3,7-dione
5. Carfilzomib, including pharmaceutically acceptable salts thereof. uhn DJ, et al, Blood, 2007, 1 1 0:3281 -3290.
Carfilzomib has the following chemical structure and name.
Figure imgf000039_0001
(S)-4-methyl-N-((S)- l -(((S)-4-methyl- l -((R)-2-methyloxiran-2-yl)- l -oxopentan-2- yl)amino)- l -oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4- phenylbutanamido)pentanamide
The 70 kilodalton heat shock proteins (Hsp70s) and 90 kilodalton heat shock proteins (Hsp90s) are a families of ubiquitously expressed heat shock proteins. Hsp70s and Hsp90s are over expressed certain cancer types. Several Hsp70s and Hsp90s inhibitors are being studied in the treatment of cancer. Suitable Hsp70s and Hsp90s inhibitors for use in combination herein include:
1 . 17-AAG(Geldanamycin), including pharmaceutically acceptable salts thereof.
J ia W et al. Blood. 2003 Sep 1 ; 102(5): 1 824-32.
17-AAG(Geldanamycin) has the following chemical structure and name.
Figure imgf000040_0001
17-(AUylamino)- 17-demethoxygeldanamycin
2. Radicicol, including pharmaceutically acceptable salts thereof. (Lee et al.,
Mol Cell Endocrinol.2002, 188,47-54) Radicicol has the following chemical structure and name.
Figure imgf000040_0002
(laR,2Z,4E,14R,15aR)-8-chloro-9,ll-dihydroxy-14-methyl-15,15a-dihydro-laH- benzo[c]oxireno[2,3-k][l]oxacyclotetradecine-6,12(7H,14H)-dione
Inhibitors of cancer metabolism - Many tumor cells show a markedly different metabolism from that of normal tissues. For example, the rate of glycolysis, the metabol process that converts glucose to pyruvate, is increased, and the pyruvate generated is reduced to lactate, rather than being further oxidized in the mitochondria via the tricarboxylic acid (TCA) cycle. This effect is often seen even under aerobic conditions and is known as the Warburg Effect. Lactate dehydrogenase A (LDH-A), an isoform of lactate dehydrogenase expressed in muscle cells, plays a pivotal role in tumor cell metabolism by performing the reduction of pyruvate to lactate, which can then be exported out of the cell. The enzyme has been shown to be upregulated in many tumor types. The alteration of glucose metabolism described in the Warburg effect is critical for growth and proliferation of cancer cells and knocking down LDH-A using RNA-i has been shown to lead to a reduction in cell proliferation and tumor growth in xenograft models.
D. A. Tennant et. al., Nature Reviews, 2010, 267. P. Leder, et. al., Cancer Cell, 2006, 9, 425.
High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions. Pharmacological inhibition of FAS affects the expression of key oncogenes involved in both cancer development and maintenance.
Alii et al. Oncogene (2005) 24, 39-46. doi: 10.1 038
Inhibitors of cancer metabolism, including inhibitors of LDH-A and inhibitors of fatty acid biosynthesis (or FAS inhibitors), are suitable for use in combination with the compounds of this invention.
In one embodiment, the cancer treatment method of the claimed invention includes the co-administration a compound of Formula (I) and/or a pharmaceutically acceptable salt, hydrate, solvate or pro-drug thereof and at least one anti-neoplastic agent, such as one selected from the group consisting of anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors,
antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, FAS inhibitors, HDAC inhibitors, LDH-A inhibitors and cell cycle signaling inhibitors.
GENERAL SYNTHETIC SCHEMES
Figure imgf000042_0001
I. a: Protection step, for example: CbzCI, NaOH, water ; I.b, I.d: Amidification step, for example: EDC, HOAt, DIEA, DMF ; l.c: Deprotection step, for example: ¾ Pd/C, EtOAc, EtOH. Many different protecting groups are available to one skilled in the art and can be used here as long as they do not interfere with the processes listed herein. Methods for the protection of amines are described in standard reference volumes, such as Greene "Protective Groups in Organic Synthesis" (published by Wiley-Interscience). For example, the amino group of Aminoacid 1 can be protected by, but not exclusively, tert- butyl carbamate (Boc), (9H-fluoren-9-yl)methyl (Fmoc) or benzyl carbamate (Cbz).
Typical conditions include (Boc^O, FmocCI or CbzCI, with or without a base like NaOH, NaHC03 or triethylamine in an organic solvent like THF, ACN, DM F, EtOAc or even in water. Protected amino-acid 2 can be transformed into the amide 3 by typical amide coupling conditions. A variety of amide coupling reagents such as EDC, HOBt, HOAt, HATU, PyBrop, etc. are commercially available and can be used here. Amide coupling reactions are generally run in solvents such as DC or DMF, utilizing an organic base like ET3N or (i-Pr)2NEt. The amino group of 3 was deprotected to afford 4. Many deprotection of the amino group are available to one skilled in the art and can be used here as long as they do not interfere with the processes listed herein. Methods for the deprotection of amines are described in standard reference volumes, such as Greene "Protective Groups in Organic Synthesis" (published by Wiley-lnterscience). They can include HC1 in dioxane, TFA in DCM, piperidine in DMF, thermal or hydrogenation conditions. Compound 4 could then be coupled to any substituted thiophene-2-carboxylic acid like any intermediate from Scheme I I and result in a final product of formula (I). A variety of amide coupling reagents such as EDC, HOBt, HOAt, HATU, PyBrop, etc. are commercially available and can be used here. Amide coupling reactions are generally run in solvents such as DCM or DMF, utilizing an organic base like EtjN or (i-Pr)2NEt.
Scheme II (x=N)
Figure imgf000043_0001
I I. a: Reductive animation step, for example: 1 ) MeOH, 2) NaCNBl-b ; l l.b : Alkylation step for example: Etl, NaH, DMF ; II.c: Hydrolysis step, for example: I N LiOH, THF.
Reductive amination of 5 with an arylamine can afford the carboxylic acid 6, which can eventually be alkylated to give 7. Subsequent saponi fication can give the carboxylic acid 8. There are a lot of conditions available to one skilled in the art to perform the reductive amination of the formyl group of 5. They can be in one-pot or sequential, using acid (for example AcOH) as a catalyst and/or a dehydrating agent (for example molecular sieves), for the imine formation. The reactions are often conducted in methanol or a chlorinated solvent like DCM or 1 ,2-dichloroethane, using a reduction agent like NaB Hi, NaCNB¾, NaBH(OAc)3 or even LAH. The alkylation reaction can be typically conducted with an alkylated agent such as an alkyl halide (EtI for example), a strong base l ike NaH in a polar solvent l ike DMF. The hydrolysis/saponification step can be conducted with a base like LiOH or NaOH in water, or in acidic conditions like ( IN HC1 for example).
Scheme III (x=0)
Figure imgf000044_0001
I II .a: Reduction step, for example: NaBH4, MeOH ; Ill.b : Esterification step for example: MeOH, H2SO4 ; lll.c: Mitsunobu reaction step, for example : ArOH, DEAD, Ph3P, TITF ; I ll.d: Bromination step, for example: CBr«i, DCM ; l ll.e: Alkylation step, for example: ArOH, CS2CO3, ACN, DMF. Reduction of the formyl group of compound 5 using for example NaBPLi can afford compound 9. Such reduction could also be conducted with other reducing agents like lithium aluminium hydride, diisobutylaluminium hydride, sodium borohydride, L- selectride, diborane, diazene or aluminum hydride. Sodium cyanoborohydride, 9-BBN- pyridine and tribut ltin hydride are also known to be selective for aldehydes.
Hydrogenation using platinum or ruthenium as catalysts could also be an option. The esterification of 9 into 10 could typically be conducted in methanol as solvent in the presence of catalytic sulfuric acid. Alcohol H) may be converted to bromo derivative 11 using CBr , hydrobromic acid or phosphorus tribromide (PBr3). This transformation can also take place using radical conditions in water instead of an organic solvent and the bromine is obtained by oxidation of hydrobromic acid with hydrogen peroxide. An incandescent light bulb is then sufficient for bromine radical generation. Final ly compound 12 can be obtained from a phenol or hydroxy-pyridine by M itsunobu reaction with 10 or alkylation with 11. Mitsunobu reactions are well known to those skilled in the art of organic synthesis. Methods and reaction conditions for such transformations are discussed in Synthesis 1981 , 1 -28. Alkylation conditions include but are not limited to using an inorganic base (like CS2CO3) and in a polar solvent like ACN or DMF.
Scheme IV
Figure imgf000045_0001
y,z = N or CH or C-alkyl
IV. a: Reduction step, for example: SnCl2, HC1, EtOH.
Reduction of the nitro group can be achieved by a wide variety of methods known to the one skilled in the art, including using SnC12 with HC1 in EtOH, or metal Fe in AcOH, or hydrazine or even hydrogenation using a catalyst such as palladium on carbon.
EXPERIMENTAL
The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way, the invention being defined by the claims. Unless otherwise noted, reagents are commercially available or are prepared according to procedures in the literature. The symbols and conventions used in the descriptions of processes, schemes, and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Example 1
Preparation of ^-5-{|(5-chIoropyridin-3-yl)amino]rnethyl}-yV-{3-cyclopcntyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamidc
Figure imgf000046_0001
fS^-Z-Amino^-cyclopentyl-A'-iZ-hydroxycthy propanamide.
A solution of fS)-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpropanoic acid (5 g, 19.43mmol), ethanolamine (1.175mL, 19.43mmol), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (7.45g, 38.9mmol), l -hydroxy-7-azabenzotriazole (5.29g, 38.9mmol), and N-methylmorpholine (8.54mL, 78mmol) was stirred in DCM (50mL) for 3h at room temperature. LCMS showed desired intermediate, LC-MS(ES) m/z = 301.3 [M+H]+. The reaction was poured into water and washed three times with water. Organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The product was then redissolved in DCM (50mL) and treated with 4M HCl/dioxane (50.0mL). The reaction stirred overnight at room temperature. LCMS showed desired product, LC-MS(ES) m/z = 201.3 [M+H]+. The reaction was then concentrated and dried under vacuum overnight to afford a white solid (3.5g).
5-Formyl-thiophene-2-carboxylic acid. as described in Renouard, Thierry; Graetzel, Michael, Tetrahedron, 2001 , 57(38), 8145 - 81 50
5-{ [(5-Chloropyridin-3-yl)amino]methyl}thiophcnc-2-carboxylic acid. A mixture of 5-formyl-2-thiophenecarboxylic acid (6.88g, 44.1 mmol) and 5-chloro-3- pyridinamine (5.667g, 44.1 mmol) was dissolved in MeOH (80mL) and heated at 50°C for 2 hours. An aliquot was removed and pumped under vacuum to dryness. NM R indicated formation of the intermediate imine. A tan precipitate was observed. The reaction was cooled to 0°C under nitrogen and solid sodium cyanoborohydride (3.32g, 52.9mmol) was added portion wise over 1 0 minutes. The ice bath was removed and the heterogeneous reaction was allowed to stir at room temperature for 3h. The reaction was quenched by adding I N HC1 until the pH = 4. The reaction was concentrated to remove the MeOH and the residue was diluted with water (not soluble) and a few drops of I N HQ to bring the pH to 3-4. The tan suspension was filtered and dried over a weekend in a Buchner funnel to provide the desired product as a tan solid ( 1 0.5g). LC/MS indicated 97% purity, LC- MS(ES) m/z = 269.2 [M+H]+.
^^-{[(S-Chloropyridin-S-y arninoJrncthylJ-TV-IS-cyclopcntyl-l-l ilhydroxyethyI)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide.
A mixture of 5- {[(5-ch!oro-3-pyridinyl)amino]methyl}-2-thiophenecarboxylic acid ( 1 .249g, 4.65mmol), (¾)-2-amino-3-cyclopentyl-N-(2-hydroxyethyl)propanamide hydrochloride ( 1 . 1 Og, 4.65mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.891 g, 4.65mmol), l -hydroxy-7-azabenzotriazole (0.712g, 4.65mmol), and
diispropylethylamine (2.435mL, 13.94mmol) in DCM (31 mL) was stirred overnight at room temperature. LC/MS indicated desired product. Added an additional 0.2 eq of 1 - ethyl-3-(3-d imethylaminopropyl)carbodi imide ( 1 9mg) and 0.2 eq of 1 - hydroxybenzotriazole (1 43mg) and stirred at room temperature for 5 hours. The reaction mixture was diluted with DCM and transferred to a separately funnel. The separated organic layer was washed with saturated aqueous solution of NaHC03. The addition of bicarbonate caused much of the product to gum up and stick to the walls of the funnel. I PA was added (25mL) to the 225mL of DCM (to give a 10% 1PA:DCM mixture). This resulted in two homogeneous layers. The layers were separated and the aqueous layer was extracted with 1 0%fPA:DCM. The organic layers were combined and washed with brine, dried over Na2S04, filtered, and concentrated to give a tan solid (2.09g). Crude was purified via chromatography on silica gel (2.5-7.5% MeOH:DCM over 40 min then 7.5- 8.5% MeOH: DCM for 20 min) and dried for 3 days at 55°C in a vacuum oven to give a pale yellow solid ( 1 .8g). Data indicated desired product : 100% purity, LC-MS(ES) m/z = 45 1 .3, 453.3 (MH+1).
Example 2
Preparation of ^-5-{[(5-chloro-2-niethyIpyridin-3-yl)amino] mcthyl}-yV-{3- l-oxopropan-2-yl}thiophene-2-carboxamide:
Figure imgf000048_0001
5-ChIoro-2-melhyl-3-nitropyridine.
To a suspension of sodium hydride (8.76 g, 60% dispersion in m ineral oil) in THF (340mL) under nitrogen atmosphere was added 2,5-dichloro-3-nitropyridine (40g, 207mmol followed by diethylmalonate (63.2mL, 412mmol dissolved in 63mL of THF) drop wise at room temperature for 20min. A vigorous evolution of gas was observed. After 2 h, additional sodium hydride (1. 1 9 g, 60% dispersion in mineral oil) was added and the reaction mixture was then stirred at room temperature for 1 .5h. The reaction mixture was concentrated under reduced pressure and diluted with 6N I-1C1 (400mL) and refluxed for 12.5 h. The resulting mixture was concentrated under reduced pressure and diluted with saturated sodium carbonate solution (400mL) until pH=9, then diluted with dichloromethane (400 mL), stirred for 1 0 min, and filtered to remove an insoluble green solid; from the filtrate, the organic layer was separated and washed with brine, dried over anhydrous Na2S04 and concentrated to afford 5-chloro-2-methyl-3-nitropyridine (26 g crude) as a brown liquid which was directly used in the next step without further purification. 5-Chloro-2-methylpyridine-3-amine.
To a solution of 5-chloro-2-methyl-3-nitropyridine (26g, 15 1 mmol) in ethyl acetate (850mL) under nitrogen atmosphere was added SnC12.2H20 (136g, 603 mmol) at room temperature. The reaction mixture was heated to 85°C for 3h. Concentration of the reaction mixture under reduced pressure afforded a pale yellow slurry which was basified with I N NaOH solution (520mL). The resulting mixture was diluted with
dichloromethane (750mL) and stirred for 10 minutes. The mixture was then fi ltered through a celite pad to remove undissolved solids. The filtrate's organic layer was separated, dried over anhydrous Na2S04 and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel ( 100-200 mesh) using 0-35% ethyl acetate in petroleum ether to afford desired 5-chloro-2-methylpyridine- 3-amine (18 g) as a brown solid.
