WO2001036404A1

WO2001036404A1 - Compounds and methods

Info

Publication number: WO2001036404A1
Application number: PCT/US2000/031580
Authority: WO
Inventors: Joseph P. Marino, Jr.; M. Dominic Ryan; Scott Kevin Thompson; Daniel Frank Veber
Original assignee: Smithkline Beecham Corporation
Priority date: 1999-11-19
Filing date: 2000-11-17
Publication date: 2001-05-25

Abstract

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.

Description

COMPOUNDS AND METHODS FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION In 1974, Folkman proposed that for tumors to grow beyond a critical size and to spread to form metastases, they must recruit endothelial cells from the surrounding stroma to form their own endogenous microcirculation in 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.

Having emerged as a promising new strategy for the treatment of cancer, 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. Moreover, 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. Finally since the "indirect attack" and "direct attack" target different cell types, there is a great potential for a more effective combination therapy.

More than 300 angiogenesis inhibitors have been discovered, of which about 31 agents are currently being tested in human trials in treatment of cancers (Thompson, et al., (1999) J Pathol 187, 503). 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. The biochemical activity of fumagillin analogs has been shown to correlate to their inhibitory effect on the proliferation of human umbillical vein endothelial cells (HUVEC). Although the mechanism of the selective action of fumagillin and related compounds on MetAP2-mediated endothelial cell cytostatic effect has not yet been established, possible roles of MetAP2 in cell proliferation have been suggested.

First, hMetAP-2-catalyzed cleavage of the initiator methionine of proteins could be essential for releasing many proteins that, after myristoylation, function as important signalling 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 α 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). This is proposed to be an indirect outcome of inhibition of MetAP2-catalyzed release of the glycine-terminal myristoylation substrate. Alternatively, 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. They catalyze the removal of the initiator methionine from newly translated polypeptides using divalent metal ions as cofactors. Two distantly related 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. Despite the difference in the gene structure, all MetAP enzymes appear to share a highly conserved catalytic scaffold termed "pita- bread" fold (Bazan, et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2473), which contains six strictly conserved residues implicated in the coordination of the metal cof actors.

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-2α) 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. 268, 10796; Li, X. & Chang, Y.-H. (1996) Biochem. & Biophys. Res. Comm. 227, 152). The N-terminal extension in these enzymes is highly charged and consists of two basic polylysine blocks and one aspartic acid block, which has been speculated to be involved in the binding of eIF-2α (Gupta, et al. (1993) in Translational Regulation of Gene Expression 2 (Ilan, J., Ed.), pp405-431, Plenum Press, New York).

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). Fumagillin and its analogs inactivate the enzymatic activity of hMetAP2 with a high specificity, which is underscored by the lack of effect of these compounds on the closely related type 1 methionine aminopeptidase (MetAPl) both in vitro and in vivo in yeast (Griffith, et al., (1997) Chem. Biol. 4, 461; Sin, et al. (1997) Proc.NatlAcad.Sci. U.S.A. 94, 6099). The extremely high potency (IC50 < 1 nM) of these inhibitors appears to be due to the irreversible modification of the active site residue, His231, of hMetAP2 (Liu, et al. (1998) Science 282, 1324). Disturbance of MetAP2 activity in vivo impairs the normal growth of yeast (Griffith, et al., (1997) Chem. Biol. 4, 461; Sin, et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6099; In-house data) as well as Drosophila (Cutforth & Gaul (1999) Mech. Dev. 82, 23). Most significantly, there appears to be a clear correlation between the inhibition effect of fumagillin related compounds against the enzymatic activity of hMetAP2 in vitro and the suppression effect of these compounds against tumor-induced angiogenesis in vivo (Griffith, et al., (1997) Chem. Biol. 4, 461).

Cancer is the second leading cause of death in the U.S., exceeded only by heart disease. Despite recent successes in therapy against some forms of neoplastic disease, other forms continue to be refractory to treatment. Thus, cancer remains a leading cause of death and morbidity in the United States and elsewhere (Bailar and Gornik (1997) N Engl J Med 336, 1569). Inhibition of hMetAP2 provides a promising mechanism for the development of novel anti- angiogenic agents in the treatment of cancers.

SUMMARY OF THE INVENTION

In one aspect, 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:

Formula (I) wherein, Rl is H, Ci^alkyl, C_2-6alkenyl, C_2-6alkynyl, C_3-7cycloalkyl-Co-₆-alkyl, Ar-Co- ₆alkyl, or Het-Co_-6alkyl, wherein when Rl is C_3- cycloalkyl-Co-₆alkyl, the C_3-7cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring; R2 is Ar, Het, or C_5-7cycloalkyl fused to an Ar or Het group; and R3 is H, C_1-6alkyl, C₂-₆alkenyl, C_2-6alkynyl, Ar-Co-₆alkyl, Het-Co-₆alkyl, or C_3- ₇cycloalkyl-C₀-₆alkyl .

In a second aspect, 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 (IA), or a pharmaceutically acceptable salt thereof:

Formula (IA) wherein,

Rl is H, Cι- alkyl, C_2-6alkenyl, C₂_₆alkynyl, C_3-7cycloalkyl-Co-₆-alkyl, Ar-C_0- ₆alkyl, or Het-Co_-6 alkyl, wherein when Rl is C₃.₇cycloalkyl-Co-₆-alkyl, the C₃_ cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring; R2 is optionally substituted C^alkyl, wherein the optional substituent is

C(O)NRΕ.", and wherein R' and R" are independently, H, optionally substituted Cι_6^ahcyl or optionally substituted C galkoxy; or R2 is Cμ

₆alkyl-OR"', wherein R"' is C_j.galkyl or Ar; or R2 is Ar, Het, or C_5- ₇cycloalkyl fused to an Ar or Het group; and R3 is H, C_1- alkyl, C_2-6alkenyl, C_2-6alkynyl, Ar-Co-βalkyl, Het-Co-₆alkyl, or C₃. cycloalkyl-Co-₆alkyl, or R2 and R3, together with the nitrogen atom to which they are attached, form an optionally substituted 4-, 5-, 6- or 7- membered saturated ring system, wherein the optional substituent is

C(O)NR'R", and wherein R' and R" are independently, H, optionally substituted C_j.galkyl or optionally substituted C_j.galkoxy.

In another aspect, 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 formula (IA), or a pharmaceutically active salt thereof.

In yet another aspect, the present invention is to pharmaceutical compositions comprising a compound of formula (I) or formula (IA) and a pharmaceutically acceptable carrier therefor. In particular, the pharmaceutical compositions of the present invention are used for treating MetAP2-mediated disease states.

