WO2002005804A1 - Composes et procedes - Google Patents

Composes et procedes Download PDF

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Publication number
WO2002005804A1
WO2002005804A1 PCT/US2001/022669 US0122669W WO0205804A1 WO 2002005804 A1 WO2002005804 A1 WO 2002005804A1 US 0122669 W US0122669 W US 0122669W WO 0205804 A1 WO0205804 A1 WO 0205804A1
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Prior art keywords
tetrahydro
amino
naphthyloxy
propanol
naphthenyl
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PCT/US2001/022669
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English (en)
Inventor
Joseph P. Marino
Scott K. Thompson
Daniel F. Veber
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Smithkline Beecham Corporation
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Priority to AU2001278951A priority Critical patent/AU2001278951A1/en
Publication of WO2002005804A1 publication Critical patent/WO2002005804A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine

Definitions

  • Compounds of this invention are non-peptide, reversible inhibitors of type 2 methionine aminopeptidase, useful in treating conditions mediated by angiogenesis, such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity.
  • angiogenesis such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity.
  • angiogenesis a process termed angiogenesis (Folkman J. (1974) Adv Cancer Res. 19; 331).
  • the new blood vessels induced by tumor cells as their life-line of oxygen and nutrients also provide exits for cancer cells to spread to other parts of the body. Inhibition of this process has been shown to effectively stop the proliferation and metastasis of solid tumors.
  • a drug that specifically inhibits this process is known as an angiogenesis inhibitor.
  • the anti-angiogenesis therapy (“indirect attack”) has several advantages over the “direct attack” strategies. All the “direct attack” approaches such as using DNA damaging drugs, antimetabolites, attacking the RAS pathway, restoring p53, activating death programs, using aggressive T-cells, injecting monoclonal antibodies and inhibiting telomerase, etc., inevitably result in the selection of resistant tumor cells. Targeting the endothelial compartment of tumors as in the "indirect attack”, however, should avoid the resistance problem because endothelial cells do not exhibit the same degree of genomic instability as tumor cells.
  • anti-angiogenic therapy generally has low toxicity due to the fact that normal endothelial cells are relatively quiescent in the body and exhibit an extremely long turnover.
  • direct attack and direct attack target different cell types, there is a great potential for a more effective combination therapy.
  • TNP-470 a semisynthetic derivative of fumagillin of Aspergillus fuigatus, is among the most potent inhibitors of angiogenesis. It acts by directly inhibiting endothelial cell growth and migration in vitro and in vivo (Ingber et al. (1990) Nature 348, 555). Fumagillin and TNP-470, have been shown to inhibit type 2 methionine aminopeptidase (hereinafter MetAP2) by irreversibly modifying its active site.
  • MetAP2 type 2 methionine aminopeptidase
  • hMetAP-2-catalyzed cleavage of the initiator methionine of proteins could be essential for releasing many proteins that, after myristoylation, function as important signaling cellular factors involved in cell proliferation.
  • Proteins known to be myristoylated include the src family tyrosine kinases, the small GTPase ARF, the HIV protein nef and the subunit of heterotrimeric G proteins.
  • a recently published study has shown that the myristoylation of nitric oxide synthase, a membrane protein involved in cell apoptosis, was blocked by fumagillin (Yoshida, et al. (1998) Cancer Res. 58(16), 3751).
  • MetAP enzymes are known to be important to the stability of proteins in vivo according to the "N-end rule" which suggests increased stability of methionine- cleaved proteins relative to their N-terminal methionine precursors (Varshavsky, A (1996) Proc. Natl. Acad. Sci. U.S.A 93, 12142). Inhibition of hMetAP2 could result in abnormal presence or absence of some cellular proteins critical to the cell cycle. Methionine aminopeptidases (MetAP) are ubiquitously distributed in all living organisms.
  • MetAP enzymes type 1 and type 2 are found in eukaryotes, which at least in yeast, are both required for normal growth; whereas only one single MetAP is found in eubacteria (type 1) and archaebacteria (type 2).
  • the N-terminal extension region distinguishes the methionine aminopeptidases in eukaryotes from those in procaryotes.