5-{[(5-Chloro-2-methyl-3-pyridinyl)amino]methyl}-2-thiophenecarboxylic acid. To a solution of 5-formyl-thiophene-2-carboxylic acid (19.7g, 126mmol) in methanol (200mL) under nitrogen atmosphere was added 5-chloro-2-methylpyrid ine-3-amine ( 1 7.99g, 126mmol). The mixture was heated to 50°C for 2h; the reaction mixture was concentrated under reduced pressure to obtain a solid. The solid material was dissolved in methanol (200mL) and cooled to 0°C then sodium cyanoborohydride (9.5 1 g, 1 51 mmol) was added over 10 minutes and the resulting mixture was stirred at room temperature for 14 h. The reaction mixture was quenched by adding IN HC1 ( l OOmL) to bring pH=4, concentrated under vacuum. The residue was diluted with water ( l OOm L). The resulting tan suspension was filtered and dried to get crude solid (28 g, crude). The solid was triturated with ethyl acetate (50mL) to get 5- {[(5-chloro-2-methyl-3- pyridinyl)amino]methyl} -2-thiophenecarboxylic acid (27g) as a pale yellow solid.
(3^-5-{[(5-ChIoro-2-methylpyridin -yl)amino]methyl}-/V-{3-cycIopcntj'l-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamidc.
A mixture of 5- { [(5-chIoro-2-methylpyridin-3-yl)amino]methyl}lhiophene-2-carboxylic acid (1 .3 14g, 4.65mmol), S -2-amino-3-cyclopentyl-N-(2-hydroxyethy l)propanamide hydrochloride (1 .1 Og, 4.65mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide ( 1 .336g, 6.97mmol), 1 -hydroxybenzotriazole (1.067g, 6.97mmol), and
diispropylethylamine (2.435mL, 1 3.94mmol) in DC (31 mL) was stirred overnight at room temperature. LC/MS indicated mostly desired product. The reaction was diluted with DCM and extracted sequentially with saturated aqueuous solution of NaHC03 and brine. The DCM was dried over Na2S04, filtered, and concentrated to give a tan solid (2.63 g). Crude was purified via chromatography on silica gel (3.5%-8.5% MeOH: DCM over 40 min). Desired fractions were isolated and concentrated to give a pale yellow solid (0.926g) with 1 00% purity by HPLC, LC-MS(ES) m/z = 465.3, 467.3 (M H+1 ).
Example 3
Preparation of (-^-S-ifi^chlorop ridin-Z- amiholmethylJ-A'-iS-c cIo ent l-l-Ii - thiophenc-2-carboxamicIe:
Figure imgf000050_0001
5-[(4-Chloropyridin-2-ylamino)methyl]t iophcnc-2-carboxylic acid.
To a suspension of 5-formylthiophene-2-carboxylic acid (22g, 141 mmol) in toluene (220mL) was added 4-chloropyridin-2-amine ( 1 8.05g, 141 mmol) and MeOH (20mL) under nitrogen atmosphere at room temperature and the resulting reaction mixture was heated to reflux using Dien-Stark condenser for 1 h. The reaction mixture was
concentrated under reduced pressure. To the resulting solid residue MeOH (220mL) was added, the mixture was cooled to 0°C and then treated with NaBH3CN ( 13.29g, 210mmol) which was added portion wise over a period of 10 min. The resulting mixture was stirred at room temperature for 1 5h. The reaction was quenched by addition of IN HCI until pH = 5-6 and concentrated under reduced pressure. The gummy residue was diluted with water ( l OOmL, note: gummy residue was insoluble) to which additional few drops of I N HCI to bring pH to 5 were added, before collection of the solid by filtration. Resulting solid was dried to afford the crude compound (30 g). The final product was obtained by recrystallizing twice from ethanol (10 volumes). 5-[(4-chloropyridin-2- ylamino)methyl]thiophene-2-carboxylic acid ( 12.5 g) was obtained as an off-white solid. S 5-{I(4-Chloropyridin-2-yl)arnino]methyl}-7V-{3-cyclopentyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophenc-2-carboxamidc.
A mixture of 5- { [(4-chloropyridin-2-yl)amino]methyl }thiophene-2-carboxylic acid ( 1 .249g, 4.65mmol), (¾)-2-amino-3-cyclopentyl-N-(2-hydroxyethyl)piOpanamide hydrochloride (1 . 1 Og, 4.65mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide ( 1 .336g, 6.97mmol), 1 -hydroxybenzotriazole ( 1 .067g, 6.97mmol), and
diispropylethylamine (2.435mL, 13.94mmol) in DCM (3 l mL) was stirred overnight at room temperature. LC/MS indicated mostly desired product. The reaction was diluted with DCM and washed sequentially with saturated aqueous solution of NaHC03 and brine, dried over Na2S04, filtered, and concentrated to give a tan solid (2.365g). LC/MS indicated 85% desired product. Crude product was purified via chromatography on silica gel (2.5-8.5% MeOH:DCM over 40 min) and placed in a vacuum oven overnight at 55°C to give a white solid ( 1.50g). Data indicated desired product: 100% purity, LC-MS(ES) m/z = 451.3, 453.3 (MH+1 ).
Exam le 4
Preparation of ^^-{[(S-chloro-pyridin^-ylJarninol rnethylJ-yV- -cyclopciityl-l- (oxetan-3-ylamino)-l-oxopropan-2-yl]thiophcnc-2-carboxamide:
Figure imgf000051_0001
(¾)-(9H-Fluorcn-9-yl)mcrhyl [3-cyclopcntyl-l-(oxclan-3-ylarnino)-l-oxopropan yl]carbamate. A solution of S 2-({ [(9H-fluoren-9-yl)methoxy]carbonyl} amino)-3- cyclopentylpropanoic acid ( 1 g, 2.64 mmol), oxetan-3-amine ( 193mg, 2.64mmol), 1 -ethyl- 3-(3-dimethylaminopropyl)carbodiimide (606mg, 3.1 6mmol), l -hydroxy-7- azabenzotriazole (430 mg, 3. 16 mmol), and N-methylmorphoiine ( 1 . 1 59mL, 1 0.54mmol), was stirred in DCM (20mL) for 1 8h at room temperature.
The reaction mixture was washed with water three times. The combined aqueous layers were extracted with DCM (2x25mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, concentrated, and vacuum pumped to a white solid ( 1 .14 g, >90% pure by LC-MS) that was used directly in the next reaction without further purification.
(S^-2-Amino -cyclopentyl-A^oxetan-3-yl)propanamide.
A solution of (¾)-(9H-fluoren-9-yl)methyl [3-cyclopentyl-l -(oxetan-3-ylamino)- l - oxopropan-2-yl]carbamate (1 .14g, 2.62mmol) and piperidine (520[iL, 5.25mmol) in THF ( 1 OmL) was stirred at room temperature for 1 8h. The reaction was checked by TLC using KMn04 stain. Desired compound was purified on silica column using
50%EtOAc/Hexanes followed by 10%MeOH/EtOAc. The desired fractions were combined, concentrated, and vacuum pumped to afford f¾ -2-amino-3-cyclopentyl-N- (oxetan-3-yl)propanamide as a yellow oil (80%pure) used as is in subsequent reaction. S^-S-iliS-Chloro-pyridin-S-y aminolmethyli-A^-fS-cyclopentyl-l-ioxctan-S-ylaniino)- l-oxopropan-2-yl]thiophene-2-carboxamide. ¾)-2-amino-3-cyclopentyl-N-(oxetan-3-yl)propanamide (200mg, 0.942mmol) and 5-{ [(5- chloropyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (253mg, 0.942mmol) were dissolved in l OmL of DMF. Triethylamine (0.394mL, 2.83mmol), 1 - hydroxybenzotriazole (144mg, 0.942mmol) and finally l -ethyl-3-(3- dimethylaminopropyl)carbodiimide ( 1 81 mg, 0.942mmol) were added and the contents allowed to stir at room temperature for 3 days. The reaction was then poured into 250mL of water and extracted (3 x 1 50mL) with ethyl acetate. The organic fractions were pooled, washed (2 x 200mL) with water and (2 x 200mL) with brine, dried over sodium sul fate, filtered and evaporated to dryness. The crude material was purified by automated flash chromatography and eluted using a 0 - 4% MeOH / DCM gradient over 40min. The desired fractions were then pooled and evaporated to dryness to give 206mg of a slightly off-white solid, LC-MS(ES) m/z = 463.2, 465.2 [M+H]+.
Example 5
Preparation of lS^-S-KiS-chloro-Z-meth lpyridin-S- aminol mct lJ-yV- - cycIopcntyl-l-(oxetan-3-ylamino)-l-oxopropan-2-yl]thiophcne-2-carboxamide:
Figure imgf000053_0001
^-5-{[(5-Chloro-2-methyIpyridin -yl)amino] methyl}- V-[3-cyclopcntyl-l-(oxctan-3- ylamino)-l-oxopropan-2-yl]thiophene-2-carboxamide.
A solution of (¾ -2-amino-3-cyclopentyl-N-(oxetan-3-yl)propanamide ( 1 13mg,
0.53 1 mmol), 5- {[(5-chloro-2-methylpyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (1 50mg, 0.531 mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (203mg, 1 .061 mmol), l -hydroxy-7-azabenzotriazole ( 144mg, 1 .061 mmol), and N- methylmorpholine (0.233 mL, 2.122mmol), was stirred in DCM (3 mL) with a couple drops of DM F for 2h at room temperature. The reaction mixture was purified directly on silica using 50-80% AcOEt/Hexanes. Desired fractions were combined and concentrated in- vacuum, transferred into submission vial using DCM. Concentrated and vacuum pumped to afford a white solid (190 mg). LCMS-showed 99% pure desired product, with a desired mass peak of LC-MS(ES) m/z = 477.2, 479.2 [M+I J+.
Example 6
Preparation of f5^-5-{[(4-chloro-pyridin-2-yl)amino] methyI}-7V-[3-cyclopcntyl-l- (oxetan-3-ylamino)-l-oxopropan-2-yl]thiophcne-2-carboxamide:
Figure imgf000054_0001
^^-{[(^Chloro-pyridin- -y aminoJmethylJ-A'-IS-cyclopcnlyl-l-ioxctan-S-ylainino)- l-oxopropan-2-yl]thiophene-2-carboxamidc.
To a solution of 5-{ [(4-chloropyridin-2-yI)amino]methyl}thiophene-2-carboxylic acid ( 190mg, 0.707mmol) in DCM ( l OOmL) were added (S -2-amino-3-cyclopentyl-N-(oxetan- 3-yl)propanamide (1 50mg, 0.707mmol), l -ethyl-3-(3-dimethyIaminopropyl)carbodiimide (163mg, 0.848mmol), l -hydroxy-7-azabenzotriazole (1 1 5mg, 0.848mmol) and N- methylmorpholine (0.233mL, 2. 121 mmol). The reaction mixture was stirred at 25°C for l Oh. The mixture was then concentrated and purified by reverse phase (eluting with 30 - 50% CH3CN/water containing 0.1 % HCOOH) to afford an off-white sol id as the desired product (257.3mg), LC-MS(ES) m/z = 463.2, 465.2 [M+H]+.
Example 7
Preparation of ( S^-A'-il-amino^-cyclopentyl-l-oxopropan- -y -S-IKS-chloropyridin- 3-yl)amino]nicthyl}thiophene-2-carboxamide:
Figure imgf000054_0002
S^-tert-Butyl (l-amino-3-cyclopentyl-l-oxopropan-2-yl)carbamate.
A mixture of (S)-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpropanoic acid (1 .388g, 5.39mmol), ammonium chloride (0.577g, 10.79mmol), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide ( 1 .551 g, 8.09mmol), 1 -hydroxy benzotriazole (1 .239g, 8.09mmol), and diisopropylethylamine (3.77mL, 21 .58mmol) was stirred overnight at room temperature. The reaction was diluted with DCM and washed sequential ly with IN HCI, water, saturated aqueous solution of NaHC03, water, and brine. The DCM solution was dried over Na2S04, filtered, and concentrated to give a white solid( l .Og). 1 H-NMR indicated desired product (>95% purity). No further purification at this stage.
^-2-Amino-3-cyclopentylpropanamidc hydrochloride. 4M HCl (9.75mL, 39.0mmol) in 1 ,4-dioxane was added to solid fSj-tert-butyl ( l -amino-3- cyclopentyl- l -oxopropan-2-yl)carbamate (l .OOg, 3.90mmol) at room temperature under nitrogen and the resulting slurry was stirred for 1 hour. The white slurry was concentrated in vacuum and the residue was pumped under high vacuum overnight to give a white solid (750.0 mgs). 1 H-NMR indicated removal of the protecting group. No further purification at this stage.
(¾^-A^-(l-Amino-3-cyclopentyl-l-oxopropan-2-yl)-5-{[(5-chloropyriclin-3- yl)amino]methyl}thiophenc-2-carboxamide.
A mixture of (S)-2-amino-3-cyclopentylpropanamide hydrochloride (250.5mg,
1 JOOmmol) , 5- { [(5-chloropyridin-3-yl)amino]methyl} thiophene-2-carboxylic acid (349mg, 1.300mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiim ide (374mg,
1 .950mmol), 1 -hydroxybenzotriazole (299mg, 1 .950mmol), and diisopropylethylamine (0.681 mL, 3.90mmol) in DCM ( l OmL) was stirred overnight at room temperature. The reaction was diluted with DCM and washed sequentially with an saturated aqueous solution of NaHC03, water, and brine. The DCM was dried over a2SC , filtered, and concentrated to give a pale yellow solid (346.6mg), which was purified via
chromatography on silica gel (2.5-9.0% MeOH:CHC13 over 40min ) and placed in a vacuum oven overnight at 55°C to give a wh ite solid (109.4mg). LC-M S(ES) m/z = 407.3, 409.3 [M+H]+
Example 8
Preparation of (S^-5-{[(5-chloropyridin-3-yl)arnino]niethyl}- V-[3-cyclopcntyl-l- (methyIamino)-l-oxopropan-2-yI]thiophene-2-carboxamide:
Figure imgf000056_0001
C5^-2-Amino-3-cyclopentyl-7V-methylpropanamide hydrochloride.
To a suspension of (¾)-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpropanoic acid ( 1 .2g, 4.68mmol) and l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.898g, 4.68mmol) in EtOAc ( 1 5mL) was added 1 -hydroxybenzotriazole (0.72g, 4.68mmol) and methylamine (9.37mL, 2M in THF). The reaction mixture was stirred at room temperature for 20h. Additional reagents ( l -ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1 - hydroxybenzotriazole, and methylamine) were added with D F (2mL) for solubility. The reaction mixture stirred at room temperature for 9 hours and was then diluted with water and EtOAc until two clear layers were observed. The aqueous layer was extracted with EtOAc (2x). The combined organic layers were washed with saturated aqueous sodium bicarbonate solution ( l x), brine ( l x), dried over MgS04, filtered, and concentrated in vacuum to afford an off-white solid (716.1 mg). The solid was readily dissolved in DCM and subjected to silica gel purification (0-60% ethyl acetate/hexanes). The desired fractions were combined, and concentrated in vacuum to afford (S)-tert-butyl (3- cycIopentyl- l -(methylamino)- l -oxopropan-2-yl)carbamate as a white solid (452.4 mg), LC-MS(ES) m/z = 171 .1 [M+H]+
To (S)-tert-butyl (3-cyclopentyl- l -(methylamino)- l -oxopropan-2-yl)cai bamate (452.4 mg, 1 .67 mmol) was added 4N HC1 in dioxane (6 niL, 24 mmol). The clear colorless solution was stirred at RT for l h. The solution was concentrated in vacuo to afford 2^-2-amino-3- cyclopentyl-N-methylpropanamide hydrochloride as a white solid (356 mg). Ή NMR (DMSO-c¾ δ 0.98 - 1. 16 (m, 2 H) 1.39 - 1 .64 (m, 4 H) 1.64 - 1 .87 (m, 5 H) 2.65 (d, J=4.55 Hz, 3 H) 3.63 (t, J=6.95 Hz, 1 H) 8.21 (br. s., 3 H) 8.53 (d, J=4.55 Hz, 1 H).