In a further aspect, the invention is to novel intermediates useful in the preparation of methionine aminopeptidase-2 inhibitors, particularly useful in the preparation of compounds of formula (I).

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that substituted bestatins of formula (I) are inhibitors of MetAP2. It has also now been discovered that selective inhibition of MetAP2 enzyme mechanisms by treatment with the inhibitors 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.

The term "Ci-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 Cι_6alkyl group may be optionally substituted independently by one or more of -OR⁴, -R⁴, or -NR⁴R5. Cøalkyl means that no alkyl group is present in the moiety. Thus, Ar-Cøalkyl is equivalent to Ar.

The term "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.

The term "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. C₂-6 lkynyl includes acetylene, 1- propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.

The term "C₃_₇cycloalkyl" as used herein at all occurrences means a cyclic radical having 3 to 7 carbons, unless otherwise limited, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.

"Ar" or "aryl" means phenyl and naphthyl, optionally substituted by one or more of Ph-Co_6alkyl, Het-Cθ-6 alkyl, C galkyl, C^.galkoxy, C\_ gmercaptyl, Ph-C()-6alkoxy, Het-C()-6alkoxy, OH, NR R⁵, Het-S-CQ-6alkyl, (CH₂)ι_₆OH, (CH₂)i-₆NR⁴R⁵, O(CH₂)i-₆NR⁴R⁵, (CH₂)₀-6CO₂R⁶, O(CH₂)!_6CO₂ R6, CF₃, OCF₃ or halogen; in addition the optional substituents Ph and Het may be optionally substituted with one or more of C\_ ₆alkyl, C galkoxy, OH, (CH )i-6NR⁴R⁵, O(CH₂)!_₆NR R⁵, CO₂R⁶, CF3, or halogen; two C^.galkyl or C galkoxy groups may be combined to form a 5-7 membered ring, saturated or unsaturated, fused onto the Ar ring. As used herein "Het" or "heterocyclic" represents a stable 5- to 7- membered monocyclic, a stable 7- to 10-membered bicyclic, or a stable 11- to 18-membered tricyclic heterocyclic ring which is either saturated or unsaturated, and which consists 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 Ph-CQ-βalkyl, Het-Cø-6 alkyl, C^alkyl, Ci _galkoxy, Cι_6mercaptyl, Ph-C₀-6alkoxy, Het-C₀-6alkoxy, OH, NR⁴R⁵, Het-S-C₀. ₆alkyl, (CH₂)i-6OH, (CH₂) !_₆NR⁴R5, O(CH₂) !_₆NR⁴R5, _(CH₂)₀_ 6^cO₂R⁶, O(CH₂) i-6CO R⁶, CF3, OCF3 or halogen; in addition the optional substituents Ph and Het may be optionally substituted with one or more of C}_ ₆alkyl, C^alkoxy, OH, (CH₂) !_₆NR⁴R5, O(CH₂)!_₆NR⁴R⁵, CO₂R⁶, CF3, or halogen; two Ci .galkyl or C1.galkoxy groups may be combined to form a 5-7 membered ring, saturated or unsaturated, fused onto the Heterocyclic ring. Examples of such 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, tetrahydropyranyl, thienyl, benzoxazolyl, benzofuranyl, benzothiophenyl, thiamoφholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazoly, as well as triazolyl, thiadiazolyl, oxadiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyridazinyl, pyrimidinyl, triazinyl and tetrazinyl which are available by routine chemical synthesis and are stable. The term heteroatom as applied herein refers to oxygen, nitrogen and sulfur.

As used herein, substituents R⁴, R^, and R^ are independently defined as H, Ci .galkyl, C3_6alkenyl, C3_galkynyl, Ar-Co-6^alkyl, Het-Co_6alkyl, or C3_7cycloalkyl-C()-6alkyl.

The term "halogen" as used herein at all occurrences means F, Cl, Br, and I.

Here and throughout this application the term CQ denotes the absence of the substituent group immediately following; for instance, in the moiety ArC()-6alkyl, when C is 0, the substituent is Ar, e.g., phenyl. Conversely, when the moiety ArCo-βalkyl is identified as a specific aromatic group, e.g., phenyl, it is understood that C is 0.

Suitably, Rl is H, Cι_-6alkyl, C₂_₆alkenyl, C₂-₆alkynyl, C₃_₇cycloalkyl- Co-₆-alkyl, Ar-C_0-6alkyl, or Het-C₀-₆ alkyl, wherein when Rl is C_3- cycloalkyl- Co-6-alkyl, the C_3-7cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring. It will be understood that when Rl is C₃. cycloalkyl-Co- ₆-alkyl, and the C₃_₇cycloalkyl group is fused to, or substituted by, an Ar or Het ring, the point of fusion or substitution can be at one or more positions on the C₃.₇cycloalkyl ring. Preferably Rl is Cι_₆alkyl or C₃.₇cycloalkyl-C₀-6- alkyl, more preferably Rl is C₃.₇cycloalkyl-C₀.₆alkyl.

Suitably, R2 is Ar, Het, or C₅.₇cycloalkyl fused to an Ar or Het group. Preferably, R2 is Ar. Suitably, R3 is H, Cι_₆alkyl, C₂-₆alkenyl, C_2-6alkynyl, Ar-Co-₆alkyl,

Het-Co-₆alkyl, or C₃_₇cycloalkyl-Co-₆alkyl.

Suitably, when Rl is C₃_₇cycloalkyl-Co-₆alkyl, and when R2 is Ar, R3 is preferably H.

It will be understood that when a moiety is "optionally substituted" the moiety may have one or more optional substituents, each optional substituent being independently selected.

Suitably, 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.

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.

Among the preferred compounds of the invention are the following compounds: (2S ,3R)-(3-Amino-2-hydroxyheptanoyl)-L- Alanine-2-Methoxy ethylamide hydrochloride;

(2R,3S)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2-Methoxy ethylamide hydrochloride;

(2R,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2-Methoxy ethylamide hydrochloride;

(2S,3S)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2-Methoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Proline-2-Methoxy ethylamide hydrochloride; (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-N-Methyl-Alanine-2-Methoxy ethylamide hydrochloride; (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-2-Methoxy ethylamide hydrochloride;

(2S ,3R)-(3-Amino-2-hydroxyheptanoyl)-2-Phenoxy ethylamide hydrochloride; (2S ,3R)-(3-Amino-2-hydroxyheptanoyl)- 1 -Naphthylene-amide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-Naphthylenemethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-2-Methoxy Ethylamide hydrochloride; (2S ,3R)-(3- Amino-4-cyclohexyl-2-hydroxy-butanoyl)-2-Methoxy Ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-Naphthylene-amide hydrochloride;

(2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxybutanoyl)-Naphthylene-amide hydrochloride; and

(2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxypentanoyl)-Naphthylene-amide hydrochloride.