  • a 64-amino acid sequence insertion (from residues 381 to 444 in hMetAP2) in the catalytic C-terminal domain distinguishes the MetAP-2 family from the MetAP- 1 family.
  • Mammalian type 2 methionine aminopeptidase has been identified as a bifunctional protein implicated by its ability to catalyze the cleavage of N-terminal methionine from nascent polypeptides (Bradshaw, et al (1998) Trends Biochem. Sci. 23, 263) and to associate with eukaryotic initiation factor 2 ⁇ (eIF-2 ⁇ ) to prevent its phosphorylation (Ray, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539). Both the genes of human and rat MetAP2 were cloned and have shown 92% sequence identity (Wu,. et al. (1993) J Biol. Chem.
  • the anti-angiogenic compounds, fumagillin and its analogs, have been shown to specifically block the exo-aminopeptidase activity of hMetAP2 without interfering with the formation of the hMetAP2 : eIF2 ⁇ complex (Griffith, et al., (1997) Chem. Biol. 4, 461; Sin, et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6099).
  • the present invention is to a compound of formula (I), or a pharmaceutically active salt thereof, and its use in treating conditions mediated by angiogenesis, such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity:
  • angiogenesis such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity:
  • Rl and R2 are, independent from one another, selected from H, C ⁇ alkyl, C3_ alkenyl, C3_ alkynyl, C3_7cycloalkyl-C ⁇ ⁇ _6-alkyl, C3_7cycloalkyl, Ar- Co_6alkyl, or Het-Co_6alkyl;wherein the C3_7cycloalkyl-C ⁇ _6 _a lkyl, C3_7cycloalkyl may be optionally fused to or substituted by an Ar or Het ring; and R3 is Ar-Co_6alkyl, or Het-C()-6alkyl.
  • the present invention is to a method of treating conditions mediated by angiogenesis, such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity by administering a compound of formula (I), or a pharmaceutically acceptable salt thereof.
  • angiogenesis such as cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity
  • the present invention is to a method of inhibiting MetAP2 in the treatment of angiogenesis-mediated diseases, all in mammals, preferably humans, comprising administering to such mammal in need thereof, a compound of formula (I), or a pharmaceutically active salt thereof.
  • the present invention is to pharmaceutical compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier therefor.
  • the pharmaceutical compositions of the present invention are used for treating MetAP2-mediated diseases.
  • substituted 1,2-aminoalcohols of formula (I) are inhibitors of MetAP2. It has also now been discovered that selective inhibition of MetAP2 enzyme mechanisms by treatment with an inhibitor of formula (I), or a pharmaceutically acceptable salt thereof, represents a novel therapeutic and preventative approach to the treatment of a variety of disease states, including, but not limited to, cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization and obesity.
  • C ⁇ _6alkyl as used herein at all occurrences means a substituted and unsubstituted, straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof.
  • Any C ⁇ _6alkyl group may be optionally substituted independently by one or more of OR4, R4, NR4R5.
  • Cgalkyl means that no alkyl group is present in the moiety.
  • Ar-CQalkyl is equivalent to Ar.
  • substituents R , R5 ? and R ⁇ are independently defined as C 2 -6alkyl, C3_6alkenyl, C3_6alkynyl, Ax-CQ. ⁇ a ⁇ ayl, Het-Co- 6alkyl, or C3_7cycloalkyl-Co_6alkyl.
  • C3-7cycloalkyl as used herein at all occurrences means substituted or unsubstituted cyclic radicals having 3 to 7 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl radicals.
  • C2-6alkenyl as used herein at all occurrences means an alkyl group of 2 to 6 carbons wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond.
  • C2-6alkenyl includes ethylene, 1-propene, 2-propene, 1 -butene, 2- butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included within the scope of this invention.
  • Any C2-6alkenyl group may be optionally substituted independently by one or more of Ph-C()-6alkyl, Het'-Co-6 alkyl, Ci.galkyl, C ⁇ alkoxy, C ⁇ _6mercaptyl, Ph-Co- ⁇ al o y, Het'-C()-6 a lkoxy, OH, NR4R5 ; Het'-S-C 0 -6alkyl, (CH 2 ) ⁇ . 6 OH, (CH 2 )I _ 6 NR4R5, O(CH 2 ) 1 _ 6 NR4R5, (CH 2 ) 0 -6CO 2 R 6 , O(CH 2 )!_ 6 CO 2 R6, (CH 2 ) ⁇ . 6 SO 2 , CF 3 , OCF 3 or halogen.
  • C2-6alkynyl as used herein at all occurrences means an alkyl group of 2 to 6 carbons wherein one carbon-carbon single bond is replaced by a carbon- carbon triple bond.
  • C 2 _6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