(3^-5-{((5-Chloropyridin-3-yl)amino]mcthyl}- V-[3-cyclopentyl-l-(mcthylarnino)-l- oxopropan-2-yl]thiophene-2-carboxamidc. To a suspension of 5-{ [(5-chloropyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid ( 130mg, 0.48mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (93 mg, 0.48 mmol), diisopropylethylamine (169 uL, 0.97 mmol) and 1 -hydroxy benzotriazole (74 mg, 0.48mmol) in EtOAc (5mL) was added S -2-amino-3-cyclopentyl-N-methylpropanamide hydrochloride ( 1 OOmg, 0.48mmol) and DMF (2mL). The solution was stirred at room temperature for 20h. The reaction was diluted with water and EtOAc. The organic layer was washed with brine, dried over MgS04, filtered, and concentrated in vacuum to afford a yellow oil (226.6 mg). This oil was readily dissolved in DMSO and subjected to reversed-phase HPLC purification ( 1 5-35% ACN/water). The desired product fractions were poured into ethyl acetate and saturated aqueous NaHC03 solution. The combined ofganics were washed with brine (l x), dried over MgS04, filtered, and concentrated in vacuum to afford a white solid ( 123.2 mg),
LC-MS(ES) m/z = 421 .3, 423.3 [M+H]+.
Example 9
Preparation of S^-S-fliS-chloropyridin-S-ylJaminolmcthylJ-A'- -cyclopentyl-l- (dimethylamino)-l-oxopropan-2-yl]thiophenc-2-carboxamidc:
Figure imgf000057_0001
( S)-(9H-Fluoren-9-yl)rnet yl(3-cyclopenfyl-l-(dimetliylaniino)-l-oxopropj)n-2- yl)carbamatc.
To a solution of (S -2-({ [(9H-fluoren-9-yl)methoxy]carbonyl} amino)-3- cyclopentylpropanoic acid ( 1 .0 g, 2.63 mmol) in DCM (l Omf) were added l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (0.605g, 3.1 5mmol), l -hydroxy-7-azabenzotriazole (0.429g, 3.1 5mmol), N-methylmorpholine (0.867mL, 7.89mmol) and dimethylamine hydrochloride (0.214g, 2.63mmol). The mixture was stirred at 25°C for 4h. The mixture was concentrated and purified with silica gel column eluting with 10 - 50% EtOAc / hexane to afford the desired product (2.497g). LC-MS(ES) m/z = 407.3 [M+ITJ+.
^- -Aitiino-S-cyclopentyl-yVjyV-dimethylpropanamidc. To a solution of ^-( H-fluoren-^y methylCS-cyclopentyl- l - dimethylamino)- !- oxopropan-2-yl)carbamate (0.887g, 2.182mmol) in THF (l OmL) was treated with piperidine (1.080mL, 10.91 mmol). The mixture was stirred at 25°C for 1 8h. The mixture was concentrated and purified with silica gel column eluting with 10 - 100%
EtOAc/hexanes followed by 0 - 5% CHCI3 / eOH containing 0.1 % NH OH. Desired fractions were collected and concentrated to afford the desired product (0.35 l g) as a colorless oil. LC-MS(ES) m/z = 185.4 [M+H]+.
^-5-{[(5-Chloropyridin -yl)amino]methyl}-/V-[3-cyclopcnlyl-l-(dimcthylaniino)-l- oxopropan-2-yl]thiophene-2-carboxamide. To a solution of 5-{[(5-chloropyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (1 1 7mg, 0.434mmol) in DCM (3mL) were added (S)-2-amino-3-cyclopentyl-N,N- dimethylpropanamide (80mg, 0.434mmol), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (lOOmg, 0.521 mmol), l-hydroxy-7-azabenzotriazole (70.8mg, 0.521 mmol) and N-methylmorpholine (0.143mL, 1.302mmol). The mixture was stirred at 25°C for l h. The mixture was concentrated and purified by reverse phase HPLC eluting with 35 - 55% acetonitrile in water containing 0.1 % HCOOH. The desired fract ions were concentrated to afford 128 mg of the desired product as an off-white solid. LC-MS(ES) m/z = 435.2, 437.2 [M+H]+.
Example 10
Preparation of C»S -5-{I(5-chloropyridin-3-yl)amino]inethyl}-7V-[3-cyclopcntyl-l- (metlioxyamino)-l-oxopropan-2-yl]thiophenc-2-carboxainide:
Figure imgf000059_0001
5)-tert-Butyl [3-cycIopentyl-l-(methoxyamino)-l-oxopropan-2-yl]carbamatc.
To a clear solution of Ci¾)-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpiOpanoic acid (l .Og, 3.89mmol), O-methylhydroxylamine hydrochloride (1.558g, 4.66mmol) and 1 - ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.894g, 4.66mmol) in DMF (6.1 mL) was added in one portion 1 -hydroxy-7-azabenzotriazole (0.635g, 4.66mmol). The reaction mixture was stirred at room temperature for 12h, then was diluted with DCM and washed with water (20 mL) and brine (20mL) before being dried over Na2S04 and filtered. Most of solvent was removed and the residual crude material was absorbed onto a dry-loading cartridge. Purification was performed by elution with EtOAc in Hexane (10 to 70%). Desired fractions were collected and concentrated to afford the desired product (920 mg). LC-MS(ES) m/z = 287.3 [M+H]+. S^-2-amino -cyclopcntyl-N-mcthoxypropanarnide hydrochloride.
To a solution of (5)-tert- butyl (3-cyclopentyl- l -(methoxyamino)- l -oxopiOpan-2- yl)carbamate (900mg, 3.14mmol) in DCM (20.75mL) was added 4N HCl (3929 μΐ, 15.71 mmol) in dioxane. Reaction mixture stirred at room temperature overnight, then concentrated down to afford the desired product (840 mg) as HCl salt. No purification at this stage. LC-MS(ES) m/z = 1 87.1 [M+H]+.
(S^-S-ifiS-chloropyridin-S-y aminolmethylJ-A^ -cyclopcntyl-l-imethoxyamino)-!- oxopropan-2-yl]thiophenc-2-carboxamidc.
To a clear solution of (S -2-amino-3-cyclopentyl-N-methoxypropanamide hydrochloride (70mg, 0.314mmol) and, 5-{[(5-chloropyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (84mg, 0.314mmol) and diisopropylethylamine (54.9μί, 0.3 14mmol) in DCM (3.1 mL) was added as one portion of solid 2-(7-Aza- l H-benzotriazole- l -yl)- l , l ,3,3- tetramethyluronium hexafluorophosphate (120mg, 0.314mmol). The reaction mixture was stirred at room temperature for 12h. The solvent was then evaporated to dryness. The residure was dissolved in DMSO (2.0mL) then subjected to reverse phase HPLC purification eluting with 10-80% CH3CN in water with 0.1 % of Formic acid. The desired fractions were collected and the solvent was removed by vacuum in order to afford the final product (24mg) as a white solid. LC-MS(ES) m/z = 437.3, 43 .3 [M+H]+.
Example 11 Preparation of ^ 5-{[(5-chloropyridin-3-yl)arnino]mcthyI}-jV-{3-cycIopentyl-l-[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxaniidc:
Figure imgf000060_0001
^-tert-Butyl {3-cyclopcntyl-l-[(3-hydroxypropyl)amino]-l-oxop opan-2- yljcarbamate.
In a 250mL round bottomed flask with stirbar were placed (S)-2-[ ert- butoxycarbonyl)amino]-3-cyclopentylpropanoic acid (5g, 19.43mmol), 1 -ethy l-3-(3- dimethylaminopropyl)carbodiimide (3.72g, 19.43mmol), and EtOAc (l OOmL). To the resulting mixture were added diisopropylethylamine (6.79mL, 38.9mmol) and 1- Iiydroxybenzotriazole (2.98g, 19.43mmol). This mixture was stirred at room temperature for 5min and then to it was added 3-amino- l -propanol (1.477mL, 19.43mmol). The reaction mixture stirred at room temperature overnight. LCMS after 16 hr showed full conversion to desired mass, although the product is not UV active. The reaction mixture was quenched by addition of water (about 50mL) and extracted with 3 x 75mL EtOAc. The combined organics were washed with IN HC1, saturated aqueous solution of
NaHC03, and brine; dried with MgS04; filtered; and concentrated by rotary evaporation to give the desired product (5.96g) as a colorless oil. LC-MS(ES) m/z = 315.3 [M+H]+. (^-S-diS-Chlorop ridin-S- aminolmcthylJ-yV-ia-c cIopcnt l-l-Ka- hydroxypropyl)amino]-l-oxopropan-2-yl} thiophene-2-carboxaniide.
In a 20mL reaction vial with stirbar were placed (S)-tert-bulyl {3-cyclopentyl- l -[(3- hydroxypropyl)amino]- l -oxopropan-2-yl}carbamate ( 1 50mg, 0.324mmol) and HCI, 4M in dioxane (3mL, 12.00mmol). The vial was capped and the reaction mixture stirred at room temperature. LCMS after 45 min showed full deprotection. The reaction mixture was then concentrated under vacuum. The residue was taken up in D F (0.5 mL).
In a separate 20mL reaction vial with stirbar were combined 5- {[(5-chloropyridin-3- yl)amino]methyl}thiophene-2-carboxylic acid (87mg, 0.324mmol), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (62.2mg, 0.324mmol), 1 -hydroxybenzotriazole (49.7mg, 0.324mmol), EtOAc ( 1 .5mL), and diisopropylethylamine (0.1 70 mL, 0.973 mmol). The reaction mixture stirred at room temperature for about 5 minutes and then to it was added the DMF solution of the deprotected amine from above. The reaction mixture stirred at room temperature overnight. LCMS after 1 9h showed full consumption of amine intermediate. The reaction mixture was quenched by addition of water ( l OmL) and extracted with 3 x 20mL of EtOAc. The combined organics were washed with brine, dried with MgS04, filtered, and concentrated by rotary evaporation to give 160mg of crude material. The crude residue was taken up in DMSO, filtered, and subjected to reverse phase HPLC purification using 0.1 %TFA/acetonitrile, 0. 1 %TFA/water conditions with a 2min hold and a 16-36%, 7.5min gradient followed with a 3 m in 1 00% organic hold time and a flow rate of 47mL/min. The desired fractions were combined and converted to the free base form by addition of saturated aqueous solution of NaHC03 and extraction with EtOAc. The combined organics were combined, washed with brine, dried with Na2S04, concentrated, and lyophilized to give the desired product as a monoliydrate (84.2 mg) as a white solid. LC-MS(ES) m/z = 465.3, 467.3 [M+H]+.
Example 12
Preparation of (S^-S-fliS-chloro-Z-methylp ridin - ^arninol rncth lJ-TV-fS- cycIopentyl-l-[(3-hydroxypropyl)amino]-l-oxopropan-2-yl}thiophenc-2- carboxamidc:
Figure imgf000062_0001
5 5-{f(5-Chloro-2-methyIpyridin-3-yI)amino]nicthyl}-iV-{3-cyclopcnlyl-l -[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamklc. In a 20 mL reaction vial with stirbar were placed (2^-tert-butyl {3-cyclopentyl- l -[(3- hydroxypropyl)amino]- l -oxopropan-2-yl}carbamate (150mg, 0.324mmol) and HCI, 4 in dioxane (3mL, 12.00mmol). The vial was capped and the reaction mixture stirred at room temperature. LCMS after 45 min showed full deprotection. The reaction mixture was concentrated under vacuum. The residue was taken up in EtOAc ( 1 .5mL) and DMF (0.5mL). To the resulting solution was added diisopropylethylam ine (0. 1 70mL,
0.973mmol), 5- { [(5-chloro-2-methylpyridin-3-yl)amino]methyl} thiophene-2-carboxylic acid (92mg, 0.324mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (62.2mg, 0.324mmol), and 1 -hydroxybenzotriazole (49.7mg, 0.324mmol). The reaction mixture stirred at room temperature overnight. The reaction mixture was then quenched by addition of water (about l OmL) and extracted with 3 x 20mL EtOAc. The combined organics were washed with brine, dried with MgS04, filtered, and concentrated by rotary evaporation to give 1 70mg of crude material. The crude residue was taken up in DMSO, filtered, and subjected to reverse phase HPLC purification, eluted using
0. 1 %TFA/acetonitrile, 0. 1 %TFA/water conditions with a 2min hold and a 13-33%, 7.5min gradient followed with a 3min 1 00% organic hold time and a flow rate of 47m L/min. The desired fractions were combined and converted to the free base form by addition of saturated aqueous solution of NaHC03 and extraction with EtOAc. The combined organics were combined, washed with brine, dried with Na2S04, concentrated, and lyophilized to give the desired product (79.5 mg). LC-MS(ES) m/z = 479.4, 481.4
[M+H]+. Example 13
Preparation of ^-S-IIi^chloropyridin-l- lJaminoJmcthylJ-A'-JS-c clo entyl-l-IiS- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide:
Figure imgf000063_0001
fS 5-{|(4-Chloropyridin-2-yl)amino]mcthyl}- V-{3-cyclopcntyl-l -| (3- hydroxypropyl)amino]- l-oxopropan-2-yI}thiophcnc-2-carboxam ide.
In a 20mL vial with stirbar were placed (SJ-tert-butyl {3-cyclopentyl- l -[(3- hydroxypropyl)amino]- l -oxopropan-2-yl} carbamate (150mg, 0.324mmol) and HCl, 4M in dioxane (2mL, 8.00mmol). The reaction mixture stirred at room temperature. LCMS after 1 hr showed reaction completion. The reaction mixture was then concentrated and the residue was taken up in EtOAc (2mL) and DMF (0.5mL). To the result ing mixture was added 5- {[(4-chloropyridin-2-yl)amino]methyl} thiophene-2-carboxylic acid (87mg, 0.324mmol), 1 -hydroxybenzotriazole (49.7mg, 0.324mmol), 1 -ethy l-3-(3- dimethylaminopropyl)carbodiimide (62.2mg, 0.324mmol), and diisopropylethylamine (0.170mL, 0.973mmol). The reaction mixture stirred at room temperature overnight. LCMS after 1 6.5 hr showed full consumption of limiting reagent. To the reaction mixture was added water (5mL) and the resulting mixture was extracted with 3 x 1 OmL EtOAc. The combined organics were washed with brine, dried with Na2S04, filtered, and concentrated by rotary evaporation. The crude residue was taken up in DMSO, filtered, and subjected to reverse phase HPLC purification using acidic: 0.1 %TFA/acetonitrile, 0.1 %TFA/water conditions with a 2min hold and a 12-32%, 7.5mingradient followed with a 3min 100% organ ic hold time and a flow rate of 47mL/min. The desired fractions were combined and converted to the free base form by addition of saturated aqueous solution of NaHC03 followed by extraction with EtOAc. The combined organics were combined, washed with brine, dried with Na2S04, concentrated, and lyophilized to give the desired product (68.6 mg) as a white solid. ). LC-MS(ES) m/z = 465.3, 467.3 [M+H]+.LCMS shows 1 00% desired material by UV (0.64 min, M+H=465.3). Example 14
Preparation of (-S' 5-{[(5-chloropyridin-3-yl)aiTiino]methyl}-yV-{3-cycloliexyl-l-|(2- hydroxyethyI)amino]-l-oxopropan-2-yl}thiophenc-2-carboxamiilc:
Figure imgf000064_0001
(SM'ert-Butyl {3-cycIohexyl-l-[(2-hydroxycthyl)amino]-l-oxopropan-2-yl}carbamate.
A solution of fS -2-((tert-butoxycarbonyl)amino)-3-cyclohexylpropanoic acid (20g, 73.7mmol, ), l -ethyl-3-(3-dimethylaminopropyl)carb'odiimide ( 1 6.96g, 88mmol), 1 - hydroxybenzotriazole ( 12.04g, 88mmol), ethanolamine (6.72mL, 1 1 l mmol) and N- methylmorpholine (32.4mL, 295mmol), stirred in DCM (300 mL) for 2h at room temperature. Reaction 'continued to stir overnight in order to push to completion, which was confirmed by LCMS, LC-MS(ES) m/z = 3 1 5.4 [M+H]+. Reaction mixture was transferred to a separatory funnel, then washed with water, saturated aqueous NaHC03 solution, brine, dried over sodium sulfate, filtered, concentrated and vacuum pumped overnight to afford a yellow oil. The product was redissolved in DCM and purified thru a silica plug using 100% EtOAc to remove baseline impurities. The filtrate was
concentrated and vacuum pumped to afford a fluffy white solid (23. 1 7g) which was used directly in the next reaction. ¾)-2-Amino-3-cyclohexyl-^V-(2-hydroxyethyl)propanamidc hydrochloride.