Methods of Preparation Compounds of the formula (I) were prepared by the methods analogous to those described in Scheme 1. Compounds analogous to Scheme 1 were either purchased or prepared according to known experimental procedures in the literature.

Scheme 1. Synthesis of Bestatin Amides

5 6 Reaction Conditions: a.) 1. f-BuOCI, .-BuOCONH₂, NaOH, H₂0,π-PrOH 2). Methyl hept-2-enoate, (DHQ)₂Phal or (DHQD)₂Phal, K₂Os0₂(OH)₄ b.) iOH, H₂Q, THF c.) R₂NH₂, DCC, HOBt, THF d.) HCI (cat.), EtOAc

Shaφless amino-hydroxylation of enoate 1 -Scheme 1 afforded either stereochemistry 2 or 3 -Scheme 1 depending on whether the dihydroquinidine or dihydroquinine ligand was employed in the reaction. LiOH hydrolysis of methyl ester 2 provided the corresponding carboxylic acid 4-Schemel, and standard peptide coupling (see, Keding, Stacy J.; Dales, Natalie A.; Lim, Sejin; Beaulieu, Danielle; Rich, Daniel H. Synth. Commun. 1998, 28(23), 4463-4470) using DCC/HOBt and an amine (such as alanine, N- methylalanine, proline, 2-methoxyethylamine, 2-phenoxyethylamine, naphthylamine, and naphthylmethylamine) gave amide 5-Schemel. The N- Boc group was subsequently deprotected to provide the amine hydrochloride 6-Scheme 1.

The n-butyl bestatines which possess all possible absolute stereochemistries (2S,3R, 2S,3S, 2R,3S, and 2R,3R) were synthesized utilizing the procedure given in Scheme 2. Swern oxidation of either 7 or 9- Scheme 2 afforded the corresponding keto-ester, and NaBH reduction provided a mixture of diastereomers 7/8 or 9/10-Scheme 2. Utilizing the procedure illustrated in Scheme 1, the methyl esters 7/8, and 9/10-Scheme 2 are converted to the corresponding amide 6-Scheme 1.

Scheme 2.

Reaction Conditions a. 1 ) oxalyl chloride, DMSO, Et₃N, CH₂CI₂ 2) NaBH₄, 10% AcOH:DMF

Novel intermediates useful in making the compounds of this invention are as follows: Structure Chemical Name

(2S,3R)-(3-[(f-butyloxycarbonyl)amino]- 2-hydroxyheptanoyl)-naphthylene-amide

(2S,3R)-(3-[(.-butyloxycarbonyl)amino]- 2-hydroxyheptanoyl)-naphthylenemethyl-amide

(2S,3R)-(3-[(f-butyloxycarbonyl)amino]-

4-cyclohexyl-2-hydroxybutanoyl)-naphthylene- amide

(2S,3R)-(3-[(f-butyloxycarbonyl)amino]-

4-phenyl-2-hydroxybutanoyl)-naphthylene- amide

(2S,3R)-(3-[(f-butyloxycarbonyl)amino]-

5-cyclohexyl-2-hydroxypentanoyl)-naphthylene-

amide

Formulation of Pharmaceutical Compositions

The pharmaceutically effective compounds of this invention (and the pharmaceutically acceptable salts thereof) 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. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are syrup, peanut oil, olive oil, water and the like. Similarly, 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.

A wide variety of pharmaceutical forms can be employed. Thus, if a solid carrier is used, 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. When a liquid carrier is used, 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.

By topical administration is meant 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. By systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration. While it is possible for an active ingredient to 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. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of 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 incoφorate any 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. By "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. In one aspect, 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.

By the term "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. It will be recognized by one of skill in the art that 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.

The term 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.

It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of this invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular mammal being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of the compound given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

EXAMPLES

The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention. In the Examples, proton NMR spectra were performed upon a Bruker 400 MHz NMR specrometer, unless otherwise indicated.

Example 1 Preparation of (2S.3R)-(3-Amino-2-hvdroxyheptanoyl)-L-Alanine-2- Methoxyethylamide Hydrochloride a) Methyl (2S.3R)-3-r(t-butyloxycarbonyl)aminol-2- hydroxyheptanoate

Following the procedure reported by Rich (Keding, S. J.; Dales, N. A.; Lim, S.; Beaulieu, D.; Rich, D. H. Synth. Commun. 1998, 28(23), 4463- 4470.), to a stirring solution of t-butyl carbamate (5.6 g, 48.4 mmol) in n- PrOH (100 ml) was added solid NaOH (1.9 g, 48.4 mmol), followed by t-butyl hypochlorite (5.4 ml, 48.4 mmol), and the mixture was stirred at 0 °C for 25 min. The homogeneous solution was warmed to room temperature ("RT") and stirred for an additional 10 min. The solution was cooled to 0 °C, and to this mixture the following reagents were added sequentially: (DHQD)₂PHAL (0.73 g, 0.93 mmol) in n-PrOH (50 ml), methyl-2-heptenoate (2.0 g, 15.6 mmol) in n-PrOH (50 ml), and K₂(OsO₂(OH)₄ (0.28 g. 0.78 mmol). The reaction was stirred for 5 h at 0 °C, poured into 250 ml of H₂O, and extracted with EtOAc. The EtOAc extracts were washed with 1 N HCl, IN NaHCO₃, and saturated aqueous NaCl. The organic layer was dried over Na₂SO₄, filtered, concentrated, and subjected to column chromatography (silica gel, EtOAc/hexane) to provide the title compound as a white solid (2.2 g, 55%). 1H-NMR (400MHz, d4-MeOH) δ 0.88 (m, 3H), 1.32-1.37 (m, 4H), 1.40 (s, 9H), 1.51-1.63 (m, 2H), 3.08 (broad singlet, IH), 3.78 (s, 3H), 3.99 (m, IH), 4.15 (broad singlet, IH), and 4.65 (m, IH). b) (2S,3R)-3-r(t-Butyloxycarbonyl)aminol-2-hydroxyheptanoic acid To a solution of the compound of Example 1(a) (77 mg, 0.28 mmol) in

30% H₂O/THF (5.5 ml) was added LiOH (21 mg, 0.49 mmol) at 0 °C. The reaction mixture was stirred for 2 h, saturated aqueous NaHCO₃ (5 ml) was added, and the mixture was concentrated to remove the THF. To the solution was added saturated aqueous NaHCO₃ (5 ml), and the resulting aqueous solution was washed with CH2CI₂ (10ml). The aqueous layer was separated, acidified to pH 2 with 3 N HCl, and then extracted with EtOAc. The EtOAc extracts were dried over Na₂SO₄, filtered, and concentrated to afford the title compound as a white solid (72 mg, 100%). 1H-NMR (400MHz, d4-MeOH) δ 0.85 (m, 3H), 1.18-1.31 (m, 5H), 1.36 (s, 9H), 1.41-1.52 (m, IH), 3.72 (m, IH), 3.93 (m, IH), and 6.20 (m, IH). c) N-Boc-L-alanyl-2-methoxyethylamide