  • Ar or "aryl” as used herein interchangeably at all occurrences mean phenyl and naphthyl, optionally substituted by one or more of Ph-Co- ⁇ alkyI, Het'-Co_6 alkyl, C ⁇ alkyl, Ci ⁇ alkoxy, C ⁇ mercaptyl, Ph-C()-6alkoxy, Het'-C()-6alkoxy, OH, NR4R5, Het'-S-C 0 -6alkyl, (CH 2 )i- 6 OH, (CH 2 )I_ 6 NR4R5, O(CH 2 )I_ 6 NR4R5, (CH 2 )o.
  • Het or "heterocyclic” as used herein interchangeably at all occurrences, mean a stable 5- to 7-membered monocyclic, a stable 7- to 10-membered bicyclic, or a stable 11- to 18-membered tricyclic heterocyclic ring all of which are either saturated or unsaturated, and which consist of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure, and may optionally be substituted with one or more of C ⁇ _ 6 arkyl, C ⁇ alkoxy, OH, (CH 2 )I-6NR 4 R5, O(CH 2 )I-6NR 4 R5, CO 2 R 6 , CF 3 , or halogen.
  • heterocycles include, but are not limited to piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, pyridinyl, pyrazinyl, oxazolidinyl, oxazolinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolidinyl, thiazolinyl, thiazolyl, quinuclidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, benzoxazolyl, furyl, pyranyl, tetrahydrofuryl, t
  • hetero or “heteroatom” as used herein interchangeably at all occurrences mean oxygen, nitrogen and sulfur.
  • the "R” group on the secondary amine in the 1,2-aminoalcohol, i.e., Rl and R2, can be defined as: H, C ⁇ alkyl, C3_ alkenyl, C3_ alkynyl, Ar-C()-6alkyl, Het-CQ. 6alkyl, C3_7cycloalkyl-C ⁇ _6-alkyl, or C3_7cycloalkyl.
  • halo or halogen as used herein interchangeably at all occurrences mean F, CI, Br, and I.
  • CQ denotes the absence of the substituent group immediately following; for instance, in the moiety ArC ⁇ - al yl, when C is 0, the substituent is Ar, e.g., phenyl.
  • Rl and R2 are, independent from one another, selected from H, C ⁇ _
  • the C3_7cycloalkyl-C ⁇ _6-alkyl and C3_7cycloalkyl substituents may be optionally fused to or substituted by an Ar or Het ring.
  • Rl and/or R2 are C3_7cycloalkyl-C ⁇ _6-alkyl, the point of attachment to the nitrogen may be either the cycloalkyl ring or the C ⁇ _6-alkyl chain.
  • Rl and R2 are independently H and C3_7cycloalkyl, optionally fused to an Ar or Het ring, more preferably C6cycloalkyl fused to an Ar ring.
  • R3 is Ar-Co_6alkyl, or Het-C ⁇ -6alkyl.
  • R3 is Ar-Co_6alkyl, morre preferably Ar, most preferably naphthyl.
  • pharmaceutically acceptable salts of formula (I) include, but are not limited to, salts with inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate, or salts with an organic acid such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p- toluenesulfonate, palmitate, salicylate, and stearate.
  • inorganic acids such as hydrochloride, sulfate, phosphate, diphosphate, hydrobromide, and nitrate
  • an organic acid such as malate, maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, methanesulfonate, p- toluenesulfonate, palmitate, salicylate, and stearate.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms.
  • the stereocenters may be (R), (S) or any combination of R and S configuration, for example, (R,R), (R,S), (S,S) or (S,R). All of these compounds are within the scope of the present invention.
  • the preferred compounds of the invention are the following compounds: 3-( 1 -Naphthyloxy)- 1 -[[( 1 S)- 1 ,2,3 ,4-tetrahydro- 1 -naphthenyl] amino]-(2S)-2- propanol;
  • Reaction Conditions a.) 1. NaH, D F, RT, 1 hr 2. R30H, 100 °C, 2-3 hr b.) R1 R2NH, EtOH, 95 °C
  • the pharmaceutically effective compounds of this invention are administered in conventional dosage forms prepared by combining a compound of this invention ("active ingredient") in an amount sufficient to treat cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization or obesity ("MetAp2-mediated disease states”) with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation.
  • the pharmaceutical carrier employed may be, for example, either a solid or liquid.
  • solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • liquid carriers are syrup, peanut oil, olive oil, water and the like.
  • the carrier or diluent may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.
  • the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
  • the amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1000 mg.
  • the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
  • the active ingredient may also be administered topically to a mammal in need of treatment or prophylaxis of MetAP2-mediated disease states.