To a solution of (¾ -tert-butyl {3-cycIohexyl- l -[(2-hydroxyethyl)am ino]- l -oxopropan-2 yl}carbamate (1 8g, 57.2mmol), in DCM (300mL) was added aqueous 4M HC1 solution(71 .6mL, 286mmol). The reaction stirred for 2h at room temperature. The reaction was concentrated and vacuum pumped overnight to afford a white sol id ( 14g), LC-MS(ES) m/z = 21 5.3 [M+H]+. (¾)-5-{[(5-Chloropyridin-3-yl)amino]mcthyl}-yV-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophenc-2-carboxamicIc.
A mixture of 5- {[(5-chloro-3-pyridinyl)amino]methyl}-2-thiophenecarboxylic acid (1.620g, 6.03mmol), (¾)-2-amino-3-cyclohexyl-N-(2-hydroxyethyI)propanamide (1.512g, 6.03mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.156g, 6.03mmol), 1 - hydroxybenzotriazole (0.923g, 6.03mmol), and diisopropylethylamine (3.16mL, 1 8.09mmol) in DCM (40mL) was stirred at room temperature for 16 hours. The reaction was diluted with DCM and a saturated aqueous NaHC03 solution then transferred to a separatory funnel. It was observed that much of the desired product had gummed up and was stuck to the walls of the separatory funnel. The upper aqueous layer was neutralized by addition of 2N HCI. 35mL of 1PA were then added to the 350mL of DCM already in the funnel. This made the brown gum dissolve and gave two homogeneous layers. The layers were separated and the aqueous layer was extracted with 50mL more of 10% 1PA:DCM. The combined IPA/DCM layers were dried over Na2S04, filtered, and concentrated to give a tan foam (2.44g). Crude product was purified via chromatography on silica gel (2.5-7.5%) MeOH:DCM). Desired Fractions were concentrated and pumped under high vacuum to give a white solid, (0.881 g). Data indicated desired product (100% purity by analytical HPLC), LC-MS(ES) m/z = 465.3, 467.4 [M+ITJ+.
Example 15
Preparation of S^-5-{[(5-chloro-2-methylpyridin-3-yl)amino] mcthyI}-Ai-{3- cycIohcxyl-l-[(2-hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamidc:
Figure imgf000065_0001
(5 -5-{[(5-Chloro-2-rncthylpyridin-3-yI)arnino]mcthyl}-^V-{3-cyclohcxyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carbox mide. A solution of 5- { [(5-chloro-2-methylpyridin-3-yl)amino]methyl} thiophene-2-carboxylic acid (2.5g, 8.84 mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.034g, 10.61 mmol), l -hydroxy-7-azabenzotriazole ( 1 .444g, 10.61 mmol), (¾)-2-amino-3- cyclohexyl-N-(2-hydroxyethyl)propanamide hydrochloride (2.21 7g, 8.84mmol) and N- methylmorpholine (3.89mL, 35.4mmol) was stirred in DCM (80mL) for 4h at room temperature. The reaction was poured into water and extracted with DCM (3x50mL). The aqueous layer formed a gummy residue which stuck to sides of the separatory funnel. The aqueous layer was then extracted again with EtOAc (3x50mL) which dissolved all the gummy residue. The combined organic layers were washed with brine, dried over Na2S04, filtered and concentrated to an off-white solid. The product was triturated with DCM, filtered, and dried overnight to afford 2.6 g of a white solid ( 1 00% pure by HPLC), LC-MS(ES) m/z = 479.3, 481 .3 [M+H]+.
Example 16 Preparation of ^-5-{[(4-chloropyridin-2-yl)arnino]methyl}-j'V-{3-cyclo exyl-l-[(2- hydroxycthyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxami(lc:
Figure imgf000066_0001
f¾)-5-{[(4-ChIoropyridin-2-yl)amino]methyl}-7V-{3-cyclohcxyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide.
To a clear solution of 5- { [(4-chloropyridin-2-yl)amino]methyl} thiophene-2-carboxylic acid (268mg, 0.997mmol) and fS -2-amino-3-cyclohexyl-N-(2-hydroxyethyl)piOpanamide hydrochloride (250mg,0.997 mmol) and diisopropylethylamine (696μ ί, 3.99mmol) in DCM (9273 μΐ/) was added solid 2-(7-aza- l H-benzotriazole- l -yl)- l , 1 ,3,3- tetramethyluronium hexafluorophosphate (41 7mg, 1 .097mmol) in one portion. The reaction was stirred overnight at room temperature, before being diluted with DCM and washed with water and brine. The organic phase was dried with Na2S04. The solvent was removed by vacuum and crude material was purified on silica gel (0-80 % MeOH in DCM). However purification was only partial. Pure fractions were combined to afford a first crop of the desired product as an off-white solid ( 1 12 mg). The impure fractions were collected and evaporated to dryness, before being dissolved in CH3CN (2.0mL) and purified by reverse phase HPLC (CH3CN in water 10-60% with 0. 1 % of formic acid). The new resulting pure fractions were combined and concentrated under vacuum to give a second crop of desired compound ( 124 mg) as a white solid. Both batches had HPLC purities>99%, LC-MS(ES) m/z = 465.2, 467.3 [M+H]+.
Example 17
Preparation of (-y -5-{[(4-chIoropyridin-2-yl)ainino]methyl}-7V-[3-cycloIiexyl-l- (methylamino)-l-oxopropan-2-yl]thiophene-2-carboxamide:
Figure imgf000067_0001
(5 -tert-Buryl [3-cyclohexyl-l-(methyIamino)-l-oxopropan-2-yl]carbamatc.
To a stirred solution of (S^-2-[(tert-butoxycarbonyl)amino]-3-cyclohexylpropanoic acid ( l Og, 36.9mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (7.06g, 36.9mmol), 1 - hydroxybenzotriazole (5.64g, 36.9mmol) in DCM (l OmL) at 0°C was added Methylamine (2 M, THF) (73.7mL, 147mmol) via syringe. The reaction was allowed to warm to room temperature after 1 hour, and stirred at room temperature overnight. The reaction m ixture was diluted with DCM (200mL) and poured into water (200mL). The organic layer was separated and washed with additional water (200mL) then dried over sodium sulfate, filtered and concentrated. The residual oil was purified by flash chromatography (0-50% EtOAc in hexanes) to afford the desired product (8.4g) as a white solid, LC-MS(ES) m/z = 285 [M+H]+. S^-2-Arnino-3-cyclohexyl-/V-rnethylpropanarnide, hydrochloride. CS ert-Butyl [3-cyclohexyl- l -(methylamino)- l -oxopropan-2-yl]carbamate (8.4g, 29.5 mmol) and HC1 (4M, dioxane) ( 1 OOmL, 400mmol) were stirred overnight at room temperature. The reaction mixture was concentrated to afford the desired product (7.48g) as a white solid, LS-MS(ES) m/z = 1 85 [M+H]+.
(^^-{[(^Chloropyridin-Z-y aminoJniethylJ-A^-lS-cyclohexyl-l- mcthylainino)-!- oxopropaii-2-yl]thiophene-2-carboxamidc.
A mixture of 5- { [(5-chloropyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (0.339g, 1 .262mmol), (¾^-2-amino-3-cyclohexyl-N-methylpropanamide, hydrochloride (0.334g, 1 .514mmol), l -ethyl-3-(3-dimethylaminopi pyl)carbodiim ide (0.242g,
1 .262mmol), 1 -hydroxy benzotriazole (0.193 g, 1 .262 mmol), and diisopropylethylamine (0.661 mL, 3.78mmol) in DCM ( l OmL) was stirred at room temperature overnight. The reaction was diluted with DCM, washed with a saturated aqueous NaHC03 solution and brine, dried over Na2S04, filtered, and concentrated to give a tan solid. Crude product was purified via chromatography on silica gel (2.5-7.5% MeOH:DCM) and placed in a vacuum oven at 50°C for 3 days to give the desired product as a white solid(23 1 .4mg), LC-MS(ES) m/z = 435.2, 437.3 [M+H]+.
Example 18 Preparation of (S^-5-{[(5-chloropyridin-3-yl)oxy] methyI}- V-{3-cyclohexyl-l-[(2- hydroxycthyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxami(le:
Figure imgf000068_0001
Methyl 5-{|(5-chloropyridin-3-yl)oxy] methyl} thiophcne-2-carboxylatc. To a solution of 5-chloropyridin-3-ol (3.64g, 28.1 mmol) in ACN (77mL) and DMF (7.73mL)\vas added Cs2C03 ( 12.47g, 38.3mmol). The reaction mixture was stirred for l Omin and cooled down in an ice bath. A solution of methyl 5-(bromomethyl)thiophene- 2-carboxylate (6.0g, 25.5mmol) in ACN (20mL) was then added dropwise using an addition funnel. The reaction mixture was stirred for 2h and was then concentrated down, before being taken back in water and EtOAc. The organic phase was washed by water and brine, then dried over Na2S04 and filtered. Crude product was filtrated through a silica gel column with 30% EtOAc in hexanes, to afford the desired product (5.3g) as a light brown solid, LC-MS (ES) m/z = 284.0, 286.0 [M+H]+.
5-{[(5-Chloropyridin-3-yl)oxy]methyl}thiophenc-2-carboxylic acid.
To a slurry of methyl 5- { [(5-chloropyridin-3-yl)oxy]methyl}thiophene-2-carboxylate ( 1 .6 g, 5.24mmol) in water (4.72mL) and THF (1 l .O l mL) was added 6N NaOH aqueous solution ( 10.49mL, 26.2mmol). The reaction mixture was stirred overnight. Most volatiles were then removed under vacuum, and the resulting solution was acidified with 6N HCI aqueous solution and was then extracted with EtOAc(2x). The combined organic extracts were washed with water then brine, dried with Na2S04 and filtered.
Concentration to dryness, followed by trituration with DCM afforded the desired product ( 1 .3g) as off white solid, LC-MS (ES) m/z = 270.0, 272.0 [M+H]+.
fS -5-{[(5-Chloropyridin-3-yl)oxy]methyl}-/V-{3-cycIohexyl-l-[(2- hydroxycthyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide.
A solution of (S -2-amino-3-cyclohexyl-N-(2-hydroxyethyl)piOpanamide, hydrochloride ( 1 .5g, 5.98mmol), 5- { [(5-chloropyridin-3-yl)oxy]methyl}thiophene-2-carboxylic acid ( 1 .61 3g, 5.98mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiim ide ( 1 .376g,
7. 1 8mmoI), l -hydroxy-7-azabenzotriazole (0.977g, 7.18mmol) and N-methylmorpholine (2.63mL, 23.93mmoI) in DCM (30mL) was stirred at room temperature for 2h. The reaction mixture was diluted with DCM and water. The separated organic layer was washed with water(2x), then brine, and dried over a2S04. A fter filtration, the solution was concentrated down with silica and purified by silica column (using 0-50% of mixture (9% eOH+l %NH4Cl+90%CHC13) in CHC13 as eluant. Desired fractions were collected and concentrated under reduced pressure to afford the desired product (2. l g) as white solid, LC-MS(ES) m/z = 466.2, 468.3 [M+H]+.
Example 19
Preparation of (^^-{[(S-chloro- -methylpyridin-S-oxylmethylJ-A'-iS-cyclohexyl-l- [(2-hydroxyethyl)amino]-l-oxopropan-2-yl}thiophcne-2-carboxamidc:
Figure imgf000070_0001
5-Chloro-2-mcthyIpyridin-3-ol.
To a solution of sulfuric acid (270mL) and water (900mL) was added 5-chloro-2- methylpyridine-3-amine (1 8g, 126mmol) at room temperature and cooled down to 0°C. A solution of NaN02 (9.58g, 139mmol, dissolved in 90mL of water) was then added drop wise over 20min. The reaction mixture was then stirred at 0°C for additional 30 min before being diluted with sulfuric acid (1 80mL) and water (720mL). The resulting mixture was then heated at 100°C for l h; then cooled down to room temperature and adjusted to pH=6 with NaOH solution. The resulting mixture was then extracted with EtOAc (2 x 500mL). The organic phases were combined, dried over Na2S04 and concentrated under reduced pressure to afford the desired product (1 7g) as a pale yellow solid, LC-MS (ES) m/z = 143.0, 145.0 [M+H]+
Methyl 5-{|(5-chloro-2-mcthylpyridin-3-yl)oxy] methyl}thiophenc-2-carboxylatc.
To a solution of methyl-5-(bromomethyl)thiophene-2-carboxylate (27.8g, 1 1 8mmol) in DMF ( 1 70mL) under nitrogen atmosphere was added at room temperature 5-chloro-2- methylpyridin-3-ol (17g, 1 1 8mmol) followed by K2C03 ( 16.36g, 1 I Smmol). The reaction mixture was stirred for 5 h, before being diluted with water (340mL) and extracted with EtOAc (600mL). The separated organic phase was dried over Na2S04, filtered and concentrated under reduced pressure to afford the desired product (27g) as an off white solid, LC-MS (ES) m/z = 297.0, 299.0 [M+H]+ 5-{[(5-Chloro-2-methyIpyridin-3-yl)oxy] methyl}thiophcne-2-carboxylic acid.
To a solution of methyl-5- { [(5-chloro-2-methylpyridin-3-yl)oxy]methyl}thiophene-2- carboxylate (27g, 91 mmol) in THF (540mL) and water (270mL) was added at room temperature lithium hydroxide mono hydride (19.04g, 453mmol). The reaction mixture was heated at 45°C for 16 h, then concentrated under reduced pressure. The obtained solid residue was diluted with water (200mL) then pH adjusted to 6 with I N HC1 aqueous solution ( l OOmL). The resulting solution was extracted with EtOAc (600 mL) and the separated organic phase was dried over Na2S04, filtered and concentrated under reduced pressure to afford the desired product (13.75 g) as a pale yellow solid, LC-M S (ES) m/z = 283.0, 285.0 [M+H]+
(S -5-{[(5-ChIoro-2-methyIpyridin-3-oxy]mcthy!}- V-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamidc.
The reaction mixture of (SJ-2-amino-3-cyclohexyl-N-(2-hydroxyethyl)propanamide, hydrochloride (66mg, 0.263mmol) and 5- { [(5-chloro-2-methylpyrid in-3- yl)oxy]methyl}thiophene-2-carboxylic acid (74.7mg, 0.263mmol), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (60.5mg, 0.3 16mmol), l -hydroxy-7-azabenzotriazole (43.0mg, 0.3 1 6mmol) and N-methylmorpholine (0. 1 16mL, 1 .053mmol) in DM F (3mL) was stirred overnight. LCMS showed completion. The reaction mixture was purified by reverse phase HPLC (0. 1 % formic acid in mobile phase, water/A CM) using 20-70% gradient, but the resulting product was not pure enough. Fractions were combined and concentrated then an additional purification was conducted by flash chromatography using a 0- 1 00% gradient of the mixture CHC13/(9% MeOH+l %NH4Cl+90%CHC13). Desired fractions were combined and concentrated to afford the desired product (69 mg) as beige solid. LC-MS (ES) m/z = 480.3, 482.3 [M+H]+ Example 20
Preparation of ^^-{[(S-chloropyridin-S-y oxylmethylJ-A^- -cyclopcntyl-l-ioxctan- 3-ylamino)-l-oxopropan-2-yI]thiophenc-2-carboxamide:
Figure imgf000072_0001
i S^-5-{[(5-Chloropyridin-3-yl)oxy]methyl}-iV-[3-cyclopentyI-l-(oxetan-3-ylamino)-l- oxopropan-2-yl]thiophene-2-carboxamidc.