To a stirring solution of /V-Boc-L-alanine (1.0 g, 5.29 mmol) in CHC1₂ (20 ml) was added l,l'-carbonyldiimidazole (1.1 g, 6.87 mmol) at RT. The solution was stirred for 30 min, cooled to 0 °C, and 2-methoxyethylamine (0.6 ml, 6.87 mmol) was added. The reaction mixture was warmed to RT, and stirred overnight. After stirring for 10 h, the solution was concentrated and the crude residue was subjected to column chromatography (silica gel, EtOAc/hexane) to give the title compound as a white solid (1.3 g, 100%). 1H- NMR (400MHz, CDC1₃) δ 1.31 (d, 3H, J=7.1 Hz), 1.40 (s, 9H), 3.30 (s, 3H), 3.41 (m, 4H), 4.15 (m, IH), 5.21 (m, IH), and 6.63 (m, IH). e) (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-alanine-2- methoxyethylamide hydrochloride

To a stirring solution of the compound of Example 1(c) (0.14 g , 0.57 mmol) in EtOAc (4 ml) was added cone. HCl (0.1 ml). The solution was stirred for 3 h at RT. The solution was then concentrated, and taken up in CHCI₂ (2 ml). Diisopropylethylamine (0.10 ml, 0.57 mmol) was added, and the solution was stirred for 10 min at RT. To this solution was added the compound of Example 1(b) (75 mg, 0.28 mmol), followed by hydroxybenzotriazole hydrate (77 mg, 0.57 mmol), and the reaction mixture was stirred at 0 °C for 30 min. Dicyclohexylcarbodiimide (0.12 g, 0.57 mmol) was added, the reaction mixture was warmed to RT, and stirred overnight. The resulting heterogeneous mixture was filtered, the filtrate was concentrated, and the crude residue was subjected to column chromatography (silica gel, EtOAc/hexane) to afford the corresponding N-Boc-carbamate as a white solid (97 mg, 87%). The N-Boc-carbamate was taken up in ethyl acetate (5ml) and cone HCl (0.1 ml) and the resulting mixture was stirred for 3 h. The reaction mixture was concentrated to provide the title compound as a white solid (69 mg, 83%). 1H-ΝMR (400MHz, d4-MeOH) δ 0.86 (t, 3H, J=6.9 Hz), 1.29-1.32 (m, 7H), 1.55 (m, IH), 1.70 (m, IH), 3.24 (s, 3H), 3.26-3.28 (m, 2H), 3.34-3.39 (m, 3H), 4.12 (d, IH, J=3.0 Hz), and 4.29 (quartet, IH, J=7.0 Hz). Example 2 Preparation of (2R,3S)-(3-Amino-2-hvdroxyheptanoyl)-L-Alanine-2- Methoxyethylamide Hydrochloride

Following the procedure of Example 1(a)- 1(d), except (DHQ^PHAL was substituted for (DHQD^PHAL in step (a), the title compound was prepared as a white solid (4 steps, 15%). 1H-NMR (400MHz, d4-MeOH) δ 0.85 (t, 3H, J=6.9 Hz), 1.26-1.32 (m, 8H), 1.55-1.57 (m, 2H), 3.28 (s, 3H), 3.30-3.33 (m, IH), 3.37-3.41 (m, 3H), 4.22 (d, IH, J=3.8 Hz), and 4.33-4.35 (m, IH).

Example 3 Preparation of (2R,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2- Methoxyethylamide Hydrochloride a) Methyl (2R.3R)-3-r(t-butyloxycarbonyl)aminol-2- hydroxyheptanoate

To a solution of oxalyl chloride (1.90 ml, 21.8 mmol) in CH₂C1₂ (50 ml) was added DMSO (3.09 ml, 43.5 mmol) at -78 °C, and the reaction mixture was stirred for 20 min. To the reaction mixture was added the compound of Example 1(a) (1.5 g, 5.45 mmol) in CH₂C1₂ (50 ml). The solution was stirred for 30 min at -78 °C, and triethylamine (7.59 ml, 54.5 mmol) was added. Stirring was continued for 30 min at -78 °C, the reaction mixture was warmed to 0 °C, and then stirred for an additional 1 h. The reaction mixture was poured into 150 ml of H₂O and extracted with CH2CI2. The organic extracts were washed with saturated aqueous NaCl, dried over Na₂SO₄, filtered and concentrated. The crude product was subjected to column chromatography (silica gel, EtOAc/hexane) to provide the keto-ester as a yellow oil (0.58 g, 38%). 1H-NMR (400MHz, d4-MeOH) δ 0.88 (m, 3H), 1.28-1.42 (m, 5H), 1.41 (s, 9H), 1.48-1.6 (m, IH), 1.85 (m, IH), 3.86 (s, 3H), 4.87 (m, IH), and 5.06 (m, IH). To the above keto-ester (0.58 g, 2.1 mmol) in 10% AcOH/DMF (6 ml) was added NaCNBH₃ (0.2 g, 3.18 mmol), and the reaction mixture was stirred at RT for 12 h. The reaction mixture was poured into 60 ml of H₂O and extracted with EtOAc. The EtOAc extracts were dried over Na₂SO₄, filtered, and concentrated to provide a 1: 1 mixture of amino alcohol diastereomers. The crude mixture was subjected to column chromatography (silica gel, EtOAc/hexane) to provide the title compound as a single stereoisomer as a white solid (0.25 g, 44%). Η-NMR (400MHz, CDC1₃) δ 0.88 (m, 3H), 1.23- 1.43 (m, 6H), 1.46 (s, 9H), 3.18 (m, IH), 3.80 (s, 3H), 3.91-4.00 (m, IH), 4.33 (m, IH), and 4.74 (m, IH). b) (2R3R)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2- Methoxyethylamide Hydrochloride Following the procedure of Example 1(b)- 1(d) except methyl (2R,3R)-

3-[(t-butyloxycarbonyl)amino]-2-hydroxyheptanoate was utilized in step (b) instead of methyl (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2-hydroxy- heptanoate, the title compound was prepared as a white solid (3 steps, 15%). 1H-NMR (400MHz, d4-MeOH) δ 0.86 (m, 3H), 1.26-1.31 (m, 7H), 1.55 (m, 2H), 3.26 (s, 3H), 3.29-3.36 (m, 2H), 3.38-3.42 (m, 3H), 4.22 (d, IH, J=3.6 Hz), and 4.33 (quartet, IH, J=7.1 Hz).