  • the amount of active ingredient required for therapeutic effect on topical administration will, of course, vary with the compound chosen, the nature and severity of the disease state being treated and the mammal undergoing treatment, and is ultimately at the discretion of the physician.
  • a suitable dose of an active ingredient is 1.5 mg to 500 mg for topical administration, the most preferred dosage being 1 mg to 100 mg, for example 5 to 25 mg administered two or three times daily.
  • topical administration non-systemic administration and includes the application of the active ingredient externally to the epidermis, to the buccal cavity and instillation of such a compound into the ear, eye and nose, and where the compound does not significantly enter the blood stream.
  • systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
  • an active ingredient may be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation.
  • the active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, e.g. from 1% to 2% by weight of the formulation although it may comprise as much as 10% w/w but preferably not in excess of 5% w/w and more preferably from 0.1% to 1% w/w of the formulation.
  • the topical formulations of the present invention both for veterinary and for human medical use, comprise an active ingredient together with one or more acceptable carrier(s) therefor and optionally any other therapeutic ingredient(s).
  • the carrier(s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
  • Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous or alcoholic solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
  • the resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100°C for half an hour.
  • the solution may be sterilized by filtration and transferred to the container by an aseptic technique.
  • bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis.
  • the basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol.
  • the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as esters or polyoxyethylene derivatives thereof.
  • suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as esters or polyoxyethylene derivatives thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
  • the active ingredient may also be administered by inhalation.
  • inhalation is meant intranasal and oral inhalation administration.
  • Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
  • the daily dosage amount of the active ingredient administered by inhalation is from about 0.1 mg to about 100 mg per day, preferably about 1 mg to about 10 mg per day.
  • this invention relates to a method of treating cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization or obesity, all in mammals, preferably humans, which comprises administering to such mammal an effective amount of a MetAP2 inhibitor, in particular, a compound of this invention.
  • a MetAP2 inhibitor in particular, a compound of this invention.
  • treating is meant either prophylactic or therapeutic therapy.
  • Such compound can be administered to such mammal in a conventional dosage form prepared by combining the compound of this invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
  • the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables.
  • the compound is administered to a mammal in need of treatment for cancer, haemangioma, proliferative retinopathy, rheumatoid arthritis, atherosclerotic neovascularization, psoriasis, ocular neovascularization or obesity, in an amount sufficient to decrease symptoms associated with these disease states.
  • the route of administration may be oral or parenteral.
  • parenteral as used herein includes intravenous, intramuscular, subcutaneous, intra-rectal, intravaginal or intraperitoneal administration.
  • the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
  • the daily parenteral dosage regimen will preferably be from about 30 mg to about 300 mg per day of active ingredient.
  • the daily oral dosage regimen will preferably be from about 100 mg to about 2000 mg per day of active ingredient.
  • Example 5 Preparation of 3-(l-Naphthyloxy)-l-cyclohexylamino-(2S)-2-propanol hydrochloride Following the procedure of Example 1(a)- 1(b) except cyclohexylamine was used in step (b) instead of (S)-l,2,3,4-tetrahydro-l-naphthyl-amine, the desired amine was obtained as a white solid (2 steps, 77%). Hydrogen chloride gas was bubbled into a solution of the amine (70 mg, 0.24 mmol) in 1:1 Et 2 0/THF (5 ml) for 15 minutes. The mixture was concentrated down and the title compound was prepared as a white solid (100%).
  • Example 10 Preparation of 3-(Benzyloxy)-l-rr(lR)-l,2,3,4-tetrahvdro-l-naphthenyllaminol-(2S)-2- propanol hydrochloride a) (2S -Benzyl glycidyl ether Following the procedure of Example 1 (a) except benzyl alcohol was utilized instead of 1-naphthol in step (a), the title compound was prepared as a white solid (30%).
