MetAp2 inhibitors and their uses

Abstract

The present invention provides novel inhibitors of MetAp2 enzyme activity and uses thereof.

Description

[Technical field]

[0001] The present invention relates generally to novel MetAp2 inhibitors and uses thereof. [Background technology]

[0002] Antiangiogenic drugs have the ability to prevent, inhibit, and cause regression of newly formed blood vessels. Multiple forms of angiogenesis inhibitors exist, ranging from endogenous proteins and small molecule cytokine antagonists to antibodies and tyrosine kinase inhibitors [1-4]. Historically, the main target of antiangiogenic drugs in clinical development has been to block the vascular endothelial growth factor (VEGF) pathway. However, other angiogenic factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and inflammatory cytokines also play critical roles in neovascularization.

[0003] One of the most effective compounds that demonstrated angiostatic activity in preclinical trials is derived from fumagillin, a natural product secreted by fungi that has anti-infective properties. Synthetic analogs of fumagillin and its bioactive metabolite fumagillol have been found to be potent antiangiogenic compounds capable of preventing the growth of new capillaries. After screening over 100 compounds, the most potent antiangiogenic analog of fumagillin was found to be chloroacetylcarbamoyl fumagillol (TNP-470) [5]. TIFF2025508363000001.tif68170

[0004] The molecular target of TNP-470 is the enzyme methionine aminopeptidase 2 (MetAP2), responsible for the removal of methionine from newly synthesized proteins. MetAP2 is overexpressed in proliferating endothelial cells, and therefore inhibition of MetAP2 may lead to selective inhibition of angiogenesis [6-10].

[0005] The activity of TNP-470 has been demonstrated in mice, rats, rabbits, hamsters, dogs, monkeys, and humans. It has also been investigated in eight clinical trials involving over 300 patients [11-19]. In these studies, TNP-470 was administered as an intravenous formulation at different dosage levels and schedules. Overall, activity was observed in over 12 tumor types. Despite its encouraging efficacy, the fumagillol analog TNP-470 has a short half-life (minutes) in plasma [17, 19], which leads to poor pharmacokinetics. In addition, it has been found that at high doses it can cause adverse side effects, such as reversible neurotoxicity, in some patients

[11] .

[0006] background 1.Folkman, J., Angiogenesis and angiogenesis inhibition: an overview.Exs, 1997.79:p.1-8. 2. Folkman, J., Fighting cancer by attacking its blood supply. Scientific American, 1996.275(3):p.150-4. 3. Folkman, J., Tumor angiogenesis and tissue factor [comment]. Nature Medicine, 1996.2(2):p.167-8. 4. Folkman, J., Angiogenesis inhibitors generated by tumors. Molecular Medicine, 1995.1(2):p.120-2. 5. Ingber, D., et al., Synthetic analogue of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature, 1990.348:p.555-557. 6.Chun,E.,et al.,Novel inhibitors targeted to methionine aminopeptidase 2(MetAP2)strongly inhibit the growth of cancers in xenografted nude model.Int J Cancer,2005.114(1):p.124-30。 7.Griffith,E.C.,et al.,Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2.Proc Natl Acad Sci USA,1998.95(26):p.15183-8。 8.Turk,B.E.,et al.,Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells.Chem Biol,1999.6(11):p.823-33。 9.Wang,J.,et al.,Tumor suppression by a rationally designed reversible inhibitor of methionine aminopeptidase-2.Cancer Res,2003.63(22):p.7861-9。 10.Zhang,Y.,et al.,Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21WAF1 / CIP1.Proc Natl Acad Sci USA,2000.97(12):p.6427-32。 11.Bhargava,P.,et al.,A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer Clin Cancer Res,1999.5:p.1989-1995。 12.Herbst,R.,et al.,Safety and pharmacokinetic effects of TNP-470,an angiogenesis inhibitor,combined with paclitaxel in patients with solid tumors:evidence for activity in non-small-cell lung cancer.J Clin Oncol,2002.20:p.4440-4447。 13.Kruger,E.,Figg,WD.,TNP-470:an angiogenesis inhibitor in clinical development for cancer.Expert Opin Investig Drugs,2000.9:p.1383-1396。 14.Kudelka,A.,et al.,A phase I study of TNP-470 administered to patients with advanced squamous cell cancer of the cervix.Clin Cancer Res 1997.3:p.1501-1505。 15.Kudelka,A.,C.Verschraegen,and E.Loyer,Complete remission of metastatic cervical cancer with the angiogenesis inhibitor TNP-470.N Engl J Med,1998.338:p.991-992。 16. Logothetis, C., et al., Phase I trial of the angiogenesis inhibitor TNP-470 for progressive androgen-independent prostate cancer. Clin Cancer Res, 2001.7: p.1198-1203。 17. Moore, J.D., et al., Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl)fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi’s sarcoma. Cancer Chemother Pharmacol, 2000.46(3): p.173-9。 18. Stadler, W., Kuzel, T., Shapiro, C., Sosman, J., Clark, J., Vogelzang, N.J., Multi-institutional study of the angiogenesis inhibitor TNP-470 in metastatic renal carcinoma. J Clin Oncol, 1999.17: p.2541-2545。 19. Tran, H., et al., Clinical and pharmacokinetic study of TNP-470, an angiogenesis inhibitor, in combination with paclitaxel and carboplatin in patients with solid tumors. Cancer Chemother Pharmacol, 2004.54: p.308-314。 20. U.S. Patent No. 5,204,345。

Summary of the Invention

[0007] Angiogenesis inhibition has been established as an important modality for tumor suppression and spread when combined with chemotherapy drugs. Although there are a wide variety of inhibitors that have reached clinical approval, many of them have been found to be inefficient or to have various side effects. Therefore, finding new angiogenesis inhibitors with high potency and drug-like properties has become a target to establish a new arena of action in cancer treatment, especially considering the lower toxicity profile of these agents compared to chemotherapy drugs.

[0008] Many studies have confirmed that MetAP2 plays an important role in the development of various types of cancer, and that specific downregulation of human MetAP2 expression by antisense oligonucleotides has a dominant effect on endothelial cell proliferation.In the context of the development of novel agents disclosed herein, the inventors have recognized the involvement of MetAP2 in lymphangiogenesis and demonstrated the dual action of MetAP2 in the formation of both blood capillaries and lymphatic capillaries.Therefore, there is a rationale for positioning MetAP2 as a useful target for the treatment of primary cancer, and metastatic disease in general.

[0009] As mentioned previously, one of the most effective known inhibitors of MetAp2 originates from the natural compound fumagillin. This molecule was isolated from Aspergillus fumigatus Fresenius and its synthetic analogue O-chloroacetylcarbamoyl fumagillol or TNP-470 (also known as AGM-1470) is one of the most potent analogues of fumagillin demonstrated in angiogenic cell models and was one of the first antiangiogenic small molecule drugs to undergo clinical trials. However, the development of this derivative was hampered by significant clinical drawbacks related to dose-dependent side effects.

[0010] A large series of fumagillin derivatives have been synthesized and their biological activity tested. While the majority of these compounds have been determined to be inactive or not sufficiently safe, a unique group of compounds disclosed herein have been determined to be safe and highly active in inhibiting endothelial cell proliferation and in abolishing MetAp2 enzyme activity.

[0011] More specifically, it has been determined that the compounds of the present invention significantly inhibit the proteolytic activity of MetAp2. Inhibition of MetAp2 enzyme activity leads to angiogenesis blockade and inhibits tumor growth. Moreover, it has been found that the presence of the compounds of the present invention in human melanoma cell lines and primary endothelial cells impairs cell proliferation. This in vitro observation correlates with in vivo results that demonstrate substantial antitumor activity in different tumor-bearing mouse models. These results support the broad range of biological effects and advantages of the compounds of the present invention.

[0012] Histological analysis showed that endothelial cell re-modulation was affected. Immunofluorescence measurement of extracted murine tumor tissue sections showed that positive cells were more sporadically and less uniformly organized as blood vessels in treated tissues compared to untreated group. Moreover, the compounds of the present invention reduced cell proliferation in treated tumors.

[0013] Considering the promising in vivo results, the compounds of the present invention have been formulated for the treatment or prevention of various diseases in various delivery forms, including nanoparticle-based encapsulation delivery. The ability to enhance the bioavailability of lipophilic drugs and improve retention and stability in vivo using doses 10-100 times lower than the free drug was determined. Cellular availability in monolayer and 3D multicellular cultures was also developed. The relatively high concentrations required to inhibit cell proliferation in vitro were found to be due to limited transport into the cells. The inhibitory effect of the encapsulated compounds on the proliferation of endothelial and A375 cells showed significant improvement in bioavailability at doses almost 20 times lower compared to the free compound. In 3D cultures, unlike 2D cell cultures, cells assemble and spatially interact, providing more physiological relevance and better drug efficacy prediction compared to monolayer cell cultures.

[0014] In summary, the data provided herein demonstrate the superiority and promising therapeutic properties of the compounds of the present invention in the prevention and treatment of cancer progression. The compounds of the present invention demonstrated inhibitory activity and generally effective inhibition of angiogenesis and tumor progression in tumor-bearing mice. These significant results were mainly attributed to the antiangiogenic and anticancer activities of the compounds.

[0015] Thus, the present invention provides novel compounds, compositions, uses, and methods of treatment and prevention.

[0016] In a first embodiment, a compound of general formula (I): The compound is of the formula TIFF2025508363000002.tif62170, where R is a carbocyclyl containing two or more ring structures.

[0017] The compounds of formula (I) contain at least six chiral carbon centers. Each of these chiral centers may be in either the (R) or (S) configuration. Thus, the compounds provided herein may be in enantiomerically pure form, or may be provided as mixtures of stereoisomers or diastereomers.

[0018] Thus, the present invention provides enantiomers of compounds of formula (I).

[0019] The present invention further provides chiral compounds of formula (I).

[0020] The present invention further provides compounds of formula (I) as MetAp2 inhibitors and further as anti-angiogenic agents.

[0021] The compounds of the present invention are further intended as MetAp2 inhibitors in methods of inhibiting MetAp2 activity or in methods of preventing or treating angiogenesis, as further disclosed herein.

[0022] The invention further provides the use of the compounds disclosed herein in the preparation of a composition, e.g., a pharmaceutical composition, which may be used experimentally or therapeutically in connection with the treatment or prevention of angiogenesis or any other disease or disorder disclosed herein.

[0023] The compounds of the present invention are compounds of formula (I) in which the "carbocyclyl" functionality (variant R in compounds of formula (I)) is any non-linear alkylene structure containing only single bonds or containing one or more double or triple bonds, or is an aromatic ring. The carbocycle is a polycyclic ring system containing more than one or at least two rings or ring systems. The carbocyclyl may be composed of more than one ring, at least one of which is a six-membered ring structure and which may be joined to another ring or ring structure in a fused, bridged or spiro fashion (which may be a six-membered ring or a ring structure of different sizes).

[0024] The carbocyclyl may be a hydrocarbon composed solely of carbon atoms or may be in the form of a heterocarbocyclyl further containing at least one heteroatom selected from the group consisting of O, N and S. The carbocyclyl may contain one or more double bonds or may be fully aromatic.

[0025] Typically, the carbocycle comprises a 6-membered ring structure that is fused, bridged or spiro-linked to another ring or ring structure to form an additional 6-membered ring. For example, carbocyclyl may be a group that is the fusion of two or three 5-membered or 6-membered rings. Alternatively, carbocyclyl may be a bridged 5-membered or 6-membered ring. Non-limiting examples of carbocyclyl groups include adamantyl and its derivatives, norbornanyl and its derivatives, norbornanyl and its derivatives, norbornenyl and its derivatives, steroidyl and its derivatives, camphoryl and camphor derivatives, camphenyl and camphene derivatives, tricyclo(2.2.1.0(2,6))heptanyl and its derivatives, tetracyclo[3.2.0.0(2,7).0(4,6)]heptanyl and its derivatives, and the like.

[0026] Adamantyl has the structure The adamantyl is derived from adamantane of TIFF2025508363000003.tif30170, which may have optional substitution and may be linked to the compound of formula (I) through any of the adamantyl carbon atoms (designated by the dashed lines). Norbornanyl is represented by the structure Norbornenyl is derived from norbornane of TIFF2025508363000004.tif30170, which may have any substitution and may be linked to the compound of formula (I) through any of the norbornanyl carbon atoms (designated by the dashed line). Norbornenyl has the structure TIFF2025508363000005.tif30170 is derived from norbornane, which may have any substitution and may be linked to the compound of formula (I) through any of the norbornenyl carbon atoms (designated by the dashed line). Steroidil may be any steroid known in the art. Camphoryl has the structure Camphor is derived from camphor of TIFF2025508363000006.tif47170, which derivatives may have any substitution and may be linked to the compound of formula (I) through any of the camphoryl carbon atoms (designated by the dashed lines). Camphenyl has the structure The camphene derivative of TIFF2025508363000007.tif39170 may have any substitution and may be linked to the compound of formula (I) through any of the camphenyl carbon atoms (designated by dashed lines).

[0027] Tricyclo(2.2.1.0(2,6))heptanyl has the structure The derivative is derived from tricyclo(2.2.1.0(2,6))heptane of TIFF2025508363000008.tif30170, which may have any substitution and may be linked to the compound of formula (I) through any of the tricyclo(2.2.1.0(2,6))heptanyl carbon atoms (designated by dashed lines).

[0028] Tetracyclo[3.2.0.0(2.7).0(4.6)]heptanyl has the structure The derivative is derived from tetracyclo[3.2.0.0(2.7).0(4.6)]heptane of TIFF2025508363000009.tif32170, which may have any substitution and may be linked to the compound of formula (I) through any of the tetracyclo[3.2.0.0(2.7].0(4,6)]heptanyl carbon atoms (designated by dashed lines).