Example 4 Preparation of (2S.3S)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2- Methoxyethylamide Hydrochloride a) Methyl (2S.3S)-3-r(t-butyloxycarbonyl)aminol-2- hvdroxyheptanoate

Following the procedure of Example 3(a) except methyl (2R,3S)-3-[(t- butyloxycarbonyl)amino]-2-hydroxyheptanoate was used in step (a) instead of methyl (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2-hydroxyheptanoate, the title compound was prepared as a white solid (2 steps, 20%). 1H-NMR (400MHz, CDC1₃) δ θ.88 (m, 3H), 1.23-1.43 (m, 6H), 1.46 (s, 9H), 3.18 (m, IH), 3.80 (s, 3H), 3.91-4.00 (m, IH), 4.33 (m, IH), and 4.74 (m, IH). b) (2S,3S)-(3-Amino-2-hydroxyheptanoyl)-L-alanine-2- methoxyethylamide hydrochloride

Following the procedure of Example 1(b)- 1(d) except methyl (2S,3S)- 3-[(/-butyloxycarbonyl)amino]-2-hydroxyheptanoate was used in step (b) instead of methyl (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2- hydroxyheptanoate, the title compound was prepared as a white solid (3 steps, 15%). Η-NMR (400MHz, d4-MeOH) δ 0.90 (t, 3H, J=7.0 Hz), 1.33-1.35 (m, 7H), 1.59 (m, IH), 3.27 (s, 3H), 3.28-3.31 (m, 2H), 3.33-3.46 (m, 3H), 4.25 (d, IH, J=3.6 Hz), and 4.39 (quartet, IH, J=7.0 Hz). Example 5 Preparation of (2S,3R)-(3-Amino-2-hvdroxyheptanoyl)-L-Proline-2- Methoxyethylamide Hydrochloride

Following the procedure of Example 1(a)- 1(d) except N-Boc-L-proline was used in step (c) instead of N-Boc-L-alanine, and N-Boc-L-proline-2- methoxyethylamide was utilized in step (d) instead of N-Boc-L-alanine-2- methoxyethylamide, the title compound was prepared as a white solid (4 steps, 42%). 1H-ΝMR (400MHz, d4-MeOH) δ 0.98 (m, 3H), 1.38-1.49 (m, 5H), 1.61-1.72 (m, IH), 1.72-2.10 (m, 5H), 2.18-2.30 (m, IH), 3.30 (m, IH), 3.37 (m, 2H), 3.42-3.48 (m, 3H), 3.61 (m, IH), 3.72 (m, 2H), 4.45-4.49 (m, IH), and 4.51 (s, IH).

Example 6 Preparation of (2S.3R)-(3-Amino-2-hvdroxyheptanoyl)-L-N-Methyl-Alanine- 2-Methoxyethylamide Hydrochloride

Following the procedure of Example 1(a)- 1(d) except N,N-Boc- methyl-L-alanine was utilized in step (c) instead of N-Boc-L-alanine, and N,N- Boc-methyl-L-alanine-2-methoxyethylamide was used instead of N-Boc-L- alanine-2-methoxyethylamide in step (d), the title compound was prepared as a white solid (5 steps, 18%). 1H-ΝMR (400MHz, d4-MeOH) δ 0.96 (m, 3H), 1.38-1.50 (m, 7H), 1.57-1.61 (m, 2H), 1.75-1.89 (m, 2H), 3.02 (s, 3H), 3.11 (m, 2H), 3.38 (s, 3H), 3.55-3.62 (m, 2H), 3.62-3.80 (m, 2H), 3.93 (m, IH), 4.50 (m, IH), and 5.25 (broad singlet, IH).

Example 7

Preparation of (2S,3R)-(3-Amino-2-hvdroxyheptanoyl)-2-Methoxyethylamide Hydrochloride

To a stirring solution of (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2- hydroxyheptanoic acid (43 mg, 0.16 mmol) in THF (1 ml) was added 2- methoxyethylamine (29 ul, 0.33 mmol), followed by hydroxybenzotriazole hydrate (45 mg, 0.33 mmol), and the reaction mixture was stirred at 0 °C for 30 min. Dicyclohexylcarbodiimide (68 mg, 0.33 mmol) in CHCI₂ (1 ml) was added, the reaction mixture was warmed to RT, and stirred overnight. The resulting heterogeneous mixture was filtered, the filtrate was concentrated, and the crude residue was subjected to column chromatography (silica gel,

EtOAc/hexane) to afford the corresponding N-Boc-carbamate as a white solid (47 mg, 88%). The N-Boc-carbamate was taken up in ethyl acetate (5ml) and cone HCl (0.1 ml) and the resulting mixture was stirred for 3 h. The reaction mixture was concentrated to provide the title compound as a white solid (35 mg, 95%). Η-NMR (400MHz, d4-MeOH) δ 0.95 (t, 3H, J=7.0 Hz), 1.38-1.40 (m, 4H), 1.61 (m, IH), 1.74 (m, IH), 3.34 (s, 3H), 3.40-3.50 (m, 5H), and 4.16 (d, IH, J=2.9 Hz).

Example 8 Preparation of (2S,3R)-(3-Amino-2-hvdroxyheptanoyl)-2-Phenoxyethylamide Hydrochloride

Following the procedure of Example 7 except 2-phenoxyethylamine was used instead of 2-methoxyethylamine, the title compound was prepared as a white solid (2 steps, 40%). 1H-NMR (400MHz, d4-MeOH) δ 0.89 (t, 3H, J=6.9 Hz), 1.14-1.17 (m, IH), 1.25-1.34 (m, 2H), 1.58-1.71 (m, IH), 1.72-1.74 (m, IH), 1.79-1.82 (m, IH), 3.43 (m, IH), 3.55-3.60 (m, IH), 3.66-3.70 (m, IH), 4.05 (t, 2H, J=5.3 Hz), 4.17 (d, IH, J=3.4 Hz), 6.88-6.90 (m, 3H), and 7.22 (m, 2H).

Example 9 Preparation of (2S.3R)-(3-Amino-2-hydroxyheptanoyl)-l -Naphthylene-amide Hydrochloride Following the procedure of Example 7 except 1 -naphthylamine was used instead of 2-methoxyethylamine, the title compound was prepared as a white solid (2 steps, 25%). Η-NMR (400MHz, d4-MeOH) δ 0.98 (t, 3H, J=7.1 Hz), 1.48-1.50 (m, 4H), 1.70-1.76 (m, IH), 1.89-1.94 (m, IH), 3.65 (m, IH), 4.51 (d, IH, J=3.2 Hz), 7.48-7.57 (m, 3H), 7.79 (d, IH, J=13.6 Hz), 7.81 (d, IH, J=13.6 Hz), 7.91 (d, IH, J=7.2 Hz), and 7.97 (d, IH, J=7.2 Hz).