  • Example 11 Preparation of 3-(Phenethyloxy)-l-rr(lR)-l,2,3,4-tetrahydro-l-naphthenyl]amino1- (2S)-2-propanol hydrochloride a) (2S)-Phenethyl glycidyl ether Following the procedure of Example 1(a) except phenethyl alcohol was utilized instead of 1-naphthol in step (a), the title compound was prepared as a white solid (30%).
  • the hMetAP2 activity can be measured by direct spectrophotometric assay methods using alternative substrates, L-metbionine-p-nitroanilide (Met-pNA) and L- methionine-7-amido-4-methylcoumarin (Met-AMC).
  • Method-pNA L-metbionine-p-nitroanilide
  • Metal-AMC L- methionine-7-amido-4-methylcoumarin
  • the formation of / nitroaniline (pNA) or 7-amido-4-mefhylcoumarin (AMC) was continuously monitored by increasing absorbance or fluorescence at 405 nm and 460 nm, respectively, on a corresponding plate reader. All assays were carried out at
  • the fluorescence or spectrophotometric plate reader was calibrated using authentic pNA and AMC from Sigma, respectively.
  • Each 50 uL assay solution contained 50 mM Hepes-Na "1" (pH 7.5), 100 mM NaCl, 10-lOOnM purified hMetAP2 enzyme, and varying amounts of Met-AMC (in 3% DMSO aqueous solution) or Met-pNA. Assays were initiated with the addition of substrate and the initial rates were corrected for the background rate determined in the absence of hMetAP2.
  • the methionine aminopeptidase activity of hMetAP2 can also be measured spectrophotometrically by monitoring the free L-amino acid formation.
  • the release of N- terminal methionine from a tripeptide (Met-Ala-Ser, Sigma) or a tetrapeptide (Met-Gly- Met-Met, Sigma) substrate was assayed using the L-amino acid oxidase (AAO) / horse radish peroxidase (HRP) couple (eq. l-3a,b).
  • a typical assay contained 50 mM Hepes-Na+ pH 7.5, 100 mM NaCl, 10 uM CoCl 2 , 1 mM o-Dianisidine or 50 uM Amplex Red, 0.5 units of HRP (Sigma), 0.035 unit of AAO (Sigma), 1 nM hMetAP2, and varying amounts of peptide substrates. Assays were initiated by the addition of hMetAP2 enzyme, and the rates were corrected for the background rate determined in the absence of hMetAP2.
  • v is the initial velocity
  • V is the maximum velocity
  • K a is the apparent Michaelis constant
  • I is the inhibitor concentration
  • A is the concentration of variable substrates.
  • the nomenclature used in the rate equations for inhibition constants is that of Cleland (1963), in which K s and Ky represent the apparent slope and intercept inhibition constants, respectively.
  • XTT a dye sensitive to the pH change of mitochondria in eukaryotic cells, is used to quantify the viability of cells in the presence of chemical compounds.
  • Cells seeded at a given number undergo approximately two divisions on average in the 72 hours of incubation. In the absence of any compound, this population of cells is in exponential growth at the end of the incubation period; the mitochondrial activity of these cells is reflected in the spectrophotometric readout (A450). Viability of a similar cell population in the presence of a given concentration of compound is assessed by comparing the A450 reading from the test well with that of the control well.
  • XTT/PMS prepared immediately before use: 8 mg XTT (Sigma X-4251) per plate is dissolved in 100 ul DMSO. 3.9 ml H 2 O is added to dissolve XTT and 20 ul of PMS stock solution (30 mg/ml) is added from frozen aliquoted stock solution (10 mg of PMS (phenazine methosulfate, Sigma P-9625) in 3.3 ml PBS without cations. These stocks are frozen at -20°C until use). 50 ul of XTT/PMS solution is added to each well and plates incubated for 90 minutes (time required may vary according to cell line, etc.) at 37°C until A 450 is >1.0.
  • Absorbance at 450 nM is determined using a 96-well UV plate reader. Percent viability of cells in each well is calculated from these data (having been corrected for background absorbance). IC50 is that concentration of compound that reduces cell viability to 50% control (untreated) viability.
  • the compounds of this invention show MetAP2 inhibitor activity having IC50 values in the range of 0.0001 to 100 uM. The full structure/activity relationship has not yet been established for the compounds of this invention. However, given the disclosure herein, one of ordinary skill in the art can utilize the present assays in order to determine which compounds of this invention are inhibitors of MetAP2 and which bind thereto with an IC50 value in the range of 0.0001 to 100 uM.