[0029] The linkage of the carbocycle to the oxygen atom may be direct or via a spacer or linker moiety, as further disclosed herein.

[0030] In some embodiments, a carbocyclyl is or includes an aromatic ring or ring structure, or a heteroaromatic ring or ring structure. The aromatic ring structure may contain between 6 and 10 carbon atoms and may or may not be fused to another aromatic or non-aromatic carbocyclic ring. The heteroaromatic ring may contain between 5 and 10 carbon atoms and one or more (1, 2, or 3) heteroatoms selected from N, O, and S.

[0031] In some embodiments, the carbocyclyl is not an aromatic ring or ring system.

[0032] Each of the carbocyclyl groups may be linked to an oxygen atom of a compound having the structure of formula (I) through any carbon atom of the carbocyclyl group. For example, when the carbocyclyl is an adamantyl, the adamantyl may be substituted to an oxygen atom of a compound of formula (I) through carbon 1 or 2 of the adamantyl ring structure. When norbornanyl and its derivatives are linked, the norbornanyl or its derivatives may be attached through carbon 1, 2 or 7 of the ring structure.

[0033] Each carbocyclyl group may be substituted on any carbon atom of the ring structure with one or more groups or atoms to provide a variety of carbocyclyl derivatives with modified (improved) toxicity and activity. Generally speaking, derivatives of any of the listed carbocyclyl groups may be alkyl derivatives, hydroxylated derivatives, aromatic derivatives, halogenated derivatives, acid derivatives (containing -COOH or other acid groups), amine derivatives, sulfonated derivatives, etc. Non-limiting examples of substitutions include -C1-C3 alkyl, -C2-C4 alkenyl, -C1-C3 alkyl halide (wherein the alkyl is substituted with one or more halogen atoms including I, Br, Cl, or F), hydroxyl, carboxyl, carboxylate, -C6-C7 alkyl, -C8-C9 alkyl, -C9-C10 alkyl, -C11 alkyl, -C12 alkyl, -C13 alkyl, -C14 alkyl, -C15 alkyl, -C16 alkyl, -C17 alkyl, -C18 alkyl, -C19 alkyl, -C20 alkyl, -C21 alkyl, -C22 alkyl, -C23 alkyl, -C24 alkyl, -C25 alkyl, -C26 alkyl, -C27 alkyl, -C28 alkyl, -C29 alkyl, -C30 alkyl, -C31 alkyl, -C20 alkyl, -C20 alkyl, -C21 alkyl, -C22 alkyl, -C23 alkyl, -C24 alkyl, -C25 alkyl, -C26 alkyl, -C27 alkyl, -C28 alkyl, -C29 alkyl, -C20 ...0 alkyl, -C20 10 Included are aryl (phenyl, naphthyl, etc.), hydroxy-C1-C3 alkyl, sulfate, sulfonate, sulfonamide, sulfonic acid, and one or more halides. When a carbon atom is said to be unsubstituted, the particular carbon atom maintains its original bond and substitution. Substitution may be by a single substituent, by two or more substituents, by multiple substituents, or the carbocyclyl group may be substituted as a whole.

[0034] In some embodiments, the variable R is attached directly to the oxygen atom of the compound of formula (I) or to a group of formula (IA): The carbocyclyl R may be linked via a spacer or linker moiety X as depicted in the compound of formula (IA), where R is as defined herein and X is a functional group connecting the carbocyclyl R with the oxygen atom of the compound of formula (I). The group X may be an atom or a group of atoms. In some embodiments, X comprises at least two atoms, for example -C=O-.

[0035] In some embodiments, X is absent and R is bonded directly to the oxygen atom.

[0036] In some embodiments, X is -C1-C5 alkylene (e.g., methylene, ethylene, propylene, etc.), which may or may not be substituted; -C2-C5 alkenylene (containing one or more double bonds), which may or may not be substituted; -C6-C7 alkylene (containing one or more double bonds), which may or may not be substituted; 10 Arylene (such as phenylene or naphthylene); -C5-C, which may be substituted or unsubstituted and contains one or more heteroatoms selected from N, O and S. 10 Heteroarylene; -C1-C5 alkylene-C6-C 10 arylene; -C1-C5 alkylene-C5-C6, which may be substituted or unsubstituted and contains one or more heteroatoms selected from N, O and S; 10 Heteroarylene; -C(=O)-C6-C6, which may be substituted or unsubstituted 10 Arylene; -C(=O)-C5-C6, which may be substituted or unsubstituted and contains one or more heteroatoms selected from N, O and S; 10 Heteroarylene; -C(=O)-C6-C6, which may be substituted or unsubstituted 10 arylene -NH-C(=S)-NH-; -C(=O)-C5-C6, which may or may not be substituted and contains one or more heteroatoms selected from N, O and S; 10Heteroarylene -NH-C(=S)-NH-; -NH-C(=S)-NH-; -C1-C5 alkylene-NH-C(=S)-NH-; -NH-C(=S)-NH-C1-C5 alkylene; -C(=O)-; -O(C=O)-; -C1-C5 alkylene-C(=O)-; -C1-C5 alkylene-O(C=O)-; -NH-C(=O)-; -NH-C(=O)-C1-C5 alkylene-C(=O)-; -SO2NH-; -NH-SO2- and the like.

[0037] As used herein, "alkylene" refers to a linear, branched, or cyclic, and in certain embodiments, linear or branched, divalent aliphatic hydrocarbon group having 1 to 5 carbon atoms (inclusive), or 1, 2, 3, 4, or 5 carbon atoms. Alkylene groups include, but are not limited to, methylene (-CH2), ethylene (-CH2CH2-), propylene (-(CH2)3-), and the like. Propylene can be linear propylene or isopropylene. Butylene can be linear butylene, secondary butylene, tertiary butylene. Pentylene can be linear pentylene, quaternary pentylene, isopentylene, secondary pentylene, and the like.

[0038] "Arylene" refers to a monocyclic or polycyclic divalent aromatic group having 6 to 10 carbon atoms and at least one aromatic ring. Arylene can include, but is not limited to, 1,2-, 1,3-, and 1,4-phenylene.

[0039] As used herein, "heteroarylene" refers to a divalent monocyclic or polycyclic aromatic ring system having 5 to 10 carbon atoms and one or more, and in some embodiments 1 to 3, heteroatoms selected from nitrogen, oxygen and sulfur in the ring(s).

[0040] The group is -C1-C5 alkylene-C6-C 10 When it includes combinations of functionalities such as arylene, each of alkylene and arylene is defined herein.

[0041] In some embodiments, X is or includes a -C1-C5 alkylene group.

[0042] In some embodiments, X is or includes -C1-C5 alkylene, -C(=O)- and / or -O(C=O)-.

[0043] In some embodiments, X is or includes -C1-C5 alkylene, which may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted by a substituent R4 as defined herein. In some embodiments, the alkylene is methylene or ethylene. In some embodiments, the alkylene is substituted by a halide atom, such as F, Cl, Br, or I.

[0044] In some embodiments, X is -C(=O)-.

[0045] In some embodiments, the compound of structure (I) or (IA) has the structure (IB): TIFF2025508363000011.tif68170(IB), where R is as defined herein.

[0046] In some embodiments, in the compound of formula (IA), X is -C(=O)-O-.

[0047] In some embodiments, the compound of formula (I) or (IA) has the designated compound (IC): TIFF2025508363000012.tif72170(IC), where R is as defined herein.

[0048] In some embodiments, in the compound of formula (IA), X is or comprises -C(=O)-C1-C5 alkylene-NH-C(=S)-NH-.

[0049] In some embodiments, the compound of formula (I) or (IA) has the formula (ID): TIFF2025508363000013.tif69170(ID), where R is as defined herein.

[0050] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted by a substituent R4, as defined herein.

[0051] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, or pentylene, which may or may not be substituted by a substituent R4, as defined herein.

[0052] In some embodiments, in the compound of formula (IA), X is or comprises -C(=O)-C1-C5 alkylene-O(C=O)-.

[0053] In some embodiments, the compound of formula (I) or (IA) has the formula (IE): TIFF2025508363000014.tif68170(IE), where R is as defined herein.

[0054] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted by a substituent R4, as defined herein.

[0055] In some embodiments, in the compound of formula (IA), X is or comprises -C(=O)-NH-C(=O)-.

[0056] In some embodiments, the compound of formula (I) or (IA) has formula (IF): TIFF2025508363000015.tif70170(IF), where R is as defined herein.

[0057] In some embodiments, in the compound of formula (IA), X is or comprises -NH-C(=O)-C1-C5 alkylene-C(=O)-.

[0058] In some embodiments, the compound of formula (I) or (IA) has formula (IG): TIFF2025508363000016.tif68170(IG), where R is as defined herein.

[0059] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted by a substituent R4, as defined herein.

[0060] In some embodiments, -C1-C5 alkylene may be ethylene or propylene, which may or may not be substituted by a substituent R4, as defined herein.

[0061] In some embodiments, the compound of formula (I) or (IA) has formula (IH): TIFF2025508363000017.tif71170(IH), where R is as defined herein.

[0062] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted by a substituent R4, as defined herein.

[0063] In some embodiments, -C1-C5 alkylene may be methylene, ethylene, propylene, butylene, or pentylene, which may or may not be substituted with halogen atoms, as defined herein.

[0064] In some embodiments, in a compound of formula (IA), X is absent.

[0065] In some embodiments, in each of the formulae disclosed herein, -C1-C5 alkylene may be selected from methylene, ethylene, propylene, butylene, and pentylene. In some embodiments, in each of the formulae disclosed herein, -C1-C5 alkylene may be unsubstituted or substituted, for example, by a halide atom.

[0066] In some embodiments, the -C1-C5 alkylene is methylene or ethylene or propylene or butylene or pentylene. In some embodiments, the -C1-C5 alkylene contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, or 5 carbon atoms.

[0067] In some embodiments, carbocyclyl R is selected from norbornanyl, norbornenyl, and adamantyl.

[0068] In some embodiments, the carbocyclyl R is an optionally substituted adamantyl. In some embodiments, the adamantyl may be linked to the oxygen atom of the compound of formula (I) via carbon 1 or 2 of the adamantyl. In some embodiments, the adamantyl has the structures (A1) and (A2): TIFF2025508363000018.tif57170 (wherein, for each of structures (A1) and (A2), independently, X is a functional group or atom that links the adamantyl group with the oxygen atom of the compound of formula (I), as disclosed herein; Each of R1, R2 and R3, independently of the others, may be -H or a halide (F, Cl, Br, I), -C1-C3 alkyl, -C2-C4 alkenyl, -C1-C3 alkyl halide, hydroxyl, carboxyl, carboxylate, -C6-C7 alkyl, -C8-C9 alkyl, -C9-C10 alkyl, -C11-C12 alkyl, -C12-C14 alkyl, -C13-C16 alkyl, -C14-C18 alkyl, -C15-C16 alkyl, -C16-C18 alkyl, -C17-C19 alkyl, -C18-C19 alkyl, -C19-C20 ... 10 aryl, hydroxyalkyl, sulfate, sulfonate, sulfonamide, and sulfonic acid).

[0069] In some embodiments, -C1-C5 alkyl is methyl or ethyl or propyl or butyl or pentyl, In some embodiments, -C1-C5 alkyl is methyl.

[0070] In some embodiments, X is absent and the adamantyl group is bonded directly to the oxygen atom of the structure of formula (I), as shown above.

[0071] In some embodiments, X is -C(=O)-; -O(C=O)-; -C1-C5 alkylene-C(=O)-; -C1-C5 alkylene-O(C=O)-; -NH-C(=O)-; or -NH-C(=O)-C1-C5 alkylene-C(=O)-, where X may or may not be substituted with an R4 variable.

[0072] In some embodiments, adamantyl has the structure (B): TIFF2025508363000019.tif52170 (where Each of R1, R2 and R3, independently of the others, may be H or -C1-C3 alkyl, -C2-C4 alkenyl, -C1-C3 alkyl halide, hydroxyl, carboxyl, carboxylate, -C6-C 10may be selected from aryl, hydroxyalkyl, and halide; TIFF2025508363000020.tif7170 is of formula (I) showing the attachment points to the oxygen atoms of the structure.

[0073] In some embodiments, one or more of R1, R2, and R3 is H or -C1-C5 alkyl selected from methyl, ethyl, propyl, butyl, and pentyl. In some embodiments, one or more of R1, R2, and R3 is methyl, ethyl, or propyl.

[0074] In some embodiments, one or more of R1, R2, and R3 is a methyl or halide atom.

[0075] In some embodiments, R4 is absent.

[0076] In some embodiments, adamantyl has the structure (C): TIFF2025508363000021.tif52170 (where Each of R1, R2 and R3, independently of the others, may be H or -C1-C3 alkyl, -C2-C4 alkenyl, -C1-C3 alkyl halide, hydroxyl, carboxyl, carboxylate, -C6-C 10 may be selected from aryl, hydroxyalkyl, and halide; TIFF2025508363000022.tif7170 is of formula (I) showing the attachment points to the oxygen atoms of the structure.

[0077] In some embodiments, one or more of R1, R2, and R3 is H or -C1-C5 alkyl selected from methyl, ethyl, propyl, butyl, and pentyl. In some embodiments, one or more of R1, R2, and R3 is methyl, ethyl, or propyl.

[0078] In some embodiments, one or more of R1, R2, and R3 is a methyl or halide atom.

[0079] In some embodiments, R4 is absent.