Example 10 Preparation of (2S,3R)-(3-Amino-2-hydroxyheptanoyl)- Naphthylenemethylamide Hydrochloride Following the procedure of Example 7 except 1-naphthylene- methylamine was used instead of 2-methoxyethylamine, the title compound was prepared as a yellow solid (2 steps, 36%). Η-NMR (400MHz, d4- MeOH) δ 0.91 (t, 3H, J=6.9 Hz), 1.29-1.41 (m, 4H), 1.58 (m, IH), 1.72 (m, IH), 3.45 (m, IH), 4.21 (d, IH, J=3.6 Hz), 4.78 (d, IH, J=14.9 Hz), 5.05 (d, IH, J=14.9 Hz), 7.42-7.46 (m, IH), 7.50-7.57 (m, 3H), 7.83 (d, IH, J=8.0 Hz), 7.90 (d, IH, J=7.3 Hz), and 8.11 (d, IH, J=8.0 Hz). Example 11 Preparation of (2S.3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-2- Methoxyethylamide Hydrochloride

Following the procedure of Example 7 except (2S,3R)-3-[(t- butyloxycarbonyl)amino]-2-hydroxy-4-phenyl-butanoic acid (commercially available from Sigma Chemical Co.) was used instead of (2S,3R)-3-[(t- butyloxycarbonyl)amino]-2-hydroxyheptanoic acid, the title compound was prepared as a white solid (2 steps, 95%). 1H-NMR (400MHz, d4-MeOH) δ 2.91 (m, 2H), 3.20 (m, IH), 3.22 (s, 3H), 3.31 (m, IH), 3.58 (m, IH), 3.91 (broad singlet, IH), 6.80 (m, IH), 7.26 (m, IH), 7.32-7.34 (m, 4H), and 7.99- 8.01 (m, 2H).

Example 12 Preparation of (2S.3R)-(3-Amino-4-cyclohexyl-2-hydroxy-butanoyl)-2- Methoxyethylamide Hydrochloride

Following the procedure of Example 7 except (2S,3R)-3-[(t- butyloxycarbonyl)amino]-2-hydroxy-4-cyclohexyl-butanoic acid (Boger, J.; Payne, L. S.; Perlow, D. S; Lohr, N. S.; Poe, M.; Blaine, E. H.; Ulm, E. H.; Schorn, T. W.; LaMont, B. I.; Tsau-Yen, L.; Kawai, M.; Rich, D. H.; Veber, D. F. 7. Med. Chem. 1985, 28(12), 1779-1790) was used instead of (2S,3R)-3- [(t-butyloxycarbonyl)amino]-2-hydroxyheptanoic acid, the title compound was prepared as a white solid (2 steps, 52%). Η-NMR (400MHz, d4-MeOH) δ 0.93-0.99 (m, 2H), 1.21-1.32 (m, 3H), 1.44-1.50 (m, 2H), 1.63-1.79 (m, 6H), 3.35 (s, 3H), 3.43-3.45 (m, 2H), 3.48-3.51 (m, 2H), 3.61 (m, IH), and 4.14 (d, IH, J=2.4 Hz).

Example 13 Preparation of (2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)- Naphthylene-amide Hydrochloride Following the procedure of Example 7 except both (2S,3R)-3-[(t- butyloxycarbonyl)amino]-2-hydroxy-4-phenyl-butanoic acid² and 1- naphthylamine were used instead of (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2- hydroxyheptanoic acid and 2-methoxyethylamine, the title compound was prepared as a white solid (2 steps, 43%). 1H-NMR (400MHz, d4-MeOH) δ 3.03 (d, 2H, J=7.3 Hz), 3.79 (m, IH), 4.27 (m, IH), 7.31 (m, 2H), 7.35-7.39 (m, 4H), 7.48-7.57 (m, 3H), 7.75 (d, IH, J=7.2 Hz), 7;.79 (d, IH, J=7.2 Hz), 7.95 (m, IH), 8.00 (m, IH), 8.15 (s, 2H), and 10.07 (s, IH). Example 14 Preparation of (2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxybutanoyl)- Naphthylene-amide Hydrochloride

Following the procedure of Example 7 except both (2S,3R)-3-[(t- butyloxycarbonyl)amino]-2-hydroxy-4-cyclohexyl-butanoic acid^{^} and 1- naphthylamine were used instead of (2S,3R)-3-[(/-butyloxycarbonyl)amino]-2- hydroxyheptanoic acid and 2-methoxyethylamine, the title compound was prepared as a white solid (2 steps, 37%). 1H-NMR (400MHz, d4-MeOH) δ 0.86-1.05 (m, 2H), 1.14-1.30 (m, 3H), 1.51-1.74 (m, 8H), 3.54 (m, IH), 4.40 (broad singlet, IH), 6.92 (broad singlet, IH), 7.51-7.57 (m, 3H), 7.73 (d, IH, J=7.3 Hz), 7.81 (d, IH, J=7.3 Hz), 7.96-8.01 (m, 4H), and 10.15 (s, IH).

Example 15 Preparation of (2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxypentanoyl)- Naphthylene-amide Hydrochloride a) Methyl 5-phenyl-2-pentenoate

To a refluxing solution of methyl (triphenylphosphoranylidene) acetate (13.10 g, 39.1 mmol) in benzene (30 ml) was added hydrocinnamaldehyde (4.91 ml, 37.2 mmol), and the reaction mixture was stirred at 80 °C overnight. The mixture was cooled to RT, and concentrated. The crude product was triturated with 10% EtOAc/hexane (125 ml), the triphenylphosphine oxide was filtered off, and the filtrate was concentrated. Purification of the crude enoate by column chromatography (silica gel, EtOAc/hexane) afforded the title compound as a clear oil (5.03 g, 75%). Η-NMR (400MHz, CDC1₃) δ 2.51 (m, 2H), 2.77 (t, 2H, J=7.3 Hz), 3.73 (s, 3H), 5.86 (d, IH, J=15.7 Hz), 7.00 (doublet of triplet, IH, J=6.9 and 15.7 Hz), 7.17-7.22 (m, 3H), and 7.26- 7.31 (m, 2H). b) (2S.3R)-3-[(t-Butyloxycarbonyl)aminol-2-hydroxy-5-phenyl- pentanoic acid Following the procedure of Example 1(a)- 1(b) except methyl 5-phenyl-