To a solution of 5- { [(5-chloropyridin-3-yl)oxy]methyl}thiophene-2-carboxylic acid (191 mg, 0.707mmol) in DCM ( 1 OOmL) were added (¾^-2-amino-3-cyclopentyl-N-(oxetan- 3-yl)propanam ide ( 1 50mg, 0.707mmol), l -ethyl-3-(3-dimethylaminopropyl)carbod iimide (163mg, 0.848mmol), l -hydroxy-7-azabenzotriazole (1 15mg, 0.848mmol) and N- methylmorpholine (0.233mL, 2. 121 mmol). The mixture was stirred at 25 °C for l Oh. The mixture was then concentrated and purified by silica gel chromatography (0 - 3%
MeOH/CHCI3) to afford the desired product as an off-white solid (1 64 mg). LC-MS (ES) m/z = 464.2, 466.2 [M+H]+
Example 21
Preparation of (S -5-{[(5-chIoropyridin-3-yl)oxy] methyl}- V-[3-cycl()pcntyl-l-(oxetan- 3-yIamino)-l-oxopropan-2-yl]thiophenc-2-carboxamide:
Figure imgf000072_0002
Ethyl 5-{[(5-chloropyridin-3-yl)(ethyl)amino]methyl}thiophene-2-ca rboxyIate. A 400 mL round-bottomed flask was charged with 5- {[(5-chloropyridin-3- yl)amino]methyl}thiophene-2-carboxylic acid (6g, 22.33mmol) in DMF (60mL) to give a yellow solution at 0°C under nitrogen. Sodium hydride 60% in mineral oil (2.77g, 69.2mmol) was added to the reaction mixture. Ethyl iodide (3.97mL, 49.1 mmol) was added to the reaction mixture. The reaction was stirred to room temperature. After overnight stirring, the reaction mixture was checked by LC S and showed some product mixed with various other alkylated products. The reaction mixture was diluted with water (200mL) and extracted with EtOAc (200mL x3 ). The EtOAc layers were dried over Na2S04, filtered, and concentrated. The residue was chromatographed on silicagel column and eluted with EtOAc and hexanes (5% to 25%) and the clean fractions were concentrated to dryness to obtain the desired product (2.5 g). LC-M S (ES) m/z = 325.2, 327.2 [M+H]+.
Lithium 5-{[(5-chloropyridin-3-yl)(ethyI)amino] methyl} thiophenc-2-carboxylate. A 50 mL round-bottomed flask was charged with ethyl 5- {[(5-chloropyridin-3- yl)(ethyl)amino]methyl}thiophene-2-carboxylate (2.5g, 7.70mmol) and I N LiOH
(7.70mL, 7.70mmol) in THE (20mL) to give a brown suspension at 25°C under nitrogen. The reaction was stirred and warmed up to 50°C overnight. The reaction was then concentrated to the desired product (2.4g). LC-MS (ES) m/z = 297. 1 , 299.1 [M+H]+.
(S^-S-f iiS-Chloropyridin-S-y oxyJ methylJ-A'-fS-cyclopcntyl-l-ioxetan-S-ylamino)-!- oxopropan-2-yl]thiophene-2-carboxarnidc.
A solution of CS -2-amino-3-cyclopentyl-N-(2-hydroxyethyl)propanamide, hydrochloride ( 1 22mg, 0.514mmol), l -ethyl-3-(3-dimethylaminopropyl)carbodiim ide ( 126 mg, 0.655 mmol), lithium 5- { [(5-chloropyridin-3-yl)(ethyl)amino]methyl} thiophene-2-carboxylate ( 1 50mg, 0.468mmol), l -hydroxy-7-azabenzotriazole (89mg, 0.655mmol) and DMF (5mL) was stirred at room temperature over the weekend. The reaction mixture was then diluted with water (20mL) and extracted with DCM (20mL). The organic layer was washed with water then dried over Na2S04, filtered, and concentrated. The residue was purified via chromatography on silica gel column using DCM and DCM with MeOH (5% to 40%) and the clean fraction were concentrated to dryness to obtain the desired product (80 mg) as a yellow solid. LC-MS (ES) m/z = 479.0, 48 1 .0 [M+H]+
Example 22 Preparation of I -5-{[(3-chlorophenyl)amino] methyl}-yV-{3-cyclohexyM-[(2- mcthoxyethyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide:
Figure imgf000074_0001
-tert-Butyl [3-cyclohcxyI-l-(methoxyethylamino)-l-oxopropa n-2-yl]carbamatc. A solution of 3-cyclohexyl-N-{ [( l , l -dimethylethyl)oxy]carbonyl}-L-alanine ( 1 0 g, 36.9 mmol), 2-methoxyethylamine (3.17 mL, 36.9 mmol), HBTU ( 14.01 g, 36.9 mmol), and Ν- methylmorpholine (6.08 mL, 55.3 mmol) stirred in Ν,Ν-Dimethylformamide (DMF) (200 m L) at RT for 2h. The reaction was monitored by LCMS to show 79% (BPC) desired product, MW = 329.3 (M+H). TLC with KMn04 stain shows product f = 0.5 and baseline spot with 1 : 1 (EtOAc:Hexanes). The reaction was poured into water and extracted with EtOAc (4 x 100 mL). The combined orgnanic layers were combined, washed with brine, dried over sodium sulfate, filtered, concentrated to an orange oil. Seemed wet. Redisolved in EtOAC and washed with brine, dried over sodium sulfate, and concentrated to a thick orange oil. Redissolved in DCM, loaded onto 80g pre-packed silica gel column. Purified using 20-40% EtOAc/Hexanes over 30 minutes. Collecting waste in tubes (since product not UV active).
LCMS-shows 100% desired product, MW = 329.3 (M+H) 1 H NM (400 MHz, DMSO-d6) d 7.78 (br. s., 1 H), 6.79 (d, J = 8.34 Hz, 1 H), 3.71 - 4.22 (m, 1 H), 3.07 - 3.42 (m, 7H), 2.46 (br. s., 2H), 1 .63 (d, J = 9.85 Hz, 4H), 1 .25 - 1 .46 (m, 1 1 H), 0.95 - 1 .28 (m, 3 H), 0.67 - 0.98 (m, 2H)
Used directly in next reaction.
(5^-2-Arnino-3-cyclohexyl-/Y-(2-methoxyethyl)propanamide hyd rochloride.
4M HCl/Dioxane (1 5.22mL, 60.9mmol) was added to a solution of fSJ-tert-bulyl [3- cyclohexyl- l -(methoxyethylamino)- l -oxopropan-2-yl]carbamate (4g, 12.1 8mmol) in DCM (50mL). The reaction stirred 2h at room temperature under nitrogen. The reaction was concentrated and vacuum pumped to afford a pale yellow foamy solid. LC-MS (ES) m/z = 229.2 [M+H]+ Sy-5-{{(3-Chlorophenyl)aminojmethyl}-A'-{3-cyclohexyI-l-[(2-methoxycthyl)arnino]- l-oxopropan-2-yl}thiophene-2-carboxamidc. A solution of 5- { [(3-chlorophenyl)amino]methyl}-2-thiophenecarboxylic acid (761 mg, 2.84mmol), f'Sj-2-amino-3-cyclohexyl-N-(2-methoxyethyl)propananiide hydrochloride (800mg, 2.369mmol), 2-(l H-benzotriazole- l -yl)- l , 1 ,3,3-tetramethyluronium
hexafluorophosphate (1078mg, 2.84mmol), and N-methylmorpholine (0.781 mL,
7. 1 1 mmol) stirred in DMF (20mL) at room temperature for 2h. The reaction was poured into water and extracted with EtOAc (3 x 20mL). The organic layers were combined, washed with brine, dried over sodium sulfate & decolorizing carbon, filtered thru a pad of celite, concentrated and vacuum pumped to afford a yellow oil. This oil was dissolved in DCM and purified on silica gel using 20%-40% EtOAc/Hexanes gradient. Desired fractions were combined, concentrated, and the resulting solid was dried in vacuum-oven to afford a pale yellow gummy solid (405mg). Solid was then dissolved in EtOAc (l OmL) and Hexanes ( l OmL) was added. Product formed on sides of round-bottomed flask.
Decanted off solution and then redissolved remaining solids in ether, concentrated, and vacuum pumped to an off-white fluffy solid. HPLC-still shows 99. 1 %. Dissolved the solid in DMSO and purified by reverse phase HPLC using 0.1 % TFA. Combined desired fractions, removed organic solvent by rotary evaporation in-vacuum. Aqueous solution was poured into a saturated aqueous solution of NaHC03, then extracted with EtOAc (3 x 20mL). The resulting combined orgnanic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated. Residue was redissolved in ether, concentrated, and vacuum pumped to a fluffy white solid. LC-MS (ES) m/z = 478.3, 480.3 [M+H]+
Example 23
Preparation of 5-{[(5-chloro-2-methylpyridin-3-yl)amino] methyl}-^V-{(S -3- cyclopentyl-l-[((7/-,55^-3-hydroxycyclobutyl)amino]-l-oxopropan-2-yl}lhiophcne-2- carboxamidc:
Figure imgf000076_0001
Mixture of tert-butyl {flS^-3-cyclopenryl-l-[((7/*,J5^-3-hydroxycyclobutyl)amino]-l- oxopropan-2-yl}carbamate and terf-butyl {(S)-3-cyclopentyl-l-[((Js,3R)-3- hydroxycyclobutyl)amino]-l-oxopropan-2-yl}carbamate. To a clear solution of (S^-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpropanoic acid (3.0g, 1 1 .66mmol), a (1 : 1 ) mixture of cis/trans 3-aminocyclobutanol ( 1 . 1 1 7g, 12.82mmol) and l -ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.68g, 13.99mmol) in
Dichloromethane (DCM) (41.2mL) was added 1 -hydroxy-7-azabenzotriazole ( 1.904g, 13.99mmol) as one portion. The reaction mixture was stirred at room temperature for 12 hours, then diluted with DCM and washed with water, an aqueous saturated solution of NaHC03 (20 mL x2) ; an aqueous saturated solution of NH4CI (20 mL x2) and brine (20mL x3). The decanted organic layer was separated and dried over Na2S04. The organic solvent was evaporated under reduced pressure to afford a wax-l ike solid which was then triturated with water. The resulted solid was collected by fi ltration to afford the titled mixture (3.7g, 1 0.77mmol, 92% yield). LC-MS (ES) m/z = 327.3 [M+H]+ Mixture of ^-2-amino^-cyclopentyl-7V-((7 -,3iS -3-hydroxycycIobutyl)propanarnidc and ^-2-amino-3-cycIopcntyl- V-((75,J ? -3-hydroxycyclobutyl)propanarnidc, bis hydrochloride.
The mixture of /erf-butyl { fSJ-3-cyclopentyl- l -[(f7r, JSJ-3-hydroxycyclobutyl)amino]- l - oxopropan-2-yl }carbamate and ri-butyl {(S)-3-cyc\open y\- \ -[((Js,3R)-3- hydroxycyclobutyl)amino]- l -oxopropan-2-yl {carbamate (3.7g, 1 0.77mmol) was then dissolved in Dichloromethane (DCM) (41 .2mL), treated with 4M HC1 in dioxane
( 14.57mL, 58.3mmol) and stirred at room temperature overnight. The solvent was then evaporated and the residue was triturated with Et20 which converted it into a white solid. The ether layer was decanted and the trituration repeated with fresh Et20. The suspension was allowed to stand for 1 0 minutes before careful removal of supernatant ether. The resulting solid was dried under high vacuum to afford the titled mixture (2.6g, 8.25mmol, 70.8% yield). LC-MS (ES) m/z = 227.2 [M+H]+
Mixture of 5-{[(5-chloro-2-methylpyridin-3-yl)arnino]mcthyl}-N-{^y)-3-cyclopcntyl-l- |(( r,J5^-3-hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-ca rboxamide and 5-{[(5-chloro-2-mcthylpyridin-3-yl)amino] methyl}-yV-{(3 -3-cyclopcntyl-l- |( 7A",5 ?)-3-hydroxycycIobulyl)aniino]-l-oxopropan-2-yl}thiophcne-2-ca rboxamide.
To a clear solution of the mixture of (¾)-2-amino-3-cyclopentyl-N-(f7r, 5S -3- hydroxycyclobutyl)propanamide and (¾ -2-amino-3-cyclopentyl-N-((7s, 3R)-3- hydroxycyclobutyl)propanamide, bis hydrochloride (600mg, 2.005mmol) and 5- {[(5- chloro-2-methylpyridin-3-yl)amino]methyl}thiophene-2-carboxylic acid (567mg, 2.005mmol) and diisopropylethylamine ( 1 .4mL, 8.02mmol) in Dichloi omethane (DCM) ( 1 8.7mL) was added as one portion of solid 0-(7-Azabenzotriazol- l -y\)-N,N,N',N'- tetramethyluronium hexafluorophosphate (91 5mg, 2.406mmol). The reaction mixture was stirred at room temperature for 1 2 hours then diluted with DCM and washed with water (20mL x3) and brine (20mL). The decanted organic phase was isolated and concentrated by evaporation under low pressure. LC-MS (ES) m/z = 491.4, 493.4 [M+ITJ+ 5-{[(5-chloro-2-methyIpyridin-3-yl)amino] methyl}^
hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophenc-2-carboxami(le.
Chiral separation of the mixture of the two diastereoisomers 5-{[(5-chloro-2- methylpyridin-3-yl)amino]methyl}-N-{fS-3-cyclopentyl-l-[( 7r,5S-3- hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide and 5-{[(5- c loro- -met ylpyridin-S-y aminojmethylJ-N-i^S^S-cyclopent l-l-f^/i.i/^-S- hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide was performed transferring the analytical conditions (on AD-H column with 30:70 EtOH:Heptane and 0.1% isopropylamine added as modifier) to a semi-prepaprative column AD-H column using a 40:60 EtOH:Heptane with 0.1%isopropylamine. Injection samples were dissolved using EtOH and methanol MeOH (up to a 3:1 ratio in volume), (a) 1 st eliiting
diastereomer (165mg) is titled desired compound, (b) 2nd eliiting diastereomer (240mg) is 5-{[(5-chloro-2-methyIpyridin-3-yl)amino]methyl}-N-{(¾-3-cycIopentyl-l-[(('/5,5R)-3- hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide. LC-MS (ES) mz = 491.4, 493.4 [M+H]+
Example 24
Preparation of 5-{[(5-chloro-2-methylpyridin-3-yl)amino)mcthyl}-7V-{(S-3- cyclopcntyl-l-fiCy^J/^^-hydroxycyclobuty aminol-l-oxopropan^-ylJthiophenc^- carboxamide:
Figure imgf000078_0001
5-{[(5-chloro-2-mcthylpyridin-3-yl)amino]methyl}-Ar-{(-? 3-cycIopcntyl-l-[((7s,5i^ hydroxycyclobutyI)amino]-l-oxopropan-2-yl}thiophenc-2-carboxamide. Chiral separation of the mixture of the two diastereoisomers 5-{[(5-chloro-2- lTiethylpyridin-S-y aminoJmethyl
Figure imgf000079_0001
hydroxycyclobutyl)amino]-l-oxopropan-2- l}thiophene-2-carboxamide and 5-{[(5- chloro-2-methylpyridin-3-yl)amino]methyl}-N- { ¾)-3-cyclopenty 1-1 -| '((Js,3R)-3- hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide was performed transferring the analytical conditions (on AD-H column with 30:70 EtOK:Heptane and 0.1% isopropylamine added as modifier) to a semi-prepaprative column AD-H column using a 40:60 EtOH:Heptane with 0.1%isopropylamine. Injection samples were dissolved using EtOH and methanol MeOH (up to a 3:1 ratio in volume), (a) 1st eluting diastereomer (165mg) is 5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}-N-{(¾)-3- cyclopent l-l-[(C7r,JS^-3-hydroxycyclobutyl)amino]-l-oxopropan-2-yl}thiophene-2- carboxamide. (b) 2nd e!uting diastereomer (240mg) is the titled desired compound. LC- MS(ES) m/z = 491.4, 493.4 [M+H]+
Example 25
Preparation of (^^-{[(S-chloro-l-methylpyridin-S-y aminoJmethylJ-jV- - cyclopentyl-l-(cyclopropyIamino)-l-oxopropan-2-yl]thiophene-2-carboxamide:
Figure imgf000079_0002
(S)-tert-Butyl [3-cyclopentyl-l-(cyclopropylamino)-l-oxopropan-2-yl]carbamate.