[0080] In some embodiments, adamantyl has the structure (D): TIFF2025508363000023.tif53170 (where Each of R1, R2 and R3, independently of the others, may be H or -C1-C3 alkyl, -C2-C4 alkenyl, -C1-C3 alkyl halide, hydroxyl, carboxyl, carboxylate, -C6-C 10 may be selected from aryl, hydroxyalkyl, and halide; TIFF2025508363000024.tif7170 is of formula (I) showing the attachment points to the oxygen atoms of the structure.

[0081] In some embodiments, one or more of R1, R2, and R3 is H or -C1-C5 alkyl selected from methyl, ethyl, propyl, butyl, and pentyl. In some embodiments, one or more of R1, R2, and R3 is methyl, ethyl, or propyl.

[0082] In some embodiments, one or more of R1, R2, and R3 is a methyl or halide atom.

[0083] In some embodiments, R4 is absent.

[0084] In some embodiments, in each of the formulas herein, the adamantyl is attached via carbon atom 1 or 2, as depicted in structure (A1) or (A2) above.

[0085] In some embodiments, the compound of formula (I) is a compound designated herein: AD-3302; AD-3281; AD-3306; AD-3283; AD-3305; and AD-3301 is selected from.

[0086] In some embodiments, the compound of formula (I) is a compound designated herein: AD-3295; AD-3294; AD-3286; AD-3287; and AD-3290 is selected from.

[0087] In some embodiments, in the compound of formula (I), R is norbornenyl or norbornanyl. In some embodiments, the carbocyclyl has the structure (E): TIFF2025508363000025.tif30170, where X is as defined herein.

[0088] In some embodiments, the carbocyclyl has the structure (F): TIFF2025508363000026.tif30170, where -C1-C5 alkylene is selected from methylene, ethylene, propylene, butylene, and pentylene. In some embodiments, alkylene is methylene, ethylene, or propylene.

[0089] In some embodiments, the carbocyclyl has the structure (G): TIFF2025508363000027.tif43170 (wherein the dashed line designates the bond of attachment to the oxygen atom of the compound of formula (I)).

[0090] In some embodiments, X is absent and the norbornenyl or norbornanyl is bonded directly to the oxygen atom of the compound of Formula (I).

[0091] In some embodiments, the compound of Formula (G) is designated herein as AD-3303.

[0092] In some embodiments, the compounds of the present invention are compounds designated herein: AD-3280; and AD-3199 It is.

[0093] Excluded from the compounds of formula (I) are compounds in which R is linear or branched alkyl, i.e., compounds that are not carbocyclyl. Also excluded are linear or branched alkyl, alkenyl, and alkynyl groups. Further excluded are optionally substituted acyl groups, such as acyl groups derived from carboxylic acids or their amide groups (e.g., alkanoyl, aroyl, aromatic heterocyclic carbonyl, carbamoyl, alkoxycarbonyl, phenoxycarbonyl, etc.), or acyl groups derived from sulfonic acids or their amide groups (e.g., benzenesulfonic acid, sulfamoyl, etc.). Also excluded are -C1-C, optionally having 1 to 3 substituents. 20and optionally substituted alkyl groups such as those shown as straight chain or branched alkyl groups. These excluded groups may be alkyl groups that are epoxidized at any position, such as methyl, ethyl, benzyl, etc. Specifically excluded are optionally substituted alkyl groups, including substituents such as amino, lower alkylamino (e.g., methylamino, ethylamino, isopropylamino, etc.), di-lower alkylamino (e.g., dimethylamino, diethylamino, etc.), nitro, halogen (e.g., fluorine, chlorine, bromine, iodine, etc.), hydroxyl, lower alkoxy (e.g., methoxy, ethoxy, etc.), cyano, carbamoyl, carboxyl, lower alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, etc.), carboxy lower alkoxy (carboxymethoxy, 2-carboxyethoxy, etc.), optionally substituted phenyl, aromatic heterocyclic groups (5-6 membered aromatic heterocyclic groups containing 1-4 heteroatoms such as nitrogen, oxygen, sulfur, etc., such as 2-furyl, 2-thienyl, 4-thiazolyl, 4-imidazolyl, 4-pyridyl, etc.). Further excluded are optionally substituted alkanoyl groups.

[0094] The present invention further comprises: TIFF2025508363000028.tif81170TIFF2025508363000029.tif165170TIFF2025508363000030.tif158170TIFF2025508363000031.tif198170TIFF2025508363000032.tif85170.

[0095] The present invention further provides Fumagillol-4-(1-adamantylamino)-4-oxobutanoate (AD-3286); Fumagillol-4-(2-adamantylamino)-4-oxobutanoate (AD-3287); and Fumagillol-5-(2-adamantylamino)-5-oxopentanoate (AD-3290).

[0096] Each of the specifically disclosed compounds above constitutes a separate embodiment of the invention. Thus, the compounds specifically disclosed herein or encompassed by the compounds of formula (I) may be used separately or in combination in preparing compositions according to the invention or in the uses and methods according to the invention. The compounds of the invention may contain one or more chiral centers. Such chiral centers may be in either the (R) or (S) configuration, or may be a mixture of (R) and (S) configurations. Thus, the compounds provided herein may be enantiomerically pure or may be stereoisomeric or diastereomeric mixtures. It should be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one skilled in the art will recognize that administration of the (R) form of the compound is equivalent to administration of the (S) form of the compound for compounds that undergo epimerization in vivo.

[0097] The compounds of the invention may be provided in the free acid or free base form, or in the form of a salt, depending on the selection of the carbocycle R and any substitutions that may be present. Pharmaceutically acceptable acid addition salts can include salts derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and the like, as well as salts derived from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are the salts of amino acids such as arginates and the like, as well as gluconates, galacturonates (see, e.g., Berge SM, et al. "Pharmaceutical Salts," J. of Pharmaceutical Science, 66:1-19 (1977)).

[0098] Acid addition salts of compounds containing a basic functionality are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in a conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner. The free base form differs slightly from each salt form in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to each free base for purposes of this invention. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge SM, et al., "Pharmaceutical Salts," J. of Pharmaceutical Science, 66:1-19 (1977)).

[0099] Base addition salts of compounds containing acidic functionality may be prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in a conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ slightly from the respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the respective free acids for purposes of the present invention.

[0100] The compounds of the present invention may be formulated or provided encapsulated or contained within nano- or microcarriers, such as nanospheres, nanocapsules, nanoparticles, microspheres, microcapsules, microparticles, etc. The carriers may be formed of any biodegradable or biostable material that allows the encapsulated active compound or any ingredient to be released at any time or at a target site. In general, the nano- or microcarriers may be lipid-coated nanoparticles, protein-coated nanoparticles, or polymer-coated nanoparticles.

[0101] In some embodiments, the carrier is comprised of one or more polymers. In some embodiments, the one or more polymers are water-soluble non-adhesive polymers. In some embodiments, the polymer is polyethylene glycol (PEG) or polyethylene oxide (PEO). In some embodiments, the polymer is polyalkylene glycol or polyalkylene oxide. In some embodiments, the one or more polymers are biodegradable polymers. In some embodiments, the one or more polymers are biocompatible polymers that are conjugates of water-soluble polymers and biodegradable polymers. In some embodiments, the biodegradable polymer is polylactic acid (PLA), poly(glycolic acid) (PGA), or poly(lactic / glycolic acid) (PLGA).

[0102] In some embodiments, the carrier is or includes PLGA. Different forms of PLGA can be selected and utilized depending on the ratio of lactide to glycolide used in polymerization. In some embodiments, PLGA is selected to have a ratio of 75:25 lactic acid to glycolic acid. Other ratios such as 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, etc. may be similarly prepared and utilized.

[0103] The compounds of the present invention may be provided in various forms of composition, each of which is selected based on the formulation, intended use and mode of administration, among others. Generally speaking, the compounds of the present invention may be provided in pharma- ceutically acceptable forms, such as pharmaceutical compositions and formulations that include one or more compounds according to the present invention and a suitable carrier.

[0104] Pharmaceutically acceptable carriers, for example, vehicles, adjuvants, excipients, or diluents, are well known to those skilled in the art and are readily available to the public. Preferably, the pharma- ceutically acceptable carrier is one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.

[0105] The choice of carrier will be determined, in part, by the particular active agent and the particular method used to administer the composition. Accordingly, suitable formulations of pharmaceutical compositions of the present invention are widely varied. The following formulations are merely exemplary and not limiting.

[0106] Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient as a solid or granules; (c) powders; (d) suspensions in a suitable liquid; and (e) suitable emulsions. Liquid formulations may contain diluents such as water and alcohol, e.g., ethanol, benzyl alcohol, and polyethylene alcohol, with or without the addition of a pharma- ceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms may be of the ordinary hard- or soft-shelled gelatin type, containing surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms may contain one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, gum arabic, gelatin, guar gum, colloidal silicon dioxide, croscarmellose, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffers, disintegrants, humectants, preservatives, flavorings, and pharmacologically compatible carriers. Lozenge forms may contain the active ingredient in a flavor, usually sucrose and gum arabic or tragacanth, similarly pastilles contain the active ingredient in an inert base such as gelatin and glycerin, or sucrose and gum arabic, and emulsions, gels, and the like contain, in addition to the active ingredient, a carrier as known in the art.

[0107] The compounds of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as pharmaceuticals for non-pressurized preparations, such as in a nebulizer or atomizer.

[0108] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which include suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. The compounds may be administered in a physiologically acceptable diluent in a pharmaceutical carrier such as a sterile liquid or mixture of liquids including water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol, isopropanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, glycerol ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers such as poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides, with or without the addition of pharma- ceutically acceptable surfactants such as soaps or detergents, suspending agents such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0109] The oil that can be used in parenteral formulation includes petroleum, animal oil, vegetable oil, or synthetic oil.Specific examples of oil include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum oil, and mineral oil.Suitable fatty acid for use in parenteral formulation includes oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid ester. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include: (a) cationic detergents, such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides; (b) anionic detergents, such as, for example, alkyl, aryl, and olefin sulfonates, alkyl sulfates, olefin sulfates, ether sulfates, and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents, such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxy-ethylene polypropylene copolymers; (d) amphoteric detergents, such as, for example, alkyl-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (3) mixtures thereof.

[0110] Parenteral formulations may contain about 0.5 to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers may be used in such formulations. To minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is in the range of about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide with propylene glycol. Parenteral formulations may be presented in single- or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier for injection, e.g., water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0111] The compounds of the present invention may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art. See Pharmaceutics and Pharmacy Practice, JBLippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

[0112] Additionally, the compounds of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases and water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray forms containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0113] The compounds of the present invention may also be formulated into compositions that can be used for ocular or dermal delivery. Ocular delivery, i.e., delivery of a compound or composition to the external surface of the eye, which anatomically includes the cornea (including epithelium, Bowman's membrane, stroma, Descemet's membrane, endothelium), conjunctiva, and sclerocorneal junction, i.e., limbus, may include providing the compounds of the present invention in a solid or semi-solid ocular insert or ocular film. The ocular film may be a solid or semi-solid viscous two-dimensional film designed to be placed in the conjunctival fornix or on the conjunctival surface. The ocular film may be configured to be placed on the surface of the eye. The ocular insert may also be a solid or semi-solid device designed to be placed in the conjunctival fornix or on the conjunctival surface.

[0114] Ophthalmic formulations include, but are not limited to, liquid formulations (e.g., solutions, suspensions) for topical administration and formulations for injection or ocular insert administration. Optionally, the ophthalmic formulation is formulated for topical administration, such as an eye drop, swab, ointment, gel, or mist (e.g., aerosol or spray). Optionally, the formulation is an eye drop.

[0115] Dermal and transdermal formulations can include, for example, gels, creams, sprays, and lotions that are applied to the skin. Other formulations include patches that are attached to the skin using an adhesive.

[0116] The compounds, compositions and formulations of the present invention are suitable for administration via any desired suitable method, for example, oral (such as buccal or sublingual), rectal, nasal, topical (such as buccal, sublingual or transdermal), vaginal or parenteral (such as subcutaneous, intramuscular, intravenous or intradermal), or any other known administration method. Each composition of the present invention may be adapted or tailored for suitable administration by combining an effective amount of one or more compounds of formula (I) with a suitable carrier and, optionally, at least one additional active or inactive agent. The compositions of the present invention may be administered to a human or non-human subject to provide for the treatment or prevention of a disease, which may be a local non-systemic disease or a systemic disease.

[0117] Typically, the disease to be treated or prevented is one associated with MetAp2 activity that the compounds of the present invention may be selected to partially or completely inhibit. In other words, the compounds of the present invention may be used in a method to block at least some of the biological effects of MetAp2 by binding to or interacting with MetAp2 and thereby abolishing a particular biological effect of MetAp2. For example, one of the biological effects of MetAp2 is to promote cell proliferation, and therefore the compounds of the present invention may be used to decrease, reduce, or inhibit cell proliferation.

[0118] Without wishing to be bound by theory, the mechanism of interaction of the compounds of the invention with MetAp2 may involve interaction with a target molecule, which may be a MetAp2 protein target, but also a coding gene or its gene product, or a regulatory protein, or a component of a signaling pathway that includes said gene or its gene product. As a result, the specific interaction of the compounds of the invention may involve either targeting or induction of changes in cellular function, or it may involve both effects.

[0119] Thus, the present invention further provides a method for reducing or diminishing or preventing biological effects associated with activity of MetAp2 in a subject, the method comprising administering to said subject an effective amount of a compound of formula (I).

[0120] In some embodiments, said reducing or decreasing or preventing comprises blocking binding to or interaction with MetAp2.

[0121] In some embodiments, the biological effect is cell proliferation.