2-pentenoate was utilized in step (a) instead of methyl-2-heptenoate, the title compound was prepared as a white solid (2 steps, 28%). Η-NMR (400 MHz, CDC1₃) δ 1.42 (s, 9H), 1.96 (m, 3H), 2.70 (m, 3H), 4.00 (m, IH), 4.20 (m, IH), 5.00 (m, IH), 7.18-7.20 (m, 3H), and 7.26-7.30 (m, 2H). c) (2S,3R)-3-r(t-Butyloxycarbonyl)aminol-2-hydroxy-5-cyclohexyl- pentanoic acid

To a parr bottle containing the compound of Example 1(b) (0.27 g, 0.9 mmol) in 10% AcOH MeOH (20 ml) was added platinum oxide (50 mg, 0.2 mmol), and the heterogeneous mixture was hydrogenated at 50 psi for 10 h. The bottle was evacuated, purged with argon, and the heterogeneous mixture was filtered through a celite plug. The filtrate was concentrated to provide the title compound as a white solid (0.25 g, 89%). Η-NMR (400 MHz, CDC1₃) δ 0.89-0.93 (m, 2H), 1.16-1.23 (m, 6H), 1.41 (s, 9H), 1.62-1.69 (m, 7H), 3.90 (m, IH), 4.19 (m, IH), and 4.94 (m, IH). d) (2S,3R)-(3-Amino-4-cvclohexyl-2-hydroxypentanoyl)- naphthylene-amide hydrochloride

Following the procedure of Example 7 except both (2S,3R)-3-[(r- butyloxycarbonyl)amino]-2-hydroxy-5-cyclohexyl-pentanoic acid and 1- naphthylamine were used instead of (2S,3R)-3-[(t-butyloxycarbonyl)amino]-2- hydroxyheptanoic acid and 2-methoxyethylamine, the title compound was prepared as a white solid (2 steps, 73%). 1H-NMR (400MHz, d4-MeOH) δ 0.87-0.92 (m, 2H), 1.13-1.18 (m, 4H), 1.21-1.35 (m, 2H), 1.60-1.81 (m, 7H), 3.42 (m, IH), 4.44 (m, IH), 6.95 (broad singlet, IH), 7.51-7.57 (m, 3H), 7.74 (d, IH, J=7.2 Hz), 7.81 (d, IH, J=7.2 Hz), 7.95-8.04 (m, 4H), and 10.17 (s, IH).

Biological Data; Direct Spectrophotometric Assays of hMetAP2

The hMetAP2 activity can be measured by direct spectrophotometric assay methods using alternative substrates, L-methionine-p-nitroanilide (Met- pNA) and L-methionine-7-amido-4-methylcoumarin (Met-AMC). The formation of -nitroaniline (pNA) or 7-amido-4-methylcoumarin (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 30 °C. The fluorscence or spectrophotometric plate reader was calibrated using authentic pNA and AMC from Sigma, respectively. For a typical 96-well plate assay, the increase in the absorbance (at 405 nm for pNA) or the fluorescence emission (λ_ex = 360 nm, λ_em = 460 nm, for AMC) of a 50 uL assay solution in each well was used to calculate the initial velocity of hMetAP2. Each 50 uL assay solution, contained 50 mM Hepes Na+ (pH 7.5), 100 rnM 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. Coupled Spectrophotometric Assays 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). The formation of hydrogen peroxide (H2O2) was continuously monitored at 450nm (absorbance increase of o-Dianisidine (Sigma) upon oxidation, Δε = 15,300 M^cπr¹)² and 30 °C in a 96- or 384- well plate reader by a method adapted from Tsunasawa, S. et al.(1997) (eq. 3a). Alternatively, formation of H2O2 was followed by monitoring the fluorescence emission increase at 587nm (Δε = 54,000 M'^cmr^-, λ_ex = 563 nm, slit width for both excitation and emission was 1.25 mm) and 30 °C using Amplex Red (Molecular Probes, Inc) (Zhou, M. et al. (1997) Anal. Biochem. 253, 162) (eq. 3b). In a total volume of 50 uL, a typical assay contained 50 mM Hepes-Na+, pH 7.5, 100 mM NaCl, 10 uM C0CI2, 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.

-Met-Ala-Ser ^' — *^■ L-Methionine + H N-Ala-Ser (1)

Co⁺⁺

AAO

L-Methionine + H,0 + O, 2-oxo-acid + NH₃ + H₂0₂ (2)

(o-Dianisidine)

Kinetic Data Analysis Data were fitted to the appropriate rate equations using Grafit computer software. Initial velocity data conforming to Michaelis-Menton kinetics were fitted to eq. 4. Inhibition patterns conforming to apparent competitive and non-competitive inhibition were fitted to eq. 5 and eq. 6, respectively. v = VA/(K_a + A) (4) v = VA/[K_a(l + I/K_is) + A] (5) v = VA/[K_a( 1 + I/K_is) + A( 1 + I/Kϋ)] (6)

In eqs 4 - 6, v is the initial velocity, V is the maximum velocity, K_a is the apparent Michaelis constant, I is the inhibitor concentration, and A is the concentration of variable substrates. The nomenclature used in the rate equations for inhibition constants is that of Cleland (1963), in which Kj_s and Kji represent the apparent slope and intercept inhibition constants, respectively. Cell growth inhibition assays

The ability of MetAP2 inhibitors to inhibit cell growth was assessed by the standard XTT microtitre assay. 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. Flat-bottomed 96-well plates are seeded with appropriate numbers of cells (4-6 x lθ3 cells/well in a volume of 200 ul) from trypsinized exponentially growing cultures. In the case of HUVECs, the wells are coated with matrigel prior to establishing the cultures. To "blank" wells is added growth medium only. Cells are incubated overnight to permit attachment. Next day, medium from wells that contain cells is replaced with 180 ul of fresh medium. Appropriate dilutions of test compounds are added to the wells, final DMSO concentration in all wells being 0.2 %. Cells plus compound are incubated for an additional 72 hr at 37°C under the normal growth conditions of the cell line used. Cells are then assayed for viability using standard XTT/PMS (prepared immediately before use: 8 mg XTT (Sigma X-4251) per plate is dissolved in 100 ul DMSO. 3.9 ml H₂O 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 A₄₅₀ 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). ICso 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.