To a solution of C>S^-2-[(tert-butoxycarbonyl)amino]-3-cyclopentylpiOpanoic acid (lOg, 38.9mmol) in Dichloromethane (DCM) (175mL) were added cyciopropylamine (4.44g, 78mmol), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (8.94g, 46.6mmol), and 1- hydroxy-7-azabenzotriazole (6.34g, 46.6mmol), The mixture was stirred at room temperature overnight then diluted with DCM and washed with saturated aqueous NaHC03, water and brine. The organic phase was decanted, and concentrated under reducing pressure to give an oil. This oil was dissolved in DMF( 10 mL). To this DMF solution, water was added slowly while stirring. A white solid formed and was collected then washed with water several times. The solid was dried in a 65°C oven for 1 8 hours to afford a white solid which was directly used in the Boc deprotection step described below.
(S -Amino-3-cyclopcnlyI- V-(2-cyclopropyl)propanamide bis-hydrocliloride.
The (¾)-tert-Butyl [3-cycIopentyl- l -(cyclopropylamino)- l -oxopropan-2-yl]carbamate obtained from the preceding step was dissolved in DCM (20mL) and 4M hydrochloric acid in dioxane ( 19.43mL, 78mmol) was added. The mixture was stirred at room temperature overnight. The solvent was then removed by distillation under reduced pressure to afford the titled compound as a white solid (7.56g, 26.7mmol, 68.6 % yield).
(^-5-{[(5-chloro-2-methyIpyridin-3-yl)amino]methyI}-/V-[3-cyclopentyl-l- (cycIopropylamino)-l-oxopropan-2-yl]thiophenc-2-carboxamidc.
A round bottom flask was charged with 5- { [(5-chloro-2-methylpyridin-3- yl)amino]methyl} thiophene-2-carboxylic acid (4.86g, 17.19mmol), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (4.28g, 22.34mmol) and l -hydroxy-7- azabenzotriazole (3.04 g, 22.34 mmol) in N,N-Dimethylformamide (DMF) (54.5mL). To ■ this mixture N-methylmorpholine (5.67mL, 51.6mmol) was added drop wise. The reaction mixture was continuously stirred at room temperature for 0.5 h. Another round bottom flask was charged with (S -Amino-3-cyclopentyl-N-(2-cyclopropyl)propanamide, bis-hydrochloride (4.0g, 1 7. 19mmol) in N,N-Dimethylformamide (DMF) (54.5mL) and N- methylmorpholine (5.67mL, 51 .6 mmol). After complete dissolution, the contents of the second flask were slowly added to the first flask. The reaction mixture was stirred at room temperature overnight. The DMF solution was carefully poured into icy water (250 mL) while stirring and a white solid precipitated. The solid was collected by filtration and washed with water (50mL x3) then Hexanes (50mL x2). The off-white solid was heated in an oven 60°C under high vacuum to obtain a solid (7.1 3g, 14.69mmol), which was redissolved in EtOAc (20mL) and stirred at reflux-using an oil bath and a condenser for 10 min. The hot EtOAc solution was allowed to cool down slowly to room temperature. The recrystal lized material was collected by filtration to afford the titled compound (6.13g, 1 3.03mmol, 76 % yield) as a white solid. LC-MS (ES) m/z = 461 .5, 463.4 [ +1TJ+
Example 26
Preparation of (7-?,Jr 3-[(S -2-(5-{[(5-chloro-2-methylpyridin-3- yl)amino]methyI}thiophene-2-carboxamido)-3-cyclopentyIpropanamido]cyclobutyl acetate:
Figure imgf000081_0001
(75',Jr 3-[^-2-(5-{[(5-chloro-2-methylpyricIin-3-yI)amino]methyl}thiophcnc-2- carboxamido)-3-cyclopentylpropanamido]cyclobutyl acetate.
A round bottom was charged with 5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N-
Figure imgf000081_0002
2-carboxamide (600mg, 1 .222mmol), triethylamine (341 μΐ , 2.444mmol) and 4-
(dimethylamino)pyridine (74.6mg, 0.61 l mmol) in Tetrahydrofuran (THF) ( 1 1 .9mL). The reaction mixture was cooled down to 0°C using an icy water bath. To this mixture, acetic anhydride ( 1 87mg, 1.833mmol) was added via syringe. The reaction m ixture was then allowed to warm up to room temperature. Disappearance of alcohol was followed by LCMS and more acetic acid had to be added for a total of 1 .8eq. After 6h of additional stirring, the reaction was worked up by addition of water followed by extraction using EtOAc (20mL x3). The decanted organic phase was washed by a saturated aqueous solution of NaHC03 then brine. The EtOAc solution was dried over Na2S04, then filtered and concentrated under low pressure. The resulting crude solid was recrystallized from hot EtOAc to afford the final desired product (440mg, 0.809mmol, 66.2 % yield). LC-MS (ES) m/z = 533.3, 535.3 [M+H]+ Example 27
Preparation of ^-^^^^-[^^-(S-iliS-chloro^-mct ylp idin-S- yl)amino]mcthyl}thiophene-2-carboxamido)-3-cyclopentylpropanamido]cyclobutyl
2,6-diaminohexanoate:
Figure imgf000082_0001
S (7-9)J5 3-[f-? -2-(5-{[(5-chloro-2-methylpyridin-3-yl)amino]m
carboxamido)-3-cyclopentylpropanamido]cyclobutyI bis[(tcrt- butoxycarbonyl)amino]hexanoate.
A solution of f¾)-2,6-bis[(tert-butoxycarbonyl)amino]hexanoic acid (300mg, 0.866mmol), 4-(dimethylamino)pyridine (159mg, 1.299mmol) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, 332mg, 1.732mmdl) n N,N- dimefhylformamide (DMF, 8.7mL) was stirred at room temperature for 4h. To this reaction mixture 5- {[(5-chloro-2-methylpyridin-3-yl)amino]methyl}-N- {fS -3- cyclopentyl-l -[(('7 -,JSJ-3-hydroxycyclobutyl)amino]- l-oxopropan-2-yl}thiophene-2- carboxamide (425mg, 0.866mmol) in DMF (2.0mL) was added. After 30 h of stirring at room temperature 2.0 additional equivalents of EDC reagent were added. The reaction mixture was continuously stirred at room temperature for another 10 hours, then poured into water and the formed solid was collected by filtration. The solid was then dried by air and redissolved in EtOAc. The solution was washed by water and brine, decanted then dried over Na2S04 and finally filtered and concentrated. The crude was finally purified by flash chromatography on silica gel column by elution with a mixture of 2 to 5% MeOH in DCM. The desired fractions were collected and concentrated to afford the titled compound (267mg, 0.31 Ommol, 35.7% yield). LC-MS (ES) m/z = 819.4, 821.4, 820.4 [M+H]+ (S)-(JS,3S)-3-[(S)-2-(5-{[(5-chloro-2-mcthy\pyr 0m-3-y\)a ^
carboxamido)-3-cycIopcntylpropanamido]cycIobutyl 2,6-dianiinohcxanoate, tri- hydrocholride.
To a solution of (S)-(JS S)-3-[(S)-2-(5-{[(5-ch\oro-2- etby\pyr din-3- yl)amino]methyl}thiophene-2-carboxamido)-3-cyclopentylpropanamido]cyclobutyl 2,6- bis[(tert-butoxycarbonyl)amino]hexanoate (640mg, 0.78 l mmol) in Dichloromethane (l O.OmL) was added 4M HCI in dioxane (3.91 mL, 15.62mmol). The reaction mixture was stirred at room temperature for 5 hours. A white solid precipitated. The reaction solvent was removed by distillation under vacuum and residual HCI was driven off by co- evaporating twice with DCM (20mL). The solid was suspended a third time in DCM
(l OmL) and collected by filtration to afford the titled compound (450mg, 0.61 l mmol, 78% yield). LC- S (ES) m/z = 619.3, 622.2 [M+H]+
Figure imgf000083_0001
7.08 (d, J=3.8 Hz, 1 H), 6.85
(d, J=2.0 Hz, 1 H), 6.43 (s, 1 H), 4.63 (t, J=5.4 Hz, 1 H), 4.57 (d, J=6.1 Hz, 2 H), 4.36 (s, 1 H), 3.37 (d, J=5.6 Hz, 2 H), 3.1 1 (d, J=6.3 Hz, 2 H), 2.33 (s, 3 H), 1.60 - 1 .84 (m, 5 H), 1 .49 - 1.59 (m, 2 H), 1.37 - 1 .48 (m, 2 H), 1.1 1 (br. s., 2 H)
Example 3 l H NM (400 MHz,
DMSO-d6) δ ppm
8.37 (d, J=8.3 Hz, 1 H), 7.98 (d, J=5.6 Hz, 2 H), 7.72 (d, J=3.8 Hz, 1 H), 7.49 (s, 1 H), 7.00 (d, J=3.8 Hz, 1 H), 6.53 - 6.67 (m, 2 H), 4.65 (s, 3 H), 4.29 - 4.44 (m, 1 H),
3.38 (d, 2 H), 3.12 (s, 2 H), 1.59 - 1.88 (m, 5 H), 1.54 (dd, J=6.7, 3.7 Hz, 2 H), 1.36 - 1.50 (m, 2 H), 1.1 1 (br. s., 2 H)
Example 4 1 H NMR (400MHz
,DMSO-d6) 5 ppm
8.77 (d, J = 6.6 Hz, 1 H), 8.43 (d, J = 8.3 Hz, 1 H), 7.96 (d, J = 2.5 Hz, 1 H), 7.76 (dd, J =3.0, 4.5 Hz, 2 H), 7.08 (d, J = 3.8 Hz, 1 H), 7.03 (t, J = 2.3 Hz, 1 H), 6.98 (t, J = 6.1 Hz, 1 H), 4.81 -4.72 (m, J = 6.6, 6.6,
13.5 Hz, 1 H), 4.71 -4.65 (m, 2 H), 4.53 (d, J = 6.1 Hz, 2 H), 4.47 - 4.39 (m, 2 H), 4.39 - 4.32 (m, 1 H), 1.88 - 1.61 (m, 5 H), 1.56 (dt, J = 3.6, 6.9 Hz, 2H), 1.45 (dd, J = 3.9, 7.2 Hz, 2 H), 1.22- 1.03 (m,2H)
Example 5 IHNMR (400MHz
,DMS0-d6) δ ppm
8.77 (d, J = 5.8 Hz, 1 H), 8.42 (d, J = 7.8 Hz, 1 H), 7.75 (d, J = 2.8 Hz, 1 H), 7.67 (s, 1 H), 7.08 (br. s., 1 H), 6.85 (s, 1 H), 6.43 (t, J = 4.9 Hz, 1 H), 4.82 - 4.72 (m, 1 H), 4.72 - 4.64 (m, 2 H), 4.57 (d, J = 5.6 Hz, 2 H), 4.42 (q, J = 6.5 Hz, 2 H), 4.38 - 4.26 (m, 1 H), 2.33 (s, 3 H), 1.87- 1.61 (m, 5 H), 1.56 (br. s., 2 H), 1.45 (br. s.,2H), 1.12 (br. s.,2H)
Example 6 IHNMR (400 MHz,
DMSO-d6) δ ppm
8.76 (d, J = 6.3 Hz, 1 H), 8.38 (d, J = 8.1 Hz, 1 H), 7.97 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 3.8 Hz, 1H), 7.48 (t, J = 6.2 Hz, 1H), 7.00 (d, J = 3.8 Hz, 1H), 6.61 (dd, J= 1.9, 5.4 Hz, 1H),
6.58 (d, J = 1.5 Hz, lH), 4.71 -4.79 (m, 1 H), 4.66- 4.71 (m, 2H), 4.64 (d, J = 6.1 Hz, 2 H),4.42 (dt, J = 6.2., 7.8 Hz, 2H), 4.37 (td, J = 3.1,4.8. Hz, lH), 1.73 (dd,J = 3.8, 9.4 Hz, 3H), 1.65 (dd, J = 5.1, 8.3 Hz, 2H), 1.50 - 1.60 (m, 2H), 1.39- 1.49 (m,2H), 1.14 (s, 2H)
Example 7 1HNMR(400 MHz,
DMSO-d6) δ ppm
8.33 (d, J=8.3 Hz, 1 H), 7.96 (d, J=2.3 Hz, 1 H), 7.76 (dd, J=9.2, 2.9 Hz, 2 H), 7.39 (s, 1 H), 7.07 (d,J=3.8Hz, 1 H), 7.03 (t, J=2.3 Hz, 1 H), 6.94 - 7.00 (m, 2 H), 4.53 (d, J=6.3 Hz, 2 H), 4.32 (d, J=3.8 Hz, 1 H), 1.41 - 1.84 (m, 9 H), 1.02- 1.18 (m, 2 H)
Example 8 lHNMR(400MHz,
DMSO-d6) δ ppm
8.42 (d, J=8.34 Hz, 1 H) 7.96 (d, J=2.27 Hz, 1 H) 7.90 (q, J=4.63 Hz, 1 H) 7.77 (d, J=2.02 Hz, 1 H) 7.75 (d, J=3.79 Hz, 1 H) 7.08 (d, J=3.54 Hz, 1 H) 7.03 (t, J=2.27 Hz, 1 H) 6.98