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Abstract

L'invention porte sur des composés qui sont des inhibiteurs réversibles, non-peptidiques de la méthionine aminopeptidase de type 2, utiles dans le traitement d'états induits par l'angiogenèse tels que le cancer, l'hémangiome, la rétinopathie proliférative, l'arthrite rhumatoïde, la néovascularisation d'athérosclérose, le psoriasis, la néovascularisation oculaire et l'obésité.
PCT/US2001/022669 2000-07-19 2001-07-19 Composes et procedes WO2002005804A1 (fr)

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Cited By (8)

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US7304082B2 (en) 1999-10-01 2007-12-04 Smithkline Beecham Corporation 1,2,4-triazole derivatives, compositions, process of making and methods of use
US7858642B2 (en) 2004-03-09 2010-12-28 Elan Pharmaceuticals, Inc. Substituted hydroxyethylamine aspartyl protease inhibitors
US7906556B2 (en) 2005-10-12 2011-03-15 Elan Pharmaceuticals, Inc. Methods of treating amyloidosis using cyclopropyl derivative aspartyl protease inhibitors
US8415319B2 (en) 2002-11-26 2013-04-09 Medtronic, Inc. Devices, systems and methods for improving memory and/or cognitive function through brain delivery of siRNA
US8957198B2 (en) 2003-02-03 2015-02-17 Medtronic, Inc. Compositions, devices and methods for treatment of Huntington's disease through intracranial delivery of sirna
US9133517B2 (en) 2005-06-28 2015-09-15 Medtronics, Inc. Methods and sequences to preferentially suppress expression of mutated huntingtin
US9273356B2 (en) 2006-05-24 2016-03-01 Medtronic, Inc. Methods and kits for linking polymorphic sequences to expanded repeat mutations
US9375440B2 (en) 2006-11-03 2016-06-28 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity

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US6242494B1 (en) * 1998-05-01 2001-06-05 Abbott Laboratories Substituted β-amino acid inhibitors of methionine aminopeptidase-2

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7304082B2 (en) 1999-10-01 2007-12-04 Smithkline Beecham Corporation 1,2,4-triazole derivatives, compositions, process of making and methods of use
US8415319B2 (en) 2002-11-26 2013-04-09 Medtronic, Inc. Devices, systems and methods for improving memory and/or cognitive function through brain delivery of siRNA
US8957198B2 (en) 2003-02-03 2015-02-17 Medtronic, Inc. Compositions, devices and methods for treatment of Huntington's disease through intracranial delivery of sirna
US7858642B2 (en) 2004-03-09 2010-12-28 Elan Pharmaceuticals, Inc. Substituted hydroxyethylamine aspartyl protease inhibitors
US9133517B2 (en) 2005-06-28 2015-09-15 Medtronics, Inc. Methods and sequences to preferentially suppress expression of mutated huntingtin
US7906556B2 (en) 2005-10-12 2011-03-15 Elan Pharmaceuticals, Inc. Methods of treating amyloidosis using cyclopropyl derivative aspartyl protease inhibitors
US9273356B2 (en) 2006-05-24 2016-03-01 Medtronic, Inc. Methods and kits for linking polymorphic sequences to expanded repeat mutations
US9375440B2 (en) 2006-11-03 2016-06-28 Medtronic, Inc. Compositions and methods for making therapies delivered by viral vectors reversible for safety and allele-specificity

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