All publications, including, but not limited to, patents and patent applications cited in this specification, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration it is believed that one skilled in the art can, given the preceding description, utilize the present invention to its fullest extent. Therefore any examples are to be construed as merely illustrative and not a limitation on the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

Claims

What is claimed is:

1. A method of inhibiting MetAP2 in mammals, comprising administering to a mammal in need of such treatment, an effective amount of a compound of formula (IA) or a pharmaceutically acceptable salt thereof:

Formula (IA) wherein,

Rl is H, Cι- alkyl, C₂-₆ lkenyl, C₂-₆alkynyl, C₃_₇cycloalkyl-Co-₆-alkyl, Ar-Co- ₆alkyl, or Het-Co-₆ alkyl, wherein when Rl is C_3-7cycloalkyl-Co-₆-alkyl, the C_3-7cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring;

R2 is optionally substituted C_j.galkyl, wherein the optional substituent is

C(O)NR'R", and wherein R' and R" are independently, H, optionally substituted C^alkyl or optionally substituted Ci.galkoxy; or R2 is C_j. ₆alkyl-OR"\ wherein R'" is C_j.galkyl or Ar; or R2 is Ar, Het, or C₅_ ₇cycloalkyl fused to an Ar or Het group; and

R3 is H, C_1-6alkyl, C₂-₆alkenyl, C₂-₆alkynyl, Ar-Co_-6alkyl, Het-Co-₆alkyl, or C₃. ₇cycloalkyl-Co-₆alkyl, or R2 and R3, together with the nitrogen atom to which they are attached, form an optionally substituted 4-, 5-, 6- or 7- membered saturated ring system, wherein the optional substituent is C(O)NR'R", and wherein R' and R" are independently, H, optionally substituted Cμgalkyl or optionally substituted C_j.galkoxy.

2. The method of claim 1, wherein the compound of formula (IA) is selected from:

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2-Methoxy ethylamide hydrochloride;

(2S,3S)-(3-Amino-2-hydroxyheptanoyl)-L-Alanine-2-Methoxy ethylamide hydrochloride; (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Proline-2-Methoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-N-Methyl-Alanine-2-Methoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-2-Methoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-2-Phenoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)- 1 -Naphthylene-amide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-Naphthylenemethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-2-Methoxy Ethylamide hydrochloride;

(2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxy-butanoyl)-2-Methoxy Ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-Naphthylene-amide hydrochloride;

(2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxypentanoyl)-Naphthylene-amide hydrochloride, or a pharmaceutically acceptable salt thereof.

3. A method for treating a disease mediated by MetAP2 in mammals, comprising administering to a mammal in need of such treatment, an effective amount of a compound of formula (IA) or a pharmaceutically acceptable salt thereof:

Formula (IA) wherein,

Rl is H, Cj_₆alkyl, C₂-₆alkenyl, C₂-₆alkynyl, C₃- cycloalkyl-Co-₆-alkyl, Ar-C₀- ₆alkyl, or Het-C₀-₆ alkyl, wherein when Rl is C₃_₇cycloalkyl-C₀-₆-alkyl, the C_3-7cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring; R2 is optionally substituted C_j.galkyl, wherein the optional substituent is

C(O)NR^rR", and wherein R' and R" are independently, H, optionally substituted C_j.galkyl or optionally substituted C_j.galkoxy; or R2 is C_j_ ₆alkyl-OR"', wherein R"' is C_μ6alkyl or Ar; or R2 is Ar, Het, or C₅. ₇cycloalkyl fused to an Ar or Het group; and R3 is H, Cj_-6alkyl, C₂-₆alkenyl, C₂-₆alkynyl, Ar-Co-₆alkyl, Het-Co-₆alkyl, or C _ ₇cycloalkyl-Co-₆alkyl, or R2 and R3, together with the nitrogen atom to which they are attached, form an optionally substituted 4-, 5-, 6- or 7-membered saturated ring system, wherein the optional substituent is C(O)NR'R", and wherein R' and R" are independently, H, optionally substituted C^alkyl or optionally substituted C_j_galkoxy.

4. The method of claim 3, wherein the compound of formula (I A) is selected from:

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-L-Proline-2-Methoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-2-Methoxy ethylamide hydrochloride;

(2S ,3R)-(3- Amino-2-hydroxyheptanoyl)-2-Phenoxy ethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-l -Naphthylene-amide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxyheptanoyl)-Naphthylenemethylamide hydrochloride;

(2S,3R)-(3-Amino-4-cyclohexyl-2-hydroxy-butanoyl)-2-Methoxy Ethylamide hydrochloride; (2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-Naphthylene-amide hydrochloride;

5. A compound of formula (I), or a pharmaceutically acceptable salt thereof:

Formula (I) wherein,

Rl is H,

C_2- alkenyl, C₂.₆alkynyl, C_3-7cycloalkyl-Co-₆-alkyl, Ar-Co- ₆alkyl, or Het-Co-βalkyl, wherein when Rl is C₃.₇cycloalkyl-Co-₆alkyl, the C₃-₇cycloalkyl group may be optionally fused to or substituted by an Ar or Het ring;

R2 is Ar, Het, or C₅. cycloalkyl fused to an Ar or Het group; and

R3 is H, Cι_₆alkyl, C _-6alkenyl, C_2-6alkynyl, Ar-C₀-₆alkyl, Het-Co-₆alkyl, or C₃_

₇cy cloalky l-Co-₆alky 1.

6. A compound selected from: (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-l -Naphthylene-amide hydrochloride; (2S,3R)-(3-Amino-2-hydroxyheptanoyl)-Naphthylenemethylamide hydrochloride;

(2S,3R)-(3-Amino-2-hydroxy-4-phenyl-butanoyl)-Naphthylene-amide hydrochloride;

7. A compound selected from: (2S,3R)-(3-[(t-butyloxycarbonyl)amino]-2-hydroxyheptanoyl)-naphthylene- amide;

(2S,3R)-(3-[(t-butyloxycarbonyl)amino]-2-hydroxyheptanoyl)- naphthylenemethyl-amide;

(2S,3R)-(3-[(t-butyloxycarbonyl)amino]-4-cyclohexyl-2-hydroxybutanoyl)- naphthylene-amide;

(2S,3R)-(3-[(t-butyloxycarbonyl)amino]-4-phenyl-2-hydroxybutanoyl)- naphthylene-amide; and

(2S,3R)-(3-[(t-butyloxycarbonyl)amino]-5-cyclohexyl-2-hydroxypentanoyl)- naphthylene-amide.

8. A process for preparing a compound of formula (I), as claimed in claim 1, comprising: a) amino-hydroxylation of a compound of formula (II)

Formula (II)

b) hydrolyzing a compound of formula (III) to afford a compound of formula (IV)

Formula (IV) c) peptide-coupling the compound of formula (IV) to give a compound of formula (V)

5

Formula (V) d) optionally deprotecting the compound of formula (V) to give a compound of formula (I), wherein the groups Rl, R2 and R3 are as defined in claim 1.