(t, J=6.19 Hz, 1 H)4.53 (d, J=5.81 Hz, 2 H) 4.33 (td, J=8.84, 5.05 Hz, 1 H) 2.56 (d, J=4.80 Hz, 3 H) 1.61 - 1.85 (m, 5 H) 1.55 (dt, J=6.82, 3.41 Hz,2H) 1.37- 1.50 (m, 2 H) 1.03- 1.15 (m, 2 H)
Example 9 1HNMR(400 MHz,
DMSO-d6) δ ppm
8.54 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 2.5 Hz, 1H), 7.75 - 7.80 (m,2H), 7.07 (d, J = 3.5 Hz, lH), 7.03 (t, J = 2.3 Hz, 1H), 6.97 (t, J = 6.2 Hz, 1H), 4.78 -4.87 (m, 1H),4.53 (d, J = 6.1 Hz, 2H), 3.05 (s, 3H), 2.83 (s, 3H), 1.66- 1.86 (m, 4H), 1.52 - 1.65 (m, 3H), 1.46 (td, J = 3.7, 7.52 Hz, 2H), 1.06- 1.18 (m,2H), 1.04 (d, J = 6.1 Hz, 1H)
Example 10 1HNMR (400 MHz,
DMSO-d6) δ ppm
7.91 (d, J=2.3 Hz, 1 H), 7.78 (d, J=2.0 Hz, 1 H), 7.68 (d, J=3.8 Hz, 1 H), 7.00 - 7.15 (m, 2 H), 4.60 (s, 2 H), 4.34 - 4.49 (m, 1 H), 3.70 (s, 3 H), 1.73 - 1.98 (m, 5 H), 1.62 - 1.73 (m, 2 H), 1.57
(dt, J=7.71,4.0 Hz, 2 H), 1.07 - 1.28 (m, 2 H)
Example 11 lHN R(400MHz,
DMSO-d6) δ ppm
8.39 (d, J=8.3 Hz, 1 H), 7.91 -8.01 (m, 2H), 7.76 (dd, J=4.4, 2.9 Hz, 2 H), 7.08 (d, J=3.8 Hz, 1 H), 7.03 (t, J=2.4 Hz, 1 H), 6.98 (t, J=6.1 Hz, 1 H), 4.53 (d, J=6.1 Hz, 2H), 4.40 (t, J=5.3 Hz, 1 H), 4.33 (dq, J=8.2, 4.9 Hz, 1 H), 3.38 (q, J=6.3 Hz, 2 H), 3.03-3.15 (m, 2 H), 1.60- 1.84 (m, 5 H), 1.49- 1.59 (m, 4 H), 1.44 (tt, J=7.4,3.4Hz, 2 H), 1.04- 1.17 (m, 2 H)
Example 12 lHNMR(400MHz,
DMSO-d6) δ ppm
8.37 (d, J=8.3 Hz, 1 H), 7.95 (t, J=5.8 Hz, 1 H), 7.75 (d, J=3.8 Hz, 1 H), 7.67 (d, J=2.0 Hz, 1 H), 7.07 (d, J=3.8 Hz, 1 H), 6.85 (d, J=2.0 Hz, 1 H), 6.43 (t, J=6.2 Hz, 1 H), 4.57 (d, J=6.1 Hz, 2 H), 4.40 (t, J=5.2 Hz, 1 H), 4.33 (dq, J=8.3, 5.0 Hz, 1 H), 3.38 (q, J=6.3 Hz, 2 H), 3.03-3.14 (m, 2 H), 2.33 (s, 3 H), 1.60
- 1.84 (m,5H), 1.49- 1.58 (m, 4 H), 1.44 (tt, J=7.4, 3.6 Hz, 2 H),1.04- 1.17 (m, 2 H)
Example 13 lH MR(400 Hz,
DMS0-d6) δ ppm
8.35 (d, J=8.3 Hz, 1 H), 7.91 -8.01 (m,2H), 7.72 (d, J=3.8 Hz, 1 H), 7.48 (t, J=6.1 Hz, 1 H), 7.00 (d, J=3.8 Hz, 1 H), 6.61 (dd, J=5.4, 1.9 Hz, 1 H), 6.59 (d, J=1.8 Hz, 1 H), 4.64 (d, J=6.1 Hz, 2 H), 4.40 (t, J=5.3 Hz, 1 H), 4.34 (dq, J=8.3, 5.0 Hz, 1 H), 3.38 (q, J=6.3 Hz, 2 H), 3.02-3.16 (m, 2 H), 1.61 - 1.84 (m, 5 H), 1.49- 1.61 (m, 4 H), 1.37 - 1.48 (m, 2 H), 1.03- 1.17 (m, 2 H)
Example 14 1H MR(400 MHz,
DMSO-d6) δ ppm
8.39 (d, J=8.6 Hz, 1 H) 7.96 (d, J=2.5 Hz, 1 H) 7.92 (t, J=5.7 Hz, 1 H) 7.76 (dd, J=7.2, 2.9 Hz, 2 H) 7.08 (d, J=3.8 Hz, 1 H)7.03 (t, J=2.3 Hz, 1 H) 6.98 (t, J=6.2 Hz, 1 H) 4.63 (t, J=5.4 Hz, 1 H) 4.53 (d, J=6.1 Hz, 2 H)4.36 -4.48 (m, 1 H)3.37(q,
J=6.1 Hz.2 H) 3.11 (m, j=5.8 Hz, 2 H) 1.47 - 1.76 (m, 7 H) 1.29 (d, J=8.3 Hz, 1 H) 1.02 - 1.23 (m, 3 H) 0.77 - 0.98 (m, 2 H)
Example 15 1HNMR (400MHz
,DMSO.d6) δ ppm
8.37 (d, J = 8.3 Hz, 1 H), 7.91 (t, J = 5.6 Hz, 1 H), 7.74 (d, J = 3.8 Hz, 1 H), 7.67 (d, J = 2.0 Hz, 1 H), 7.08 (d, J = 3.8 Hz, 1 H), 6.86 (d, J = 2.0 Hz, 1 H), 6.43 (t, J = 6.1 Hz, 1 H), 4.62 (t, J = 5.4 Hz, 1 H), 4.57 (d, J = 6.1 Hz,2H), 4.47 - 4.30 (m, 1 H), 3.37 (q, J = 6.1 Hz, 2 H), 3.33 (s, 1 H), 3.19 -3.02 (m, 2 H), 2.33 (s, 3 H), 1.75 - 1.47 (m, 6H), 1.29 (br. s., 1 H), 1.22 - 1.01 (m, 3 H), 0.99-0.75 (m,'2H)
Example 16 lHNMR(400MHz,
DMSO-d6) δ ppm
8.35 (d, J=8.34 Hz, 1 H) 7.98 (d, J=5.56 Hz, 1 H) 7.90 (t, J=5.68 Hz, 1 H) 7.72 (d, J=3.79 Hz, 1 H) 7.48 (t, J=6.19 Hz, 1 H) 7.00 (d, J=3.54 Hz, 1 H) 6.53 - 6.66 (m, 2 H) 4.57 - 4.68 (m, 2
H) 4.30 -4.50 (m, 1 H)3.37 (q, J=6.15 Hz, 2 H) 3.01 -
3.17 (m, 2 H) 1.47- 1.75 (m, 6 H) 1.27 (br. s., 1 H)
1.18 (br. s., 1 H) 1.10 (d, J=9.09 Hz, 2 H) 0.90 (d, J=12.13 Hz, 2H)
Example 17 1HNMR (400MHz
,DMSO-d6) δ ppm
8.41 (d, J=8.3 Hz, 1 H), 7.96 (d, J=2.3 Hz, 1 H), 7.87 (d, J=4.8 Hz, 1 H), 7.76 (dd, J=6.2, 2.9 Hz, 2 H), 7.08 (d, J=3.8 Hz, 1 H), 7.03 (s, 1 H), 6.98 (s, 1 H), 4.53 (d, J=6.1 Hz, 2H), 4.39 (td, J=5.0, 3.4 Hz, 1 H), 2.56 (d, J=4.5 Hz, 3 H), 1.51 - 1.73 (m, 7 H), 1.23 - 1.32 (m, 1 H), 1.11 (d, J=8.3 Hz, 3 H), 0.92 (br. s., 2 H)
Example 18 lHNMR (400MHz
,DMSO-d6) δ ppm
8.51 (d, J = 8.34 Hz, lH), 8.29- 8.36 (m, 1 H), 8.25 (d, J = 1.77 Hz, 1H), 7.95 (t, J = 5.56 Hz, 1H), 7.82 (d, J = 3.79 Hz, lH), 7.70-7.76 (m, l H), 7.25 (d, J = 3.79 Hz, 1H), 5.45 (s, 21-0,4.64 (t, J = 5.43 Hz, 1H), 4.41 - 4.51 (m, 1H), 3.36-3.42
(m, 21-1), 3.05 - 3.20 (m, 2H), 1.52 - 1.75 (m, 7H), 1.23 - 1.36 (m, 1H), 1.04- 1.22 (m, 3H), 0.79-0.98 (m, 211)
Example 19 IHNMR (400MHz
,DMSO-d6) δ ppm
8.52 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 2.0 Hz, 1H), 7.96 (I, J = 5.6 Hz, 1H), 7.82 (d, J = 3.8 Hz, 1H),7.67 (d, J = 1.8 Hz, HI), 7.23 (d, J = 3.8 Hz, ll-[),5.43 (s, 2H), 4.64 (br. s., 1H), 4.41 -4.51 (m, lH), 3.36 - 3.43 (m, 2H), 3.06 -3.19 (m, 2H), 2.35 (5,31-1), 1.52 - 1.75 (m, 7H), 1.31 (d, J = 3.5 Hz, 1 H), 1.04 - 1.22 (m, 3H), 0.79 - 0.99 (m, 2H)
Example 20 IH MR (400MHz
,DMSO-cl6) δ ppm
8.80 (d, J = 6.6 Hz, l H), 8.55 (d, J = 8.1 Hz, 1H), 8.34 (d, J = 2.5 Hz, lH), 8.25 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 3.8 Hz, 1H), 7.72 - 7.77 (m, 1 H), 7.25 (d, J = 3.8 Hz, HI), 5.45 (s, 2H),4.74 - 4.83 (m, 1H), 4.64 - 4.73 (m, 2H), 4.36 - 4.46 (m, 3H), 1.64 - 1.83
(m, 5H), 1.53- 1.61 (m, 21-1), 1.41 - 1.49 (m, 2H), 1.15 (d, J = 11.87 Hz, 2H)
Example 21 1H MR (400MHz
,DMSO-d6) δ ppm
8.41 (d, J=8.3 Hz, 1 H), 8.07 (d, J=2.5 Hz, 11-0, 7.94 (t, J=5.6 Hz, 1 H), 7.84 (d, J=1.8 Hz, 1 H), 7.76 (d, J=3.8 Hz, 1 H), 7.19 ((, J=2.3 Hz, 1 H), 7.05 (d, J=3.8 Hz, 1 H), 4.78 (s, 2 H), 4.72 - 4.53 (m, 1 H), 4.43 - 4.27 (m, 1 H), 3.51 (q, J=7.0 Hz, 2 H), 3.42 - 3.28 (m, 211), 3.21 -3.01 (m, 2 H), 1.81 - 1.39 (m, 9 H), 1.21 -0.97(m, 5 H).
Example 22 1H NMR (400MHz
,DMSO-d6) δ ppm
8.37 (d, J = 8.3 Hz, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.74 (d, J = 3.8 Hz, 1H), 7.06- 7.11 (in, 1H), 7.01 - 7.06 (m, 1H), 6.70 (t, J = 6.1 Hz, 1 H), 6.62 (t, J = 2.2 Hz, 1H), 6.57 (t, J = 1.8 Hz, 1H), 6.55 (d, J = 1.8 Hz, lH),4.47(d, J = 6.1 Hz, 2H), 4.38 - 4.45 (m, 1H), 3.33 (s, 3H),3.29- 3.33 (m, 2H),3.16-3.22 (m, 2H),
1.42 - 1.77 (m,7H), 1.29 (br. s., IH), 1.03 - 1.17 (m, 3H), 0.74 - 0.96 (m, 2H)
Example 23 1HNMR (400MHz
,DMSO-d6)5ppm
8.34 (d, J = 8.3 Hz, IH), 8.24 (d, J = 6.8 Hz, IH), 7.75 (d, J = 3.8 Hz, I H), 7.67 (d, J = 2.0 Hz, IH), 7.07 (d, J = 3.8 Hz, IH), 6.85 (d, J = 2.3 Hz, IH), 6.42 (t, J = 6.1 Hz, IH), 4.99 (d, J=5.6 Hz, IH), 4.57 (d, J = 5.8 Hz, 2H), 4.29-4.41 (m, IH), 4.28 - 4.20 (m, IH), 4.16 (d, J=7.1 Hz, 1 H), 2.33 (s, 3H),2.20-1.98 (m, 41-1), 1.81-1.43 (m, 9 H), 1.22- 1.01 (m, 2H)
Example 24 lHNMR (400MHz
,DMSO-d6) δ ppm
8.33 (d, J = 8.3 Hz, IH), 8.17 (d, J = 7.6 Hz, IH), 7.74 (d, J = 3.8 Hz, IH), 7.67 (d, J = 2.0 Hz, IH), 7.07 (d, J = 3.8 Hz, IH), 6.85 (d, J = 2.0 Hz, IH), 6.42(1, J = 6.1 Hz, IH), 5.04 (d, J = 5.8 Hz, IH), 4.57 (d, J = 5.8 Hz, 2H), 4.33 (td, J = 9.1, 5.1 Hz, IH), 3.72 -3.85
(m, 1H),3.52-3.71 (m, 1H), 2.40-2.49 (m, 2H), 2.33 (s, 3I-I), 1.64- 1.79 (m, 6H), 1.52 - 1.63 (m, 3H), 1.36 - 1.50 (m, 2H), 1.01 - 1.20 (m, 2H)
Example 25 1HN R (400MHz
,DMSO-d6) δ ppm
8.35 (d, J = 8.3 Hz, 1H),
8.06 (d, J = 4.3 Hz, 1H), 7.74 (d, J = 3.8 Hz, 1H), 7.67 (d, J = 2.0 Hz, 1H),
7.07 (d, J = 3.5 Hz, 1 H), 6.85 (d, J = 2.0 Hz, 1H), 6.42 (t, J = 5.9 Hz, 1H), 4.57 (d, J = 5.8 Hz, 2H), 4.29 (td, J = 8.9, 5.4 Hz, 1H), 2.61 (td, J = 7.5, 3.8 Hz, 1H), 2.33 (s, 3H), 1.66 - 1.78 (m, 3H), 1.52 - 1.64 (m, 3H), 1.33 - 1.49 (m,2H), 0.98- 1.20 (m,2H), 0.50-0.68 (m, 2H), 0.30 - 0.48 (m, 2H)
Example 26 1HNMR (400MHz
,DMSO-d6) δ ppm
8.40 (d, J = 7.1 Hz, 1H), 8.37 (d, J = 8.3 Hz, IH), 7.75 (d, J = 3.8 Hz, IH), 7.67 (d, J = 2.0 Hz, 1 H), 7.07 (d, J = 3.8 Hz, 1 H), 6.85 (d, J = 2.0 Hz, 1 H), 6.42 (t, J = 6.1 Hz, 1H), 5.02
(t, J = 5.9 Hz, 1H), 4.57 (d, J = 6.1 Hz, 2H), 4.17-4.42 (m, 2H), 2.19- 2.37 (m, 6H),2.00 (s,3H), 1.51 - 1.81 (m, 6H), 1.44 (tt, J = 7.3, 3.7 Hz, 2H), 0.92- 1.20 (m, 2H)
Example 27 1HNMR (400MHz
,DMSO-d6) δ ppm
7.83 (s, 1H), 7.66 - 7.77 (m,
NH2
1H), 7.05 -7.16 (m,2H), 5.64 (d, J = 1.5 Hz, 1H), 5.10 (br. s., 1H),4.61 (s, 2H), 4.29 (br. s., 2H), 3.95 (s, 1H), 2.75 (t, J = 7.5 Hz, 2H),2.38 (s, 7H), 1.72 (s, 3H), 1.75 (s, 2H), 1.55 (d, J=7.6 Hz, 6Ή), 1.40 (br. s., 4H), 1.08 (br. s., 2H)
BIOCHEMICAL ASSAY FOR LSD-1 ACTIVITY
Wipl Dephosphorylation Assay with Fluorescein Diphosphate (FDP) Substrate
Recombinant Wipl protein (Wipl(A2-K420)) was expressed in Sf9 cells, lysed in 0.5% CHAPS, purified over 1.) Nickel Column, 2.) Superdex 200 pool3, then 3.) MonoQ p2 , and finally stored at -20°C in storage buffer (50mM Tris-HCl (pH 8.0), 20% Glycerol, 0.2M aCl,lmM TCEP, ImM CHAPS, 0.25mM Imidazole). Assay reaction was carried out in reaction buffer containing 50mM Tris pH7.5, 30mM MgCl2, 0.05mg/mL BSA, 0.05% CHAPS ,and ImM DTT. Fluorescein diphosphate (FDP, tetiaammonium salt; Invitrogen) solution was prepared as l OniM stock made in 50mM Tris (pH7.5), and stored at -20°C.
For the assay, compounds were serially-diluted from a maximum final concentration of 5μΜ, 3-fold drops for 1 1 points to a minimum final concentration of 0.085nM, and ΟΑ μί^ of compounds in DMSO were added to Non-binding 384 well plate (Corning).
Subsequently, buffer and Wip l enzyme (I nM final) were added for a 15min
preincubation. Reaction was then started with the addition of FDP substrate (50μΜ final) in buffer. The reaction was allowed to proceed for2 hours at room temperature.
Fluorescence was read using a Viewlux Microplate Imager (Perkin Elmer) Fluorescein 480/540 protocol. Readout in Relative Fluorescent units (RFUs) were compared with vehicle ( 1 % DMSO) treated control wells. Curves were analyzed using Activity Base and XLfit, and XC50 results expressed as pIC50 values.