[0122] The present invention further provides a method of preventing or treating angiogenesis, an angiogenesis-associated disease, or an angiogenesis-dependent disease in a subject, the method comprising administering to the subject an effective amount of a compound according to formula (I).

[0123] In some embodiments, the method is for preventing or treating angiogenesis, ie, for preventing or reducing or slowing the process involving the growth of new blood vessels from pre-existing blood vessels.

[0124] In some embodiments, the method is for preventing or treating angiogenesis-related diseases, i.e. diseases that may result or be caused by increased neovascularization or angiogenic processes, such diseases may include intraocular neovascularization, diseases or conditions associated with intraocular neovascularization, and cancer.

[0125] The cancer prevented or treated by the use of the compounds of the present invention may be any malignant proliferative disease or disorder, such as blastoma, carcinoma, lymphoma, leukemia, sarcoma, mesothelioma, glioma, germinoma, choriocarcinoma, melanoma, glioblastoma, malignant lymphoma, and any other neoplastic disease or disorder. In some embodiments, the cancers that can be prevented or treated using the compound of the present invention include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and head and neck cancer.Solid cancer can include, for example, breast cancer, prostate cancer, sarcoma, and skin cancer.

[0126] In some embodiments, the methods of the invention are for preventing or treating angiogenesis-dependent diseases such as intraocular neovascular disease, wounds, chronic ulcers, ischemic stroke, myocardial infarction, angina pectoris, peripheral arterial disease, critical limb ischemia, diabetic foot ulcers, and vascular dementia.

[0127] The present invention further provides a method for preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I).

[0128] The present invention further provides a method for preventing or treating pulmonary and hepatic fibrosis in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I).

[0129] In some embodiments, the fibrosis is selected from the formation of scar tissue or tissue lesions that cause chronic and progressive impairment of organs in the subject's body.

[0130] The present invention further provides a method for preventing or treating a disease in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I), wherein the disease is selected from angiogenesis, intraocular angiogenesis, intraocular neovascular disease, wounds, chronic ulcers, ischemic stroke, myocardial infarction, angina pectoris, peripheral arterial disease, critical limb ischemia, diabetic foot ulcer, cerebrovascular dementia, cancer, pulmonary fibrosis, hepatic fibrosis, endometriosis, arthritis (e.g. rheumatoid arthritis), autoimmune diseases, obesity and microsporidiosis.

[0131] The present invention further provides a method of preventing or treating at least one intraocular or cutaneous disease or condition in a subject, the disease or condition being associated with MetAp2 activity, the method comprising intraocular or cutaneous delivery to the subject of a composition comprising at least one MetAp2 inhibitor according to the present invention.

[0132] In some embodiments of the compositions and methods of the present invention, the compound of formula (I) is a compound selected from the compounds specified herein: AD-3281, AD-3306, AD-3201, and AD-3306.

[0133] The term "treatment" as used herein includes both prevention and treatment of disease, disorder or illness. The term generally refers to administering a therapeutic amount of a compound or a composition containing the compound, which is effective to ameliorate the undesirable symptoms associated with the disease, to prevent the onset of such symptoms before they occur, to slow the progression of the disease, to slow the worsening of symptoms, to enhance the onset of remission periods, to slow the irreversible damage caused in the advanced chronic stage of the disease, to delay the onset of the advanced stage, to reduce the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the onset of the disease or a combination of two or more of the above.

[0134] The "effective amount" of a compound or a composition comprising the compound as the object of this specification is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending on, among other things, the type and severity of the disease to be treated and the treatment regimen. The effective amount is typically determined in a properly designed clinical trial (dose ranging test), and a person skilled in the art will know how to properly carry out such a test to determine the effective amount. As is generally known, the effective amount depends on various factors, including various pharmacological parameters such as the affinity of the ligand to the receptor, distribution profile in the body, and half-life in the body, as well as unwanted side effects, if any, age and sex.

[0135] The invention further provides kits comprising a compound of the invention and instructions for use. [Brief description of the drawings]

[0136] For a better understanding of the subject matter disclosed herein, and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0137] [Figure 1]1A-G demonstrate the inhibitory effect of compounds of the invention on MetAp2. Compound AD-3201 is TNP-470. [Diagram 2] 2A-C show the overall effect of different compounds on HUVEC. [Diagram 3] Figure 3 demonstrates that laser irradiation in mice induces CNV. Compound AD3281 led to 46% at the selected dose (12 μg / eye). Eylea led to 59% at the given dose and time. [Figure 4] 4A-D show the chemical synthesis and characterization of AD-3281. A. Synthesis diagram of AD-3281, fumagillin under basic condition was converted to fumagillol, then AD-3281 was obtained after esterification reaction. B. Mass spectrometry (MS) of AD-3281. C. H NMR spectrum of AD-3281. D. HPLC of AD-3281 and TNP-470, early peak represents TNP-470, pink peak shows retention time of AD-3281 at about 15 min. Detection was performed at 205 nm. [Diagram 5] Figures 5A and B demonstrate basal MetAp1 and MetAp2 expression in endothelial and cancer cells. (A, B) Western blot analysis to determine the expression of (A) MetAp2 and (B) MetAp1 in HUVEC, A375, and MDA-MB-231. MetAp levels were found to be higher in cancer cells than in HUVEC. n=3. [Figure 6]Figure 6A and B demonstrate how AD-3281 inhibits the activity of MetAp2 in an enzyme assay. A. AD-3281 affected the enzyme activity of rhMetAp2 and reduced the activity of MetAp2 in cancer cells. L-Met-AMC was added as a substrate to test the enzyme activity of MetAp2. Addition of 2.5 μM AD-3281 inhibited the enzyme activity of rhMetAp2 by 95%, and by 64% and 80% in A375 and MDA-MB-231 cancer cells, respectively, during 1 hour. B. Measurement of the activity of MetAp2 after 10 minutes. AD-3281 showed a reduction in MetAp2 activity compared to the control group. n=3. *p<0.05, **p<0.01. Results are expressed as mean ± SEM. [Figure 7] Figure 7A-E show the effect of AD-3281 on endothelial and cancer cell function. (A, B) AD-3281 impairs the ability of cells to proliferate. Cells were treated with different concentrations of AD-3281 for 72 hours, after which MTT assays were performed to quantify their proliferation. A dose-dependent reduction is observed compared to the control. A. HUVEC, n=6. B. A375, n=7. (C, D) The effect of increasing concentrations of encapsulated AD-3281 on cell viability. C. A375 left 72 hours, right 96 hours incubation. D. HUVEC, n=5. E. AD-3281 affects A375 cell viability in 3D compared to untreated control cells, cell viability was measured using WST1 assay after 96 hours incubation. n=4. *p<0.05, **p<0.01, ***p<0.001. Results are expressed as mean ± SEM. [Figure 8]FIG. 8A-H shows how AD-3281 suppresses A375 and MDA-MB-231 xenograft growth in treated mice. (A, B) AD-3281 affects tumor volume in treated groups compared to control groups. A. Mice injected SC with A375 treated at 15 mg / kg and 30 mg / kg every other day. n=7. B. Effect of AD-3281 on MDA-MB-231 treated at 7.5 mg / kg and 15 mg / kg every other day. n=4. (C, D) Average weight of tumors extracted from treated and untreated mice. C. A375, D. MDA-MB-231. E. Representative images of tumors extracted from untreated and treated mice after 18 and 15 days of treatment in mice injected with A375 and MDA-MB-231, respectively. F. Immunofluorescence staining for MetAp2 (red), CD31 (green), and DAPI (blue). Histological examination of tumors extracted from treated and untreated mice stained with GH&E. H. Immunofluorescence staining for Ki67 (green) and DAPI (blue). *p<0.05, **p<0.01, ***p<0.001. Results are expressed as mean ± SEM. Scale bar 100 μm. [Figure 9] 9A-E depict the preparation and characterization of PLGA nanoparticles. A. Illustrated synthesis process of encapsulated AD-3281 in PLGA nanoparticles by emulsification-evaporation method. (B) TEM image of PLGA nanoparticles. (C) Zeta potential of nanoparticles. (D) Typical DLS measurement of PLGA nanoparticles; graphical representation. (E) Summary of loading measurement parameters. [Figure 10]Figure 10A and B show basal MetAp2 expression and enzyme activity in endothelial cells and cancer cells in response to treatment. (A) Western blot analysis to determine the expression of MetAp2 HUVEC, A375, and MDA-MB-231. MetAp2 levels were found to be higher in cancer cells than in HUVEC. n=3. (B) AD-3281 affected the enzyme activity of rhMetAp2 and reduced the activity of MetAp2 in cancer cells. L-Met-AMC was added as a substrate to test the enzyme activity of MetAp2. The addition of 2.5 μM AD-3281 inhibited the enzyme activity of rhMetAp2 by 95%, and by 64% and 80% in A375 and MDA-MB-231 cancer cells, respectively, during 1 hour. Bottom, measurement of MetAp2 activity after 10 minutes of reaction. AD-3281 showed a reduction in MetAp2 activity compared to the control group. n=3. *p<0.05, **p<0.01. Results are expressed as mean±SEM. [Figure 11] Figure 11 shows basal protein expression of MeAp1. Western blot analysis of MeAp1 from cell lysates and quantification of the resulting signal. [Figure 12] 12A and B show how AD-3281 inhibits the activity of MetAp2 in an enzymatic assay. (A) AD-3281 suppressed HUVEC and A375 cell proliferation. Cells were treated with different concentrations of AD-3281 for 72 hours, after which MTT assays were performed to quantify their proliferation. A dose-dependent reduction is observed compared to the control. n=6-7. (B) Representative microscopy brightfield images (transformed to black and white) of HUVEC tube formation showing impaired tubes in 100 μM AD-3281 cultures. [Figure 13] Figure 13 depicts HUVEC tube formation. Examples of images obtained by bright field microscopy. HUVECs treated with AD-3281 were allowed to form tubes and images were taken after 12 hours and compared to untreated controls. [Figure 14]14A and B show how AD-3281 suppresses A375 and MDA-MB-231 xenograft growth in treated mice. (A) Mice injected SC with A375 were treated with 15 mg / kg and 30 mg / kg every other day. n=7. The graph shows the average weight of tumors extracted from treated and untreated mice, and below tumors excised on day 18 are depicted. (B) Effect of AD-3281 on MDA-MB-231 tumors treated with 7.5 mg / kg and 15 mg / kg every other day. n=4. The graph shows tumor weight at the end point, and below are representative images of tumors extracted from untreated and treated mice after 15 days of treatment. [Figure 15] Figures 15A-C show immunofluorescence staining of excised A375 xenografts treated with 30 mg / kg qod. (a) Sections reacted with antibodies against MetAp2 (red), CD31 (green), and DAPI (blue). (b) Immunofluorescence staining for Ki67 (green) and DAPI (blue). (c) Histological examination of tumors extracted from treated and untreated mice stained with H&E. [Figure 16] Figure 16 shows immunofluorescence staining of MDA-MB-231 resected tumor (7 kg / mg). MetAp2 (red), CD31 (green), and DAPI (blue). Scale bar 100 μm. [Figure 17] 17A-E show the preparation and characterization of PLGA nanoparticles. (a) Illustrated synthesis process of encapsulated AD-3281 in PLGA nanoparticles by emulsification-evaporation method. (b) TEM image of PLGA nanoparticles. (c) Zeta potential of nanoparticles. (d) Typical DLS measurement of PLGA nanoparticles; graphical representation. (e) Summary of loading measurement parameters. [Figure 18]FIG. 18A-C. Cellular uptake of PLGA nanoparticles. A375 cells were exposed for 0 min, 4 h, and 7 h to encapsulate 6-coumarin in PLGA nanoparticles. (a) FACS dot plot analysis showing the cell population containing nanoparticles labeled with 6-coumarin. Bar plots show the normalized average uptake of fluorescent nanoparticles. n=6. (b) MTT proliferation assay performed on A375 after 72 and 96 h exposure to encapsulated AD-3281. (c) Viability of 3D spheroids after exposure to free or encapsulated AD-3281 measured with WST-8 reagent. [Figure 19] FIG. 19 shows ERG studies in mice demonstrating no visual field defects with low dose (LD): 12 μg / eye and high dose (HD): 48 μg / eye of AD328. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] Fumagillol-1-adamantyl acetate (AD-3281) - Procedure A 100 mg fumagillol (350 μmol) was dissolved in 30 ml dichloromethane in a 50 ml round bottom flask. 68 mg (350 μmol) 1-adamantane acetic acid was added followed by 22 mg (180 μmol) 4-(dimethylamino)pyridine (DMAP). The solution was magnetically stirred and 96 mg (0.5 mmol) N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC HCl) was added. The solution was stirred for 8 hours. To the mixture, 100 ml dichloromethane and 25 ml methanol were added and the compound was washed in a separatory funnel with 0.1 N HCl (twice), then 0.1 N sodium bicarbonate (twice), then water. The resulting solution was dried over MgSO4, filtered and evaporated to dryness. It was then purified by column chromatography on silica gel using dichloromethane and increasing concentrations of methanol.

[0139] Fumagillol-1-adamantyl acetate (AD-3281) - Procedure B In a 50 ml round bottom flask, 57 mg fumagillol (200 μmol) was dissolved in 25 ml dichloromethane. 220 μmol of carboxyl bulk derivative was added, followed by 10 mg (80 μmol) 4-(dimethylamino)pyridine (DMAP). The solution was magnetically stirred and 80 mg (400 μmol) N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC HCl) was added. The solution was stirred for 8 hours. To the mixture, 100 ml dichloromethane and 25 ml methanol were added and the compound was washed in a separatory funnel with 0.1 N HCl (twice), then 0.1 N sodium bicarbonate (twice), then water. The resulting solution was dried over MgSO4, filtered and evaporated to dryness. It was then purified by column chromatography on silica gel using increasing concentrations of dichloromethane.

[0140] Fumagillol-1-adamantyl acetate (AD-3281) - Procedure C Synthesis of AD-3281 initiated by Fumagillol synthesis: 5 mmole Fumagillin (dicyclohexylamine salt) was dissolved in 200 ml ether in a 1000 ml round bottom flask. 200 ml NaOH (1N) was added and the solution was magnetically stirred at room temperature overnight. The reaction solution was transferred to a separatory funnel and the phases were separated. The upper ether layer was washed three times with 100 ml brine. It was dried with MgSO4 and evaporated to dryness. The synthesis of Fumagillol-1-adamantyl acetate (AD-3281) was carried out as follows: 1.0 mmole Fumagillol and 1.2 mmole 1-adamantane acetic acid were dissolved in 30 ml dichloromethane in a 50 ml round bottom flask. To the stirred solution was added 0.6 mmole 4-(dimethylamino)pyridine followed by 3 mmole N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixture was stirred overnight and then transferred to a separatory funnel. 70 ml dichloromethane and 50 ml methanol were added. The solution was washed twice with 50 ml HCl (0.1N), twice with 50 ml sodium bicarbonate (0.1N) and then with 50 ml water. It was dried over MgSO4 and evaporated to dryness. The residue was purified on a silica gel column using a dichloromethane:methanol gradient.

[0141] Fumagillol-1-adamantyl carboxylate (AD-3283) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol 1-adamantyl carboxylic acid.

[0142] Fumagillol-4-(1-adamantylamino)-4-oxobutanoate (AD-3286) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol 4-(1-adamantylamino)-4-oxobutanoic acid.

[0143] Fumagillol-4-(2-adamantylamino)-4-oxobutanoate (AD-3287) This compound was prepared using general procedure A from 100 μmol fumagillol and 110 μmol 4-(2-adamantylamino)-4-oxobutanoic acid.

[0144] Fumagillol-{[(2-adamantylamino)carbonothioyl]amino}acetate (AD-3295) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol {[(2-adamantylamino)carbonothioyl]amino}acetic acid.

[0145] Fumagillol-6-{[(1-adamantylamino)carbonothioyl]amino}hexanoate (AD-3294) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol {[(2-adamantylamino)carbonothioyl]amino}hexanoic acid.

[0146] Fumagillol-5-(2-adamantylamino)-5-oxopentanoate (AD-3290) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol (2-adamantylamino)-5-oxopentanoic acid.

[0147] Fumagillol-(3-bromo-1-adamantyl) acetate (AD-3301) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol (3-bromo-1-adamantyl)acetic acid.

[0148] Fumagillol-(3,5-dimethyl-1-adamantyl) acetate (AD-3302) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol (3,5-dimethyl-1-adamantyl)acetic acid.

[0149] Fumagillol-bicyclo[2.2.1]hept-5-ene-2-carboxylate (AD-3303) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol bicyclo[2.2.1]hept-5-ene-2-carboxylic acid.

[0150] Fumagillol-chloro(3,5,7-trimethyl-1-adamantyl)acetate (AD-3305) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol chloro(3,5,7-trimethyl-1-adamantyl)acetic acid.

[0151] Fumagillol-3,5,7-trimethyladamantane-1-carboxylate (AD-3306) This compound was prepared using general procedure A from 200 μmol fumagillol and 220 μmol 3,5,7-trimethyladamantane-1-carboxylic acid.

[0152] Enzyme inhibition assay of activity Quantitative MetAP2 activity assay by N-terminal truncation of the substrate 7-amino-4-methyl-coumarin was performed as follows: MetAP2 activity was measured using the fluorescent peptide substrate L-Met-AMC (Santa Cruz Biotechnology, Inc, sc-207807) as described in Garrabrant et al. (Angiogenesis 7:91-96, 2004). Briefly, reactions (100 μl final) were initiated by adding 0.5 μg MetAP2 or 5 μg HUVEC homogenate to reaction buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1 mM CoCl2, 1 mg / ml PEG6000, and 250 μM L-Met-AMC substrate. Assays were prepared in black flat-bottom 96-well microtiter plates and fluorescence was measured every 20 seconds over a period of 60 minutes at 25° C. using a microplate fluorometer (Bioteck, SynergyHT) with excitation and emission wavelengths set at 360 nm and 440 nm, respectively. As a control, inhibition of the enzyme was obtained by incubating the enzyme with TNP-470 for 15 minutes before addition of the substrate.

[0153] HUVEC proliferation assay Human umbilical vein endothelial cells (HUVEC) or human melanoma cell line (A375) were used to test the ability of compounds to inhibit endothelial cell proliferation. 1500 A375 cells / well or 2000 HUVEC cells / well, respectively, were seeded in a 96-well plate and allowed to adhere to the plate for a few minutes. Compounds were then added to the cells at concentrations ranging from 0.05 to 10 μM (<0.1% DMSO). Cells were then incubated for an additional 72 hours and MTT was performed (2-3 hours at 37°C). The dye was solubilized in DMSO and absorbance was determined at 570 nm.

[0154] The compounds described herein, among which the compounds of the present invention are: TIFF2025508363000033.tif68170TIFF2025508363000034.tif212170TIFF2025508363000035.tif191170TIFF2025508363000036.tif187170.

[0155] Experimental setup 1 Enzyme assays The inhibitory effects of the compounds were tested in the MetAp2 enzyme assay, and the most active compounds were compounds designated herein as AD-3281 and AD-3306 (FIGS. 1A-B, E-G).

[0156] Compound AD-3201 showed similar (and slightly better) activity compared to TNP-470 in inhibiting the catalytic activity of MetAp2. A concentration of 0.5 μM inhibited 75% of the activity, and 2.5 μM produced 97% inhibition (95% for TNP-470). Compound AD-3306 was nearly identical to TNP-470 (Figure 1C-D).

[0157] Cell assay results The ability to inhibit cell proliferation in endothelial and cancer cells was assessed using the MTT assay. The overall effects of the different compounds on HUVEC and A375 viability were relatively similar to TNP-470 and fumagillol (Figure 2A-C).

[0158] Laser induced CNV with AD3281 compared to EYLEA Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme that is overexpressed in activated endothelium. We have previously demonstrated that MetAp2 can be used as a target to regress choroidal neovascularization (CNV) in mice (1). Biochemical inhibition of MetAp2 can be performed with small molecules. Recently, we have identified another novel MetAp2 inhibitor, AD3281, which shows efficacy comparable to that of TNP-470. In this work, we evaluated the new MetAp2 inhibitor against Eylea, the standard of care for the treatment of "wet" AMD. To further stabilize and solubilize the compound in the vitreous.

[0159] method Laser-induced CNV was created by a previously described technique with some modifications. C57Bl / 6J mice (6–8 weeks old) were anesthetized by intraperitoneal injection of a mixture of 85% ketamine and 15% xylazine. A mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride was applied to both eyes to dilate the pupils. Lesions were induced around the optic nerve with a diode-pumped solid-state laser (0.1 s; spot size 100 μm; power 120 mW) through a Nidek slit-lamp delivery system while a handheld cover slide was used as a contact lens. Only lesions that developed a subretinal bubble or focal serous retinal detachment were used for the experiments. For this purpose, four burns per eye were performed, leaving space around the optic nerve head. Intravitreal injections of treatment drugs were performed using a PLI-100 Pico-Injector after laser treatment. After 7 days, mice were euthanized, eyes were enucleated and fixed in 4% paraformaldehyde for 60 min, the cornea and lens were removed, and the entire retina was carefully dissected from the choroid of the eyecup.

[0160] Blood vessels were labeled with a 1:200 dilution of Alexa Fluor 488 or isolectin IB4 conjugated with lectin-FITC. Eyecups were flat-mounted in Aquamount with the pleura facing down and the choroid facing up. Fluorescence images of the choroidal flat-mounts were acquired. CNV areas (expressed in μm2) in the choroidal flat-mounts were assessed using ImageJ software.

[0161] Compound concentration: 12 mg / ml (26 mM), PBS 0.2% Solutol 30% Hydroxypropyl beta cyclodextrin.

[0162] The results are shown in FIG.

[0163] Synthesis of AD-3281 The synthesis of fumagillol derivatives was carried out as follows: 5.0 mmole fumagillin (dicyclohexylamine salt) was dissolved in 200 ml ether in a 1000 ml round bottom flask. 200 ml NaOH (1N) was added and the solution was magnetically stirred at room temperature overnight. The reaction solution was transferred to a separatory funnel and the phases were separated. The upper ether layer was washed three times with 100 ml brine. It was dried over MgSO4 and evaporated to dryness. 1.0 mmole fumagillol and 1.2 mmole 1-adamantane acetic acid were dissolved in 30 ml dichloromethane in a 50 ml round bottom flask. To the stirred solution was added 0.6 mmole 4-(dimethylamino)pyridine followed by 3.0 mmole N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride. The mixture was stirred overnight and then transferred to a separatory funnel. 70 ml dichloromethane and 50 ml methanol were added. The solution was washed twice with 50 ml HCl (0.1 N), twice with 50 ml sodium bicarbonate (0.1 N), then with 50 ml water. It was dried over MgSO4 and evaporated to dryness. The residue was purified on a silica gel column using a dichloromethane:methanol gradient.

[0164] Characterization of AD-3281 H 1 -NMR (CDCl3) spectroscopic analysis (Bruker 500MH, Hebrew University of Jerusalem, service of the core facility) was performed to determine the molecular structures of the novel inhibitors.

[0165] High performance liquid chromatography (HPLC) for the determination of AD-3281 AD-3281 was detected as a peak using HPLC (System Gold Microbore, Beckman Coulter) at 15 min using 50% ACN in water. The flow rate was 1 ml / min with a sample injection volume of 10 μL onto a Kinetex 5u EVO column (C18, 150×4.6 mm). The temperature was set at 20° C. and detection was monitored at 205 nm wavelength.

[0166] cell culture Human umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA) and grown in PeproGrow endothelial cell medium supplemented with MacroV with 1% penicillin / streptomycin. MDA-MB-231 and A375 were purchased from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) medium with 1% penicillin / streptomycin. Cells were kept humidified and incubated at 37°C and 5% CO2. All cells were characterized as mycoplasma-free using the EZ-pcr Mycoplasma Test Kit (Biological Industries) before use.

[0167] Western blot Cell extracts were isolated with RIPA buffer containing a protease inhibitor cocktail (Sigma, S8820) for 30 min on ice. Lysates were centrifuged and supernatants were collected. The amount of isolated protein was determined using a BCA Protein Assay kit (Pierce™, Thermo Fisher Scientific, Cambridge, MA, USA). Proteins (15 μg protein) were separated by 12.5% ​​Tris-glycine SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Membranes were incubated in blocking buffer for 2 h and then incubated overnight at 4° C. with anti-MetAp2 or anti-MetAp1 antibodies, Ab134124 or Ab185540 (Abcam, Cambridge, UK), respectively, in TBST containing 5% BSA. Membranes were washed three times for 5 min in TBST and then incubated for 1 h with a 1:5000 dilution of goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Ab97080, Abcam). Ab49900 or Ab124979 for β-actin or cofilin, respectively (Abcam), were used as loading controls.

[0168] MetAp2 enzyme activity assay A375 and MDA-MB-231 were subcultured with trypsin, then centrifuged and counted. To obtain homogenates containing equal cell numbers, cells were resuspended in an appropriate volume of cold RIPA containing a protease inhibitor cocktail. Insoluble cell components were removed by centrifugation at 15,000 RPM for 10 minutes at 4°C. The protein content of the supernatant was determined according to the Bradford protein assay using BSA as a standard.

[0169] Enzyme assays were performed using 5 μg protein per sample as previously described. To test the inhibitory effect of AD-3281, AD-3281 was also added to 0.33 μg recombinant human methionine aminopeptidase 2 obtained from bio-techne (USA, 3795-ZN) under the same conditions, and samples were incubated with the inhibitor for 15 min at RT before adding the substrate. The reaction was initiated by adding 250 μM L-Met-AMC as a substrate. The fluorescence increase due to substrate degradation during the enzyme assay was measured every 20 s for 1 h 30 min at 37°C using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA). The assay was carried out in 96-well plates on ice and was performed in assay buffer (pH 7.5) containing 50 mM HEPES, 0.1 mM CoCl2, 100 mM NaCl and 1 mg / mL PEG6000 in a final volume of 100 μL.

[0170] Cell growth and proliferation assays A375 were seeded (2000 cells / well) and exposed to TNP-470 to adjust the concentration of AD-3281 (1-100 μM), and the cells were incubated at 37°C for 72 h. After incubation, MTT (Sigma Aldrich, St. Louis City, MO, USA) was added to each well (0.5 mg / mL) for viability detection, and incubated at 37°C and 5% CO2 for 40 min. The absorbance was measured at 540 nm using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA). The proliferation of HUVEC was measured under the same conditions. HUVEC were seeded (3000 cells / well) in 96-well plates.

[0171] To further verify the inhibitory activity of encapsulated AD-3281 in PLGA nanoparticles, HUVECs and A375s were exposed to different concentrations of nanoparticles corresponding to 50-1000 nM free AD-3281 (0.2-4.16 mg / ml nanoparticles) and empty nanoparticles were added as controls. The effect of AD-3281 on HUVEC growth was evaluated after 5 days.

[0172] Spheroid formation using multiwell arrays 3D Petri Dish 35-well arrays (Microtissues Inc., RI, USA) were used to create 2% agarose hydrogel microwells. Each well contained 5,000 cells of A375 cells. Thirty minutes after seeding, 1 ml of medium was added and the templates were incubated at 37°C for 24 hours. Spheroids were treated with 10 μM and 50 μM of free and encapsulated AD-3281, respectively, and after 96 hours of incubation, the viability of the spheroids was measured using the WST1 assay. The absorbance was measured at 450 nm using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA).

[0173] Animal models Animal studies were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine of the Hebrew University and followed the guidelines for the use of laboratory animals. Six to eight-week-old male Foxn1 nu mice were cultured at 5 × 10 6A375 and MDA-MB-231 cells / mouse were injected SC. Mice were treated IP with 30 mg / kg and 15 mg / kg AD-3281 every other day, and 15 mg / kg and 7.5 mg / kg AD-3281 every other day in A375 and MDA-MB-231 injected mice, respectively. Tumor growth was measured with digital calipers. At the end of the experiment after 18 or 15 days, mice were sacrificed, tumors were surgically removed, weighed, volume was measured, and histological description was performed. AD-3281 was dissolved in 10:10:80 chromophore:ethanol:saline.

[0174] Preparation of AD-3281-loaded nanoparticles Nanoparticles were prepared using emulsion evaporation method. AD-3281 was loaded into 100 mg of PLGA with 50:50 lactic acid-glycolic acid ratio. PLGA polymer was dissolved in 10 ml of ACN with 0.01% Tween 80, and AD-3281 was added to the dissolved polymer. The organic phase was added to the aqueous Solutol solution under stirring with a head stirrer for 10 minutes. The solution was then transferred to a dry round-bottom flask and connected to a rotary evaporator. After the solvent was completely evaporated, the nanoparticles were centrifuged at 10,000 rpm for 10 minutes, resuspended in 20% trehalose, and lyophilized.

[0175] Coumarin-6 as a labeling agent was loaded into PLGA nanoparticles using the same protocol. Empty vehicles were prepared using the same techniques and conditions without the addition of AD-3281 or Coumarin-6.

[0176] Physiochemical characterization of nanoparticles The size and charge of the nanoparticles were measured with a Malvern Zetasizer (Malvern Instruments, UK) and dynamic light scattering (DLS), which provided the average size and size range, and charge. All measurements were carried out at 25° C. The nanoparticles were dispersed in deionized distilled water.

[0177] To examine the morphology of the nanoparticles, transmission electron microscopy (TEM) images were taken. Samples were stained with 2.5% uranyl acetate. Briefly, 5 μl of sample was placed on a formvar / carbon coated copper 200 mesh grid (EMS) and mixed with 5 μl NV (NANOVAN, Nanoprobes, NY, USA) for 5-10 seconds, excess stain was wiped off, and the grid was dried. The grid was observed with a Jeol® JEM-1400 Plus TEM (Jeol®, Tokyo, Japan) equipped with an ORIUS SC600 CCD camera (Gatan®, Abingdon, UK) and the Gatan Microscopy Suite program (DigitalMicrograph, Gatan®, UK).

[0178] Cancer cell uptake To evaluate the uptake of PLGA nanoparticles by A375, 6-coumarin was used as a labeling agent and encapsulated in PLGA polymer. A375 were seeded at 300,000 cells / well in 6-well plates. After 24 hours, fluorescent nanoparticles (10 mg / ml) were suspended using an ultrasonic bath for 5 minutes and 75 μl was added to the seeded cells. After 0, 4, 7, and 24 hours, the cells were washed three times with cold phosphate-buffered saline (PBS) after incubation with the particles, then detached with trypsin, washed again, fixed with 4% paraformaldehyde, and analyzed by FACS (BD LSRFortessa™).

[0179] statistical analysis Results are expressed as mean ± SEM. Studies involving more than three groups were compared and analyzed using one-way analysis of variance (ANOVA), and significant differences were detected using Tukey's multiple comparison post-hoc test. Differences were considered statistically significant when p<0.05.

[0180] result Synthesis and characterization of AD-3281 Fifteen fumagillol derivatives with the fumagillol backbone and various side chains were synthesized and tested. Among the various analogs, AD-3281 represents the most potent compound. The AD-3281 synthesis started with fumagillin, and alkaline hydrolysis of fumagillin under basic conditions yielded fumagillol. The synthesis was then completed by esterification with 1-adamantaneacetic acid. (Figure 4A) shows the synthesis of AD-3281. The chemical structure of the new inhibitor was confirmed by mass spectrometry and H NMR (Figure 4B, C). The retention time of AD-3281 was determined by high performance liquid chromatography (HPLC) (Figure 4D).

[0181] MetAp2 expression and inhibition Basal cellular protein expression of MetAp2 was determined using Western blot. Compared to endothelial cells, cancer cells showed higher MetAp2 expression. Higher levels of MetAp2 were observed in A375 cancer cells compared to MDA-MB-231 (Figure 5).

[0182] To test the inhibition of MetAp2 enzyme activity in response to AD-3281, the compound was added to human recombinant MetAp2, and the fumagillol analogue TNP-470 was used as a positive control. N-terminal methionine cleavage activity was compared. We found that 2.5 μM AD-3281 led to a 95% reduction in MetAp2 activity compared to untreated controls.

[0183] MetAp2 activity in A375 and MDA-MB-231 was also measured under the same conditions after treatment with 2.5 μM AD-3281. In this assay, we found that AD-3281 reduced enzyme activity by 64% and 80%, respectively, in both cell lines during 1 hour (FIG. 6A). Significant inhibition was obtained after 10 minutes of AD-3281 exposure in both cancer cell lines (FIG. 6B).

[0184] AD-3281 affects proliferation of endothelial and cancer cells To test the antiangiogenic activity of AD-3281, MTT viability assay was performed to evaluate the inhibitory effect on endothelial cell proliferation. HUVEC cells were seeded and treated with 1 μM and 10 μM AD-3281. After 72 h of incubation, AD-3281 showed a significant reduction of HUVEC proliferation by 40% and 50%, respectively (Figure 7A).

[0185] To further examine the inhibitory activity of AD-3281 on cancer cell proliferation, MTT assays were also performed on A375 cancer cells. The cells were exposed to different concentrations of AD-3281 (1-100 μM), and AD-3281 caused a significant dose-dependent reduction in cancer cell proliferation by 37-63%, respectively (Figure 7B).

[0186] To increase the bioavailability to cells and to facilitate the degradation of the hydrophobic new compound, we formulated AD-3281 into PLGA nanoparticles using an emulsion evaporation method. We evaluated the effect of encapsulated AD-3281 on the proliferation of endothelial cells. HUVECs were treated qod with PLGA-AD-3281 (equivalent to 500 nM-1000 nM AD-3281) and the growth of HUVECs was evaluated after 5 days using an MTT assay. Treated cells showed about 31% inhibition of cell proliferation compared to cells exposed to vehicle only. Similarly, the effect of AD-3281 nanoformulation on the proliferation of A375 cancer cells was tested. After 72 hours, a dose-dependent inhibition was observed (22-40%) in cells treated with encapsulated AD-3281 (equivalent to 500 nM-1000 nM AD-3281). An incubation period of 96 h showed an effective reduction of 82–92% in A375 cell growth (Figure 7C,D). Nanoparticle interaction with cells was examined using an uptake assay, confirming polymer nanoparticle uptake by A375 at a level of approximately 20% after 7 h of incubation.

[0187] The inhibitory activity of AD-3281 on A375 cells was evaluated in 3D multicellular spheroids, which exhibit spatial cell-cell interactions, proliferation, and nutrient gradients due to drug diffusion similar to in vivo. Therefore, it was expected that 3D spheroids would be a better predictor of in vivo performance. Spheroid viability was measured using the WST1 assay; spheroids were exposed to 10 μM equivalents of AD-3281 and 50 μM free inhibitor. After 96 hours, spheroids treated with encapsulated inhibitor induced a 51% reduction in spheroid viability compared to spheroids treated with vehicle alone. Spheroids treated with free inhibitor did not show a significant reduction in spheroid viability (Figure 7E).

[0188] AD-3281 inhibits tumor growth In vivo studies were performed in two cancer xenograft models. Mice were cultured at 5×10 6 A375 cancer cells were injected subcutaneously (sc) and tumors grew to approximately 100 mm 3 Once the tumors reached a size of 100 mm, different intraperitoneal (IP) administrations of AD-3281 were initiated at 15 mg / kg and 30 mg / kg qod. After 8 days, AD-3281 caused a significant inhibition of about 70% in tumor growth in the treated group compared to the control, and tumor growth at day 18 was inhibited by 99% (Figure 8A). At the end, the average weight of tumors extracted from the non-treated group at day 18 was 1 g, and the treated group had an average weight of about 0.1 g (Figure 8C). Immunofluorescence staining of sections obtained from the extracted tumors revealed that MetAp2 and CD31 were co-localized and expressed in both treated and non-treated tumors. However, in treated samples, the co-localization seemed to be of a smaller degree, and in the non-treated group, intact blood vessels were more clearly observed (Figure 8F, G). Furthermore, AD-3281-treated tumors showed less cell proliferation than untreated tumors, as detected by the nuclear marker Ki-67 (FIG. 8H).

[0189] In the scMDA-MB-231 xenograft study, tumors were also inhibited when treated with 7.5 mg / kg and 15 mg / kg qod of AD-3281. Treatment was effective already after 5 days, and after 15 days, 97% volume inhibition was observed in the 7.5 mg / kg treatment group, while three tumors appeared to be completely eradicated in the 15 mg / kg treatment group (Figure 8B). Extracted tumors had an average weight of 1 g in the untreated group and average weights of 0.05 and 0.001 g in the 7.5 mg / kg and 15 mg / kg treatment groups, respectively (Figure 8D). Immunohistology of MDA-MB-231 tumor-bearing mice showed a trend toward reduced angiogenesis similar to melanoma.

[0190] Consideration Tumor growth and metastasis depend on the angiogenesis process, and inhibition of angiogenesis has been established as an important modality for tumor suppression and spread when combined with chemotherapy drugs. Although there are a wide variety of inhibitors that have reached clinical approval, many of them are not efficient enough or contain various side effects. Therefore, finding new angiogenesis inhibitors with high potency and drug-like properties may open new avenues for cancer treatment, especially considering the lower toxicity profile of these agents compared to chemotherapy.

[0191] Many studies have confirmed that MetAp2 plays an important role in the development of various types of cancer, and that specific downregulation of human MetAp2 expression by antisense oligonucleotides has a dominant effect on endothelial cell proliferation. In our recent paper, we found the involvement of MetAP2 in lymphangiogenesis and demonstrated the dual action of MetAp2 in the formation of both blood capillaries and lymphatic capillaries. Therefore, there is a rationale for positioning MetAp2 as a useful target for the treatment of primary cancer and metastatic disease.

[0192] One of the most effective known inhibitors of MetAp2 originates from the natural compound fumagillin. This small molecule was isolated from Aspergillus fumigatus Fresenius, and its synthetic analog O-(chloroacetylcarbamoyl) fumagillol or TNP-470 (also called AGM-1470) is one of the most potent analogs of fumagillin demonstrated in angiogenic cell models and was one of the first antiangiogenic small molecule drugs to undergo clinical trials. However, the development of this derivative was hampered by significant clinical drawbacks related to dose-dependent side effects. The high potency of fumagillol derivatives motivated us and others to search for new, safe compounds with high activity.

[0193] The selected lead compound, AD-3281, was the most active in inhibiting endothelial cell proliferation and abolished MetAp2 enzyme activity.

[0194] It has been established that MetAp2 is overexpressed in the tumor microenvironment, and most significantly in endothelium. We found that MetAp2 is also highly expressed in cancer cells at a level comparable to that in endothelial cells, and that AD-3281 significantly inhibited the proteolytic activity of MetAp2 in an enzyme assay (Figure 6). It was shown that inhibition of MetAp2 enzyme activity leads to angiogenesis blockade and inhibits tumor growth.

[0195] Furthermore, we aimed to verify the effect of AD-3281 on cell functionality in cancer cells and endothelial cells. It is known that MetAp2 inhibition in blood vessels regulates cell proliferation through cell cycle arrest in late G1 phase. We found that the presence of AD-3281 in human melanoma cell line A375 and primary endothelial cells HUVEC impaired cell proliferation within a concentration range of 1-100 μM. The relatively high concentration required for inhibition was attributed to poor water solubility. In many cases, transfer to particulate carriers may trigger cellular uptake via endocytosis, thus improving drug access to cells. Therefore, to increase cellular availability, we devised a nanoformulation based on PLGA biodegradable polymer with an average size of 194.0 ± 88.06 nm. Our previous studies have demonstrated the ability to enhance the bioavailability of lipophilic drugs and improve retention and stability in vivo using doses 10-100 times lower than the free drug. Similarly, the inhibitory effect of encapsulated AD-3281 on proliferation of endothelial cells and A375 cells showed a significant improvement in the bioavailability of AD-3281 at doses almost 20 times lower compared to the free molecule (Figure 7). Unlike 2D cell cultures, 3D cell assemblies spatially interact, providing more physiological relevance and better drug efficacy predictions compared to monolayer cell cultures. To generate robust spheroid growth, we use laboratory-designed 3D-printed multiwell arrays that can produce uniform 3D cultures with high reproducibility. In the spheroid model, encapsulated AD-3281 was found to significantly inhibit spheroid growth, while free AD-3281 showed a slight reduction in spheroid viability. This may be explained by the improved penetration of AD-3281 in a particulate PLGA nanocarrier format that allows for high capacity inter- and intracellular delivery.

[0196] The in vitro observations correlated with our in vivo results, and AD-3281 showed substantial antitumor effects, which were observed in two tumor-bearing mouse models. This indicates the broad range of biological effects of AD-3281. Histological analysis showed that endothelial cell remodulation was affected in AD-3281-treated mice. Immunofluorescence measurements of extracted murine tumor tissue sections showed that in the blood vessels of treated tissues, positive cells were more sporadically organized and less uniformly organized as blood vessels compared to the untreated group. Moreover, AD-3281 reduced cell proliferation in treated tumors, which was detected by the nuclear marker Ki-67.

[0197] Taken together, our data indicate that our new inhibitor AD-3281 has promising therapeutic properties in the treatment of cancer progression, and this novel inhibitor demonstrated an effective inhibitory role in angiogenesis and tumor progression in tumor-bearing mice. These significant results were mainly attributed to antiangiogenic and anticancer activity. Our results with AD-3281 highlight that AD-3281 is a great potential compound for treating highly vascularized tumors, which may be useful as a long-term maintenance drug to prevent tumor recurrence in cancer patients.

[0198] Experimental setup 2 cell culture Human umbilical vein endothelial cells were purchased from Lonza (Walkersville, MD, USA) and grown in PeproGrow endothelial cell medium supplemented with MacroV with 1% penicillin / streptomycin. MDA-MB-231 and A375 were purchased from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) medium with 1% penicillin / streptomycin. Cells were kept humidified and incubated at 37°C and 5% CO2. All cells were characterized as mycoplasma-free using the EZ-pcr Mycoplasma Test Kit (Biological Industries) before use.

[0199] Western blot Cell extracts were isolated with RIPA buffer containing a protease inhibitor cocktail (Sigma, S8820) for 30 min on ice. Lysates were centrifuged and supernatants were collected. The amount of isolated protein was determined using a BCA Protein Assay kit (Pierce™, Thermo Fisher Scientific, Cambridge, MA, USA). Proteins (15 μg protein) were separated by 12.5% ​​Tris-glycine SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA, USA). Membranes were incubated in blocking buffer for 2 h and then incubated overnight at 4° C. with anti-MetAp2 or anti-MetAp1 antibodies, Ab134124 or Ab185540 (Abcam, Cambridge, UK), respectively, in TBST containing 5% BSA. Membranes were washed three times for 5 min in TBST and then incubated for 1 h with a 1:5000 dilution of goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Ab97080, Abcam). Ab49900 or Ab124979 for β-actin or cofilin, respectively (Abcam), were used as loading controls.

[0200] MetAp2 enzyme activity assay A375 and MDA-MB-231 were subcultured with trypsin, then centrifuged and counted. To obtain homogenates containing equal cell numbers, cells were resuspended in an appropriate volume of cold RIPA containing a protease inhibitor cocktail. Insoluble cell components were removed by centrifugation at 15,000 RPM for 10 minutes at 4°C. The protein content of the supernatant was determined according to the Bradford protein assay with BSA as standard. Enzyme assays were performed using 5 μg protein per sample as previously described. To test the inhibitory effect of AD-3281, under the same conditions, AD-3281 was also added to 0.33 μg recombinant human methionine aminopeptidase 2 obtained from bio-techne (USA, 3795-ZN), and samples were incubated with the inhibitor for 15 minutes at RT before adding the substrate. The reaction was initiated by adding 250 μM L-Met-AMC as substrate. The fluorescence increase due to substrate degradation during the enzymatic assay was measured every 20 seconds using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA) for 1 hour and 30 minutes at 37° C. The assay was performed in a 96-well plate on ice in an assay buffer (pH 7.5) containing 50 mM HEPES, 0.1 mM CoCl2, 100 mM NaCl and 1 mg / mL PEG6000 in a final volume of 100 μL.

[0201] Cell growth and proliferation assays A375 were seeded (2000 cells / well) and exposed to TNP-470 to adjust the concentration of AD-3281 (1-100 μM). The cells were incubated at 37°C for 72 h. After incubation, MTT (Sigma Aldrich, St. Louis City, MO, USA) was added to each well (0.5 mg / mL) for viability detection and incubated at 37°C and 5% CO2 for 40 min. The absorbance was measured at 540 nm using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA). The proliferation of HUVEC was measured under the same conditions. HUVEC were seeded (3000 cells / well) in 96-well plates.

[0202] To further verify the inhibitory activity of encapsulated AD-3281 in PLGA nanoparticles, HUVECs and A375s were exposed to different concentrations of nanoparticles corresponding to 50-1000 nM free AD-3281 (0.2-4.16 mg / ml nanoparticles) and empty nanoparticles were added as controls. The effect of AD-3281 on HUVEC growth was evaluated after 5 days.

[0203] Tube formation assay To evaluate the angiogenic effects of AD-3281, an endothelial tube formation assay was performed. HUVECs were maintained in serum-free Dulbecco's modified Eagle's medium, harvested, and seeded onto 0.1% gelatin-coated 96-well plates and monitored for 12 hours using a microscope. Images were taken of six separate wells and analyzed using the Incucyte® Live-Cell Analysis Systems.

[0204] Spheroid formation using multiwell arrays 3D Petri Dish 35-well arrays (Microtissues Inc., RI, USA) were used to create 2% agarose hydrogel microwells. Each well contained 5,000 cells of A375 cells. Thirty minutes after seeding, 1 ml of medium was added and the templates were incubated at 37°C for 24 hours. Spheroids were treated with 10 μM and 50 μM of free and encapsulated AD-3281, respectively, and after 96 hours of incubation, the viability of the spheroids was measured using the WST1 assay. The absorbance was measured at 450 nm using a plate reader (Wallac 1420 VICTOR plate reader, Perkin-Elmer Life Sciences, USA).

[0205] Animal models Animal studies were approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine of the Hebrew University and followed the guidelines for the use of laboratory animals. Six to eight-week-old male Foxn1 nu mice were cultured at 5 × 10 6A375 and MDA-MB-231 cells / mouse were injected SC. Mice were treated IP with 30 mg / kg and 15 mg / kg AD-3281 qod, and 15 mg / kg and 7.5 mg / kg AD-3281 (10:10:80 Cremophor EL:ethanol:saline) qod for A375 and MDA-MB-231 injected mice, respectively. Tumor growth was measured with digital calipers. At the end of the experiment after 18 or 15 days, mice were sacrificed and tumors were surgically removed, weighed, volumes were measured, and histological descriptions were performed. Tumor tissues were excised at termination and analyzed by immunohistology on paraffin-embedded sections using anti-Ki-67 (Abcam, catalog no. ab15580), anti-CD31 (Abcam, catalog no. ab28364), and anti-MetAp-2 (Ab134124, Abcam, Cambridge, UK) using standard protocols ( Figure S ).

[0206] Preparation of AD-3281-loaded nanoparticles Nanoparticles were prepared using the emulsion evaporation method. AD-3281 was loaded onto 100 mg of PLGA (50:50, acid-terminated, Sigma-Aldrich, cat:719900) with a 50:50 lactic acid-glycolic acid ratio. PLGA polymer was dissolved in 10 ml of ACN with 0.01% Tween 80, AD-3281 was added to the dissolved polymer with stirring for 10 min using an overhead stirrer, and the organic phase was added to the aqueous Solutol solution. The solution was then transferred to a round-bottom flask and connected to a rotary evaporator. After the solvent was completely evaporated, the nanoparticles were centrifuged at 10,000 rpm for 10 min, resuspended in 20% trehalose, and lyophilized. (Steps are shown in Figure 9a). Coumarin-6 (0.1% w / w) was used to fluorescently label PLGA nanoparticles using the same protocol. An empty vehicle was prepared using the same techniques and conditions without the addition of any loading compound.

[0207] Physiochemical characterization of nanoparticles The size and charge of the nanoparticles were measured with a Malvern Zetasizer (Malvern Instruments, UK) and dynamic light scattering (DLS), which provided the average size and size range, and charge. All measurements were carried out at 25° C. The nanoparticles were dispersed in deionized distilled water.

[0208] To examine the morphology of the nanoparticles, transmission electron microscopy (TEM) images were taken. Samples were stained with 2.5% uranyl acetate. Briefly, 5 μl of sample was placed on a formvar / carbon coated copper 200 mesh grid (EMS) and mixed with 5 μl NV (NANOVAN, Nanoprobes, NY, USA) for 5-10 seconds, excess stain was wiped off, and the grid was dried. The grid was observed with a Jeol® JEM-1400 Plus TEM (Jeol®, Tokyo, Japan) equipped with an ORIUS SC600 CCD camera (Gatan®, Abingdon, UK) and the Gatan Microscopy Suite program (DigitalMicrograph, Gatan®, UK).

[0209] Cancer cell uptake To evaluate the uptake of PLGA nanoparticles by A375, 6-coumarin was used as a labeling agent and encapsulated in PLGA polymer. A375 were seeded at 300,000 cells / well in 6-well plates. After 24 hours, fluorescent nanoparticles (10 mg / ml) were suspended using an ultrasonic bath for 5 minutes and 75 μl was added to the seeded cells. After 0, 4, 7, and 24 hours, the cells were washed three times with cold phosphate-buffered saline (PBS) after incubation with the particles, then detached with trypsin, washed again, fixed with 4% paraformaldehyde, and analyzed by FACS (BD LSRFortessa™).

[0210] statistical analysis Results are expressed as mean ± SEM. Studies involving more than three groups were compared and analyzed using one-way analysis of variance (ANOVA), and significant differences were detected using Tukey's multiple comparison post-hoc test. Differences were considered statistically significant when p<0.05.

[0211] result MetAp2 expression and inhibition Basal cellular protein expression of MetAp2 was determined using Western blot. Compared to endothelial cells, cancer cells showed higher expression of MetAp2. Higher levels of MetAp2 were observed in A375 cancer cells compared to MDA-MB-231 (Figure 10a). Levels of MetAp1 were found to be similar in both cancer cell lines, with slightly higher expression levels in HUVEC cells (Figure 11).

[0212] To test the inhibition of MetAp2 enzyme activity in response to AD-3281, the compound was added to human recombinant MetAp2, and the fumagillol analogue TNP-470 was used as a positive control. N-terminal methionine cleavage activity was compared. We found that 2.5 μM AD-3281 led to a 95% reduction in MetAp2 activity compared to untreated controls (Figure 10b).

[0213] MetAp2 activity in A375 and MDA-MB-231 was also measured under the same conditions after treatment with 2.5 μM AD-3281. In this assay, we found that AD-3281 reduced enzyme activity by 64% and 80%, respectively, in both cell lines over the course of 1 hour (FIG. 10b). Significant inhibition was obtained after 10 minutes of AD-3281 exposure in both cancer cell lines.

[0214] AD-3281 affects proliferation of endothelial and cancer cells and disrupts tube formation To test the antiangiogenic activity of AD-3281, MTT viability assay was performed to evaluate the inhibitory effect on endothelial cell proliferation. HUVEC cells were seeded and treated with 1 μM and 10 μM AD-3281. After 72 h of incubation, AD-3281 showed a significant reduction of 40% and 50%, respectively, in HUVEC proliferation (FIG. 12a). To further examine the inhibitory activity of AD-3281 on cancer cell proliferation, MTT assay was also performed on A375 cancer cells. The cells were exposed to different concentrations of AD-3281 (1-100 μM), resulting in a significant dose-dependent reduction of 37-63%, respectively, in cancer cell proliferation (FIG. 11a). The antiangiogenic activity of AD-3281 was evaluated using a tube formation assay with HUVEC. AD-3281 at a concentration of 100 μM was shown to prevent tube formation, while 50 μM appeared to form narrower tubes compared to untreated controls, but did not prevent network formation (FIG. 12b, FIG. 13).

[0215] AD-3281 inhibits tumor growth In vivo studies were performed in two cancer xenograft models. Mice were cultured at 5×10 6 A375 cancer cells were injected subcutaneously (sc) and tumors grew to approximately 100 mm 3Once the tumors reached a size of 100 μg / mL, different intraperitoneal (IP) administrations of AD-3281 were initiated at 15 mg / kg and 30 mg / kg qod. After 8 days, AD-3281 caused a significant inhibition of about 70% in tumor growth in the treated group compared to the control, and tumor growth was completely inhibited (99%) at day 18 (Figure 14). At the end, the average weight of tumors extracted from the non-treated group at day 18 was 1 g, and the treated group had an average weight of about 0.1 g (Figure 14a). Immunofluorescence staining of sections obtained from the extracted tumors revealed that MetAp2 and CD31 were co-localized and expressed in both treated and non-treated tumors. However, in the treated samples, the co-localization seemed to be of a smaller degree, and in the non-treated group, intact blood vessels were more clearly observed (Figure 15). Furthermore, AD-3281-treated tumors showed less cell proliferation than untreated tumors, as detected by the nuclear marker Ki-67 (FIG. 15).

[0216] In the scMDA-MB-231 xenograft study, tumors were also inhibited when treated with AD-3281 at 7.5 mg / kg and 15 mg / kg qod. Treatment was effective already after 5 days, and after 15 days, 97% volume inhibition was observed in the 7.5 mg / kg treatment group, while three tumors appeared to be completely eradicated in the 15 mg / kg treatment group (Figure 14b). Extracted tumors had an average weight of 1 g in the untreated group. In the 7.5 mg / kg and 15 mg / kg treatment groups, the average weights were 0.05 and 0.001 g, respectively. Immunohistology of MDA-MB-231 tumor-bearing mice showed a trend toward reduced angiogenesis similar to melanoma (Figure 16). All treated mice had no signs of systemic adverse effects and no weight loss was detected (>10%).

[0217] Encapsulation of AD-3281 in PLGA nanoparticles To develop solid particles for solubilization and encapsulation of AD-3281 that could increase solubility and cellular bioavailability, we formulated AD-3281 into PLGA nanoparticles using an emulsion evaporation method (Figure 17). PLGA nanoparticles contained 0.1-0.3% (w / w) free AD-3281 as determined by high performance liquid chromatography (HPLC) (Figure 17). Particle size was determined by dynamic light scattering (DLS) and Z potential, the zeta potential of PLGA nanoparticles, was measured with a Zeta-sizer DLS (Malvern Instruments, UK). The morphology of the nanoparticles was determined using TEM and showed a uniform spherical structure. For TEM analysis, samples were placed on glow discharge carbon coated 300 mesh copper TEM grids (Ted Pella, Inc.). After blotting, samples were negatively stained with 2% aqueous solution of uranyl acetate for 30 seconds and air dried. The samples were examined with a FEI Tecnai 12 G2TWIN TEM operated at 120 kV.

[0218] Nanoparticle uptake We evaluated the uptake of polymeric nanoparticles by A375. A375 were incubated with 6-coumarin encapsulated PLGA nanoparticles for 0 min, 4 h, 7 h and 24 h. Cells were washed, resuspended in 0.2 ml FACS buffer (PBS containing 1% FCS and 0.1% sodium azide) and analyzed using flow cytometry (Beckman Coulter). Analysis was performed using FCS Express 5Flow research edition (De Novo software). The assay confirmed maximum uptake after 7 h of incubation by using a fluorescence activated cell analyzer (Figure 17).

[0219] Antiproliferative activity of AD-3281 encapsulated nanoparticles We evaluated the effect of encapsulated AD-3281 on the proliferation of endothelial cells. HUVECs were treated qod with PLGA-AD-3281 (corresponding to 500 nM-1000 nM AD-3281) and the growth of HUVECs was evaluated after 5 days using MTT assay. Treated cells showed about 31% inhibition of cell proliferation compared to cells exposed to vehicle only. Similarly, the effect of AD-3281 nanoformulation on the proliferation of A375 cancer cells was tested. After 72 hours, a dose-dependent inhibition was observed (22-40%) in cells treated with encapsulated AD-3281 (corresponding to 500 nM-1000 nM AD-3281). A 96 hour incubation period showed an effective reduction of 82-92% in the growth of A375 cells (Figure 18b). The interaction of nanoparticles with cells was examined using an uptake assay, which confirmed polymer nanoparticle uptake by A375 at a level of about 20% after 7 h of incubation (Figure 18b).

[0220] AD-3281 inhibits the growth of A375 spheroids The inhibitory activity of AD-3281 on A375 cells was evaluated in 3D multicellular spheroids, which exhibit spatial cell-cell interactions, proliferation, and nutrient gradients due to drug diffusion as in vivo. Therefore, it was expected that 3D spheroids would be a better predictor of in vivo performance. Spheroid viability was measured using the WST1 assay; spheroids were exposed to 10 μM equivalents of AD-3281 and 50 μM free inhibitor. After 96 hours, spheroids treated with encapsulated inhibitor induced a 51% reduction in spheroid viability compared to spheroids treated with vehicle alone. Spheroids treated with free inhibitor did not show a significant reduction in spheroid viability (Figure 18b).

[0221] Consideration Tumor growth and metastasis depend on the angiogenesis process. Inhibition of angiogenesis has been established as an important modality for tumor suppression and spread when combined with chemotherapy drugs. Although there are a wide variety of inhibitors that have reached clinical approval, many of them are not efficient enough or contain various side effects. Therefore, finding new angiogenesis inhibitors with high potency and drug-like properties may open new avenues for cancer treatment, especially considering the lower toxicity profile of these agents compared to chemotherapy.

[0222] Many studies have confirmed that MetAp2 plays an important role in the development of various types of cancer, and that specific downregulation of the expression of human MetAp2 by antisense oligonucleotides has a dominant effect on endothelial cell proliferation. In our recent paper, we found the involvement of MetAP2 in lymphangiogenesis and demonstrated the dual action of MetAp2 in the formation of both blood capillaries and lymphatic capillaries. Therefore, there is a rationale for positioning MetAp2 as a useful target for the treatment of primary cancer and metastatic disease.

[0223] One of the most effective known inhibitors of MetAp2 originates from the natural compound fumagillin. This small molecule was isolated from Aspergillus fumigatus Fresenius, and its synthetic analog O-(chloroacetylcarbamoyl) fumagillol or TNP-470 (also called AGM-1470) is one of the most potent analogs of fumagillin demonstrated in angiogenic cell models and was one of the first antiangiogenic small molecule drugs to undergo clinical trials. However, the development of this derivative was hampered by significant clinical drawbacks related to dose-dependent side effects. The high potency of fumagillol derivatives motivated us and others to search for new, safe compounds with high activity.

[0224] The present inventors synthesized a series of derivatives based on the structure of fumagillin. The selected lead compound AD-3281 is the most active in inhibiting endothelial cell proliferation and abolishes MetAp2 enzyme activity. It has been established that MetAp2 is overexpressed in the tumor microenvironment, and most significantly in endothelium. The present inventors found that MetAp2 is also highly expressed in cancer cells at a level comparable to that of endothelial cells, and that AD-3281 significantly inhibited the proteolytic activity of MetAp2 in enzyme assays (Figure 10). It was shown that inhibition of MetAp2 enzyme activity leads to angiogenesis blockade and inhibits tumor growth.

[0225] Furthermore, we aimed to verify the effect of AD-3281 on cell functionality in cancer cells and endothelial cells. It is known that MetAp2 inhibition in vascular endothelium regulates cell proliferation through cell cycle arrest in late G1 phase. We found that the presence of AD-3281 in human melanoma cell line A375 and primary endothelial cells HUVEC impaired cell proliferation within the concentration range of 1-100 μM and endothelial tube formation at 100 μM. The in vitro observations correlated with our in vivo results, and AD-3281 showed substantial antitumor effects, which were observed in two tumor-bearing mouse models. This indicates a wide range of biological effects of AD-3281. Histological analysis showed that endothelial cell remodulation was affected in mice treated with AD-3281. Immunofluorescence analysis of extracted murine tumor tissue sections showed that positive cells were more sporadically and less uniformly organized as blood vessels in treated tissues compared to untreated groups. Moreover, AD-3281 reduced cell proliferation in treated tumors, as detected by the nuclear marker Ki-67.

[0226] Given the promising in vivo results, AD-3281 may be a great candidate for further development towards clinical trials. However, the desired clinically translatable formulation would be one that maximizes AD-3281 solubilization while using minimal amounts of organic solvents that may lead to adverse effects. Therefore, we developed a nanoparticle-based encapsulation of the active compound, utilizing an established emulsification technique that produces biodegradable PLGA nanoparticles loaded with AD-3281 (Figure 17) with an average size of about 200 nm, which is typically used for injectable particles for cancer therapy. Our previous studies have demonstrated the ability to enhance the bioavailability of lipophilic drugs and improve retention and stability in vivo, using doses 10-100 times lower than the free drug. We aimed to explore cellular availability in monolayer cultures and in 3D multicellular cultures. In many cases, the transfer of a molecule from its free form to a particulate carrier may trigger cellular uptake via endocytosis, thus improving drug access to cells. Consistently, we found that the relatively high concentrations required to inhibit cell proliferation in vitro were due to limited transport into the cells. The inhibitory effect of encapsulated AD-3281 on proliferation of endothelial cells and A375 cells showed a significant improvement in the bioavailability of AD-3281 at a dose nearly 20-fold lower than the free molecule (Figure 18). Unlike 2D cell cultures, 3D cell assemblies spatially interact, providing more physiological relevance and better drug efficacy predictions compared to monolayer cell cultures. To generate robust spheroid growth, we use laboratory-designed 3D-printed multiwell arrays that can generate uniform 3D cultures with high reproducibility. In the spheroid model, encapsulated AD-3281 was found to significantly inhibit spheroid growth, while free AD-3281 showed a slight reduction in spheroid viability. This may be explained by the improved penetration of AD-3281 in a particulate PLGA nanocarrier format that allows for high capacity inter- and intracellular delivery.

[0227] Taken together, our data indicate that our new inhibitor AD-3281 has promising therapeutic properties in the treatment of cancer progression, and this novel inhibitor demonstrated an effective inhibitory role in angiogenesis and tumor progression in tumor-bearing mice. These significant results were mainly attributed to antiangiogenic and anticancer activity. Our results with AD-3281 highlight that AD-3281 is a great potential compound for treating highly vascularized tumors, which may be useful as a long-term maintenance drug to prevent tumor recurrence in cancer patients.

[0228] ERG studies in mice revealed high safety of the injected compound up to 4.6 μg / μl, nearly four times the active dose in the CNV model (FIG. 19).

Claims

1. General formula (I): wherein R is a carbocyclyl containing two or more ring structures and is selected from adamantyl or a derivative thereof; norbornanyl or a derivative thereof; norbornenyl or a derivative thereof; norbornenyl or a derivative thereof; steroidyl or a derivative thereof; camphoryl or camphor derivatives; camphenyl or camphene derivatives; tricyclo(2.2.1.0(2,6))heptanyl or a derivative thereof; or tetracyclo[3.2.0.0(2,7).0(4,6)]heptanyl or a derivative thereof.

2. 2. The compound of claim 1, wherein the variant R is attached to an oxygen atom of the compound of formula (I) directly or via a spacer or linker moiety X.

3. The compound has formula (IA): (IA) 3. The compound of claim 2, wherein R is the carbocyclyl and X is a functional group bridging the carbocyclyl R and the oxygen atom of the structure of formula (I).

4. 4. The compound of claim 3, wherein X is absent and R is directly bonded to the oxygen atom.

5. The compound of structure (I) or (IA) may be a compound of structure (IB): (IB) 10. The compound of claim 1, wherein R is the carbocyclyl.

6. The compound of formula (I) or (IA) is a compound designated (IC): (IC) 6. The compound according to any one of claims 1 to 5, wherein R is the carbocyclyl.

7. The compound of formula (I) or (IA) has the formula (ID): (ID) 2. The compound of claim 1, wherein R is a carbocyclyl.

8. The compound of formula (I) or (IA) may be of formula (IE): (IE) 2. The compound of claim 1, wherein R is a carbocyclyl.

9. The compound of formula (I) or (IA) may be a compound of formula (IF): (IF) 2. The compound of claim 1, wherein R is a carbocyclyl.

10. The compound of formula (I) or (IA) may be a compound of formula (IG): (IG) 2. The compound of claim 1, wherein R is a carbocyclyl.

11. The compound of formula (I) or (IA) may be of formula (IH): (IH) 2. The compound of claim 1, wherein R is a carbocyclyl.

12. R is structure (A1) or (A2): Adamantyl of wherein, for each of structures (A1) and (A2), independently: X is a functional group or atom that connects the adamantyl group with the oxygen atom of the compound of formula (I); R １ , R ２ and R ３ each independently of the other is —H, or a halide (F, Cl, Br, I), —C １ ~C ３ Alkyl, —C ２ ~C ４ Alkenyl, -C １ ~C ３ Alkyl halide, hydroxyl, carboxyl, carboxylate, -C ６ ~C １０ selected from aryl, hydroxyalkyl, sulfate, sulfonate, sulfonamide, and sulfonic acid; The compound of claim 1.

13. The adamantyl has the structure (B): (where, R １ , R ２ and R ３ Each of is, independently of the other, H or —C １ ~C ３ Alkyl, —C ２ ~C ４ Alkenyl, -C １ ~C ３ Alkyl halide, hydroxyl, carboxyl, carboxylate, -C ６ ~C １０ selected from aryl, hydroxyalkyl, and halide; indicates the point of attachment to the oxygen atom of the structure of formula (I).

14. The adamantyl has the structure (C): (where, R １ , R ２ and R ３ Each of is, independently of the other, H or —C １ ~C ３ Alkyl, —C ２ ~C ４ Alkenyl, -C １ ~C ３ Alkyl halide, hydroxyl, carboxyl, carboxylate, -C ６ ~C １０ selected from aryl, hydroxyalkyl, and halide; indicates the point of attachment to the oxygen atom of the structure of formula (I).

15. The adamantyl has the structure (D): (where, R １ , R ２ and R ３ Each of is, independently of the other, H or —C １ ~C ３ Alkyl, —C ２ ~C ４ Alkenyl, -C １ ~C ３ Alkyl halide, hydroxyl, carboxyl, carboxylate, -C ６ ~C １０ selected from aryl, hydroxyalkyl, and halide; indicates the point of attachment to the oxygen atom of the structure of formula (I).

16. The compound of formula (I) is a compound designated herein: AD-3302; AD-3281; AD-3306; AD-3283; AD-3305; AD-3301; AD-3295; AD-3294; AD-3286; AD-3287; and AD-3290 2. The compound of claim 1 selected from:

17. The carbocyclyl has the structure (E): Or, the carbocyclyl has structure (F): wherein said -C 1 -C 5 alkylene is selected from methylene, ethylene, propylene, butylene and pentylene; or The carbocyclyl has the structure (G):

10. The compound of claim 1, wherein the dashed line designates the bond of attachment to the oxygen atom.

18. A composition comprising the compound of claim 1.

19. 10. A pharmaceutical composition comprising one or more compounds of claim 1 for administration by oral, buccal, sublingual, rectal, nasal, topical, transdermal, intravaginal, parenteral, subcutaneous, intramuscular, intravenous or intradermal administration.

20. 10. A pharmaceutical composition comprising one or more compounds according to claim 1 for use in a method for treating or preventing a disease associated with MetAp2 activity.

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