Metap2 inhibitors and methods of treating obesity

Abstract

To provide modified or polymer conjugated MetAP2 inhibitors.SOLUTION: The present invention relates to methods of preventing, inducing, causing or increasing weight loss, treating obesity and / or treating metabolic syndrome utilizing the modified or polymer conjugated MetAP2 inhibitors. The present invention also relates to methods of improving insulin sensitivity and glycemic control, reducing insulin levels and / or improving leptin sensitivity utilizing the modified or polymer conjugated MetAP2 inhibitors.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 810,468, filed on April 10, 2013, and U.S. Provisional Patent Application No. 61 / 925,918, filed on January 10, 2014. The entire content of each of these applications is hereby incorporated by reference into this specification.

Background Art

[0002] Obesity is a chronic disease and a major health concern in modern society. According to the U.S. Centers for Disease Control and Prevention (CDC), the United States is in the midst of an obesity epidemic. In the United States, approximately 65% of adults are overweight, 30% of adults are obese, and more than 5 million adults are classified as morbidly obese. Additionally, more than 10 million are near that criterion and at risk of obesity - related health problems. This problem is on the increase, and obesity in children and adolescence has doubled in the past 20 years.

[0003] Existing methods for treating obesity include diet and exercise; very low calorie diet therapy; behavioral therapy; drug therapy including appetite suppressants, thermogenic drugs, and agents that inhibit food absorption; mechanical devices such as jaw wiring, waist cords, and balloons; and surgery. However, these existing treatment methods are not very effective. Adherence to energy-restricted diet therapy is a problem, generally ineffective, and drug therapy has only limited effectiveness in long-term weight management. In many cases, toxicity and side effects are preventing the development of potential anti-obesity drug candidates. The metabolic syndrome (Sutherland et al., Metabolic Syndrome and Related Disorders 2:82-104 (2004); Esposito et al., Nutr. Metab. Cardiovasc. Dis. 14:228-232 (2004)) is associated with obesity and is characterized by a cluster of metabolic risk factors including: 1) abdominal obesity (excessive adipose tissue in and around the abdomen); 2) atherogenic dyslipidemia (high triglycerides; low HDL cholesterol and high LDL cholesterol); 3) elevated blood pressure; 4) insulin resistance or impaired glucose tolerance; 5) a prothrombotic state (e.g., high fibrinogen or high plasminogen activator inhibitor-1 in the blood); and 6) a proinflammatory state (e.g., elevated CRP in the blood). The metabolic syndrome is increasing in prevalence in developed countries and is closely associated with the risk of coronary heart disease (Malik et al., Circulation 110:1245-1250 (2004); Irabarren et al., J. Am. Coll. Cardiol. 48:1800-1807 (2006)).

[0004] Cardiovascular metabolic syndrome includes obesity-related metabolic disorders and atherosclerosis. Cardiovascular metabolic disorders also promote the calcification of arteries and heart valves, and as a result, can lead to acute myocardial infarction and aortic stenosis, which are devastating clinical complications. Furthermore, diabetes causes chronic kidney disease, which also leads to ectopic calcification of the cardiovascular system and acute myocardial infarction. Collectively, some of the major components of cardiovascular metabolic syndrome that develop through interrelated mechanisms promote each other through local or systemic inflammation. Additionally, the lack of patient compliance with prescribed drug therapies presents a major challenge in the global insurance healthcare community. In the United States alone, the estimated avoidable healthcare expenditure in 2009 was $300 billion. With the expiration of patents for large new drugs, the depletion of distribution routes, and cost containment by users, closing this compliance gap is "a must do" for pharmaceutical companies.

[0005] Therefore, there is a need for novel compounds and methods for inducing, triggering, and / or increasing weight loss, and for treating obesity and metabolic syndrome. The present invention addresses these needs. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0006] The present invention provides a method for inducing or causing weight loss in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention for inducing or causing weight loss. In certain embodiments, the subject is overweight or obese. In certain embodiments, inducing or causing weight loss is increasing weight loss.

[0007] The present invention also provides a method for treating obesity, metabolic syndrome and / or related co-morbidities in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analogue or derivative thereof, according to a rational schedule, to treat or alleviate these diseases and conditions.

[0008] The present invention also provides a method for improving insulin sensitivity and blood glucose control, reducing insulin levels and / or improving leptin sensitivity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention, or a pharmaceutically acceptable salt, prodrug, metabolite, analogue or derivative thereof, according to a rational schedule, to treat or alleviate these diseases and conditions.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In this specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, this specification, including definitions, will control. Furthermore, the materials, methods and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Mode for Carrying Out the Invention

[0011] The compound of the present invention The present invention provides a drug conjugate composition comprising a modified active moiety, a conjugate moiety, and a cleavable linker, wherein cleavage of the linker occurs substantially in a target tissue to produce a modified active moiety with reduced efflux from the target tissue compared to the unmodified active moiety. The present invention also provides a composition comprising a modified active moiety.

[0012] The conjugate moiety used depends on the biological requirements, such as the pharmacokinetic and pharmacodynamic properties of the active moiety, and knowledge of the disease state, in addition to the physicochemical properties of both the conjugate moiety and the active moiety. One of ordinary skill in the art will be able to select an appropriate conjugate moiety based on the above considerations. The conjugate moiety is used to deliver a small molecule active moiety, or an active moiety of a larger molecule, such as a protein, peptide, or oligonucleotide.

[0013] The conjugate moiety improves the delivery of the active moiety to the target. The conjugate moiety is selected to maximize the bioavailability of the active moiety; optimize the onset, duration, and rate of delivery of the active moiety; and maintain the concentration of the active moiety within the target tissue in the therapeutic range for as long as required for effective treatment. The conjugate moiety can also assist in minimizing the harmful side effects of the active moiety. Thus, the conjugate moiety prolongs the pharmacological activity of the active moiety, stabilizes labile active moieties from chemical and proteolytic degradation, minimizes side effects, increases solubility, and delivers the active moiety to specific cells or tissues.

[0014] Other characteristics to be considered for the complex moiety are that the complex moiety has minimal immunogenicity and toxicity, or is non-immunogenic and non-toxic. The molecular weight of the complex moiety should be large enough to avoid rapid removal by renal ultrafiltration and small enough to prevent unwanted accumulation in the body. In certain embodiments, the complex moiety is hydrophilic and biodegradable. Non-biodegradable complex moieties are also suitable in the compositions and methods of the present invention. The complex moiety should be able to carry the required amount of the active moiety and prevent premature metabolism of the active moiety during its movement to the target tissue.

[0015] Preferred complexes include synthetic polymers and natural product-related polymers, including all forms of polymers, namely peptides, polysaccharides, polynucleic acids, antibodies and aptamers. In a preferred embodiment, the complex is a synthetic polymer. Preferred polymers of the present invention are described in U.S. Patent Nos. 4,997,878 to Bock et al., 5,037,883 to Kopecek et al., 5,258,453 to Kopecek et al., 6,464,850 to Zhang et al., and 6,803,438 to Brochini et al., the entire contents of each of which are incorporated herein by reference. Additional preferred polymers are described in Subr et al., J Controlled Release, 18, 123-132 (1992). In some embodiments, the method of