Wipl Dephosphorylation Assay with phospho-P38 Substrate
Recombinant full-length Wip l protein (FL-Wip l ) was expressed in Sf9 cells, lysed in 0.5% CHAPS, purified over 1 .) NiNTA column, then 2.)Superdex 200 pool4, and finally stored at -20°C in storage buffer (50mM Tris-HCl (pH 8.0), 20% Glycerol, 0.1 5M NaCl, 0. 1 M EGTA, l mM TCEP). Recombinant GST-tagged full-length p38 was expressed in Sf9 cells, lysed, purified over 1.) NiNTA column, 2.)Superdex 200 pool 3. Recovered protein was then treated with recombinant M K6 (U. Dundee; DU 1 671 ) at 1 /50 molar ratio to achieve phosphorylation of p38 (T1 80/Y 182). Following reaction with M K6, p38 underwent a buffer exchange and republication over a Nickel column, and was then stored at -80°C in storage buffer (50mM Tris-HCl (pH 8.0), 0.1 5M NaCl, 0. 1 mM EGTA, 1 mM TCEP).
For the assay, compounds were serially-diluted from a maximum final concentration of 50μΜ, 3-fold drops for 1 1 points to a minimum final concentration of 0.85nM, and 0.1 μί of compounds in DMSO (or neat DMSO for vehicle control) were added to Non-binding 384 well plate (Corning). A final concentration of 15nM FL-Wip l was added to the assay plate in assay buffer (50mM Tri ( pH 7.5), 30mM MgCl2, 0.1 mM EGTA, O. l mg/mL BSA, 1 mM CHAPS, 1 mM DTT) for a 1 5min preincubation. Reaction was then started with the addition of 8nM phosphorylated GST-p38 (Τ180ΛΊ 82). The reaction was allowed to proceed for 90 m inutes at room temperature. A mixture of detection reagents were added containing final concentrations of 50 mM EDTA, 1 :300 phospho-p38 MAPK (Thr l 80/ Tyrl 82) rabbit mAB (D3F9), 5 ug mL anti-GST-IgG conjugated to SureLight
Allophycocyanin, and I nM LANCE E-W1024 labelled anti rabbit IgG. The reaction was covered and incubated for 60 m inutes at room temperature. Finally, fluorescence was read using a Viewlux Microplate Imager (Perkin Elmer) or alternatively an Envision
MultiLabel Reader (Perkin Elmer). Readout in Relative Fluorescent units ( FUs) were compared with vehicle ( 1 % DMSO) treated control wells. Curves were analyzed using ActivityBase and XLfit, and XC50 results expressed as pIC50 values.
Cellular Assay for p53-Transcriptional Activation
A p53-Luciferase reporter BacMam construct was made that contains a p53 response element ((CCTGGACTTGCCTGGCCTG) 15) upstream of a minimal promoter derived from HSV TK and the firefly Luciferase open reading frame. The construct was cloned into a BacMam expression vector, BacMam virus was cultured from DH 1 Obac cells, and titer was determined. MX-1 breast carcinoma cell line was obtained from the NCI and cultured as recommended in RPMI+10% fetal bovine serum (FBS).
For the assay, MX- 1 cells were trypsinized and viable cells were counted and diluted in OptiMEM (Invitrogen)+ 5% FBS to achieve 10,000 cells per 48uL (2.1 x 105 cells/mL). For transduction of the p53-luciferase reporter, BacMam was added to diluted cells such that the Multiplicity of Tranduction (MOT) is 5 pfu/cell. Transduced MX- 1 cells were then seeded 48uL/well into 384 well black polystyrene cell culture mici oplates (Greiner) and placed in tissue culture incubator at 37°C and 5% C02 for 24 hours. The following day, compounds were serially-diluted by 3-fold drops for 10 point curves. Initial compound serial dilutions in DMSO were then diluted 20-fold further in cell media, and 2.0μί of compound dilutions were added to cells (0.2% DMSO final). The dilution curve spanned from a maximum final concentration of 20μΜ to a minimum final concentration of 1 .OnM. Microplates were then returned to tissue culture incubator at 37°C and 5%C02. After an additional 24 hours incubation, plates were removed from the incubator and allowed to sit on bench-top for 1 5 minutes at room temperature. At this point, 25μΙ≤ of resuspended Bright Glo (Promega) solution was added to all wel ls, and plates were shaken for 2 minutes at room temperature, and luminescence was read on the En Vision 2100 plate reader. ). Readout in Relative luminescence units (RLUs) were compared with vehicle (0.2% DMSO) treated control wells. Curves were analyzed using XLfit. Results were expressed as several parameters including: 1 .) maximum-fold induction, 2.) concentration of 2-fold induction, 3.) concentration of 3-fold induction, and 4.) XC50.
Wipl Cellular Outgrowth Assay
Cell lines used for the cellular outgrowth assay included MX- 1 breast carcinoma line (NCI), A549 lung carcinoma (ATCC), Molt3 T-Cell leukemia (ATCC), and HN5 head and neck carcinoma cell line (ICR-UK). All cel l lines were cultured according to
recommended conditions. Cell lines were split in duplicate T-75 flasks two to three days prior to assay set-up in ratios which yielded70-80% confluence at time o f harvest for plate seeding. Cells were trypsinized, viable cell concentrations were determined using a using ViCell XR (Beckman Coulter), and cells were resuspended to yield 2,000 cells per ml. Into 96 well black polystyrene cell culture microplates (Nalgene) 96 ds cell suspension was added and seeded cells were allowed to adhere overnight in 37°C + 5% C02 incubator.
Compounds were diluted in U-bottom 384-well plate over 10 points in DMSO.
Compound dilutions in DMSO or neat DMSO (for vehicle control) were then diluted 20- fold with media (RPMI- 1640 + 1 0% FBS) in an intermediate compound plate and mixed thoroughly. Titrations were 3-fold for a 1 0 point series with a maximum final concentration of 30μΜ and a minimum final concentration of 1 .5nM. From the intermediate plate, 4 μ1≤ were transferred to each well of the 48 μ ΐ/well of cells (total volume on cells, 50 μΐβ; 0.2% DMSO). Microplates were then returned to tissue culture incubator at 37°C and 5%C02 for an additional 7 day incubation. For reference, on the day of compound addition (Time-0), one additional plate for each cell type is developed with the addition of 50μ ΐ3 CellTiter-Glo (Promega), plates are shaken gently for 2 minutes, allowed to react for 20 minutes, and luminescence is read on a Tecan Safire (Tecan). This T=0 value indicates the signal of the starting number of cells. At day 7, plates are harvested by the addition of 50^ils Cell Titer-Glo (Promega) to all wells as described above. Readout in Relative luminescence units (RLUs) were compared with vehicle (0.2% DMSO) treated control wells. Curves were analysed using XLfit and reported as IC50s. Biological Data
Exemplified compounds of the present invention were tested according to the above assays and were found to be inhibitors of WIP l . The plCso values ranged from about 5.3-8.5. The IC50 values of the present compounds range from about 3 to 5000 nM. The IC50 values of the more active compounds range from about 3 to 100 nM. The most active compounds are under 10 nM.
The plC50 of compounds of Examples 1 -3 and 6-9 ranges from 7.5 to 8.0.
The pIC50 of compounds of Examples 14, 1 7, 4 ranges from 8.0-8.5.
Exemplified enzymatic activity (Wip l Dephosphorylation assay with Fluorescein Diphosphate (FDP) Substrate)
Figure imgf000100_0001

Claims

What is claimed
1. A compound of Formula (I);
Figure imgf000101_0001
(I) wherein
Ri is hydrogen, Ci-C4alkoxy, Ci-C4alkyl, substituted Ci-C4alkyl, C3-Cycycloalkyl, or C3- Cyheterocycloalkyl, wherein said C3-Cycycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -0(0 )Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl;
R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C3alkyl, substituted Ci-C3alkyl, Ci- C4alkoxy, hydroxyl, amino, substituted amino, C3-Cycycloalkyl, cyano, ester, carboxylic acid, and C3-C6heterocycloalkyl; R3 is hydrogen, Ci-C6alkyl, substituted Ci-C6alkyl, or R3 is absent when X is O or S;
R4 is cyclohexyl or cyclopentyl;
X is N, O or S; or a pharmaceutically acceptable salt or prodrug thereof.
2. A compound of claim 1, which is represented by Formula (II):
Figure imgf000101_0002
(II); wherein
Ri is hydrogen, Ci-C4alkoxy, Ci-C4alkyl, substituted Ci-C4alkyl, C3-Cycycloalkyl, or C3- Cyheterocycloalkyl, wherein said C3-Cycycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -0(0 )Ra, wherein Ra is C i -C6alkyl or substituted C 1 -C6alkyl;
R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C3alkyl, substituted Ci-C3alkyl, Ci- C4alkoxy, hydroxyl, amino, substituted amino, C3-Cycycloalkyl, cyano, ester, carboxylic acid, and C3-C6heterocycloalkyl; R3 is hydrogen, Ci-C6alkyl, substituted Ci-C6alkyl, or R3 is absent when X is O or S;
R4 is cyclohexyl or cyclopentyl;
X is N, O or S; or a pharmaceutically acceptable salt thereof.
3. A compound of claim 1, which is represented by Formula (III):
Figure imgf000102_0001
(in);
wherein
Ri is hydrogen, Ci-C4alkoxy, Ci-C4alkyl, substituted Ci-C4alkyl, C3-Cycycloalkyl, or C3- Cyheterocycloalkyl, wherein said C3-Cycycloalkyl may be substituted with one to three substituents selected from the group consisting of hydroxyl and -0(0 )Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl;
R2 is an aryl or heteroaryl ring, which may be substituted with one to four substituents selected from the group consisting of halo, Ci-C3alkyl, substituted Ci-C3alkyl, Ci- C4alkoxy, hydroxyl, amino, substituted amino, C3-Cycycloalkyl, cyano, ester, carboxylic acid, and Cs-Ceheterocycloalkyl;
R3 is hydrogen, Ci-C6alkyl or substituted Ci-Cealky;
R4 is cyclohexyl or cyclopentyl; or a pharmaceutically acceptable salt thereof.
4. A compound according to any one of claims 1-3, wherein Ri is Ci-C4alkyl or substituted Ci-C4alkyl.
5. A compound according to any one of claims 1-3, wherein Ri is C3-C7cycloalkyl.
6. A compound according to any one of claims 1-3, wherein Ri is cyclopropyl or cyclobutyl, wherein said cyclopropyl and cyclobutyl may be substituted with hydroxyl or -0(0)Ra, wherein Ra is Ci-C6alkyl or substituted Ci-C6alkyl.
7. A compound according to any one of claims 1-6, wherein R2 is phenyl, which may be substituted with one to four substituents selected from the group consisting of halogen, Ci-C3alkyl, substituted Ci-C3alkyl, Ci-C4alkoxy, hydroxyl, amino, substituted amino, C3- C7cycloalkyl, C3-C7heterocycloalkyl, cyano, ester, and carboxylic acid.
8. A compound according to any one of claims 1-6, wherein R2 is pyridinyl, which may be substituted with one to three substituents selected from the group consisting of halo, Ci-C3alkyl, and substituted Ci-C3alkyl.
9. A compound according to any one of claims 1-8, wherein R3 is hydrogen.
10. A compound according to any one of the above claims, wherein R4 is cyclopentyl.
11. A compound according to claim 1 , which is selected from the group consisting of:
(S)-5 - { [(5 -chloropyridin-3 -yl)amino]methyl} -N- {3 -cyclopentyl- 1 - [(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide; (S)-5- {[(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N- {3-cyclopentyl- 1 -[(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5- {[(4-chloropyridin-2-yl)amino]methyl} -N- {3-cyclopentyl- 1 -[(2- hydroxyethyl)amino] - 1 -oxopropan-2-yl } thiophene-2-carboxamide;
(S)-5- {[(5-chloro-pyridin-3-yl)amino]methyl} -N-[3-cyclopentyl- 1 -(oxetan-3-ylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide; (S)-5-{ [(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N- [3 -cyclopentyl- 1 -(oxetan-3- ylamino)- 1 -oxopropan-2-yl]thiophene-2-carboxamide;
(S)-5- {[(4-chloro-pyridin-2-yl)amino]methyl} -N-[3-cyclopentyl- 1 -(oxetan-3-ylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S) -N-{ 1 -amino-3 -cyclopentyl- 1 -oxopropan-2-yl)-5 - { [(5 -chloropyridin-3 - yl)amino]methyl}thiophene-2-carboxamide;
(S)-5 - { [(5 -chloropyridin-3 -yl)amino]methyl} -N- [3 -cyclopentyl- 1 -(methylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S)-5 - { [(5 -chloropyridin-3 -yl)amino]methyl} -N- [3 -cyclopentyl- 1 -(dimethylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S) -5 - { [(5 -chloropyridin-3 -yl)amino]methy 1} -N-[3 -cyclopentyl- 1 -(methoxyamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S) -5 - { [(5 -chloropyridin-3 -yl)amino]methy 1} -N- {3 -cyclopentyl- 1 - [(3 - hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5-{ [(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N- {3-cyclopentyl-l -[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(¾^-5-{[(4-chloropyridin-2-yl)amino]methyl}-N-{3-cyclopentyl-l-[(3- hydroxypropyl)amino]-l-oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5 -{ [(5 -chloropyridin-3 -yl)amino]methyl} -N- {3 -cyclohexyl- 1 - [(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5-{ [(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N- {3 -cyclohexyl- 1 -[(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide;
(¾)-5-{[(4-chloropyridin-2-yl)amino]methyl}-N-{3-cyclohexyl-l-[(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5-{ [(4-chloropyridin-2-yl)amino]methyl} -N- [3 -cyclohexyl- 1 -(methylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide; (S)-5- {[(5-chloropyridin-3-yl)oxy]methyl} -N- {3-cyclohexyl- 1 -[(2-hydroxyethyl)amino]- l-oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5- {[(5-chloro-2-methylpyridin-3-oxy]methyl} -N- {3-cyclohexyl- 1 -[(2- hydroxyethyl)amino]- 1 -oxopropan-2-yl}thiophene-2-carboxamide;
(S)-5 - { [(5 -chloropyridin-3 -yl)oxy]methyl} -N- [3 -cyclopentyl- 1 -(oxetan-3 -ylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S)-5 -{ [(5 -chloropyridin-3 -yl)oxy]methyl} -N- [3 -cyclopentyl- 1 -(oxetan-3 -ylamino)- 1 - oxopropan-2-yl]thiophene-2-carboxamide;
(S)-5- {[(3-chlorophenyl)amino]methyl} -N- {3-cyclohexyl- 1 -[(2-methoxyethyl)amino]- 1 - oxopropan-2-yl}thiophene-2-carboxamide;
5-{[(5-chloro-2-methylpyridin-3-yl)amm^
hydroxy cyclobutyl)amino] - 1 -oxopropan-2-yl} thiophene-2-carboxamide; 5-{[(5-chloro-2-methylpyridin-3-yl)amm^
hydroxy cyclobutyl)amino] - 1 -oxopropan-2-yl} thiophene-2-carboxamide;
(S)-5- { [(5-chloro-2-methylpyridin-3-yl)amino]methyl} -N- [3 -cyclopentyl- 1 - (cyclopropylamino)- 1 -oxopropan-2-yl]thiophene-2-carboxamide;
(7 3r^-3-[(¾)-2-(5-{[(5-chloro-2-m
carboxamido)-3-cyclopentylpropanamido]cyclobutyl acetate; and S ^5",J^ 3-[fS 2-(5-{[(5-chloro-2-methylpyridin-3-yl)amino]methyl}th^
carboxamido)-3-cyclopentylpropanamido]cyclobutyl 2,6-diaminohexanoate; or a pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition comprising a compound according to any one of the above claims or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
13. A method of treating cancer which comprises administering to a human in need thereof an effective amount of a compound as described in any one of claims 1-11.
14. A method of claim 13 wherein the cancer is selected from the group consisting of brain, glioblastomas, leukemias, lymphomas, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, meduUoblastoma, neuroblastoma, small cell lung, endometrium, cervix, esophagus, colon, gastric, bladder, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, mesothelioma, sarcoma, osteosarcoma, giant cell tumor of bone and thyroid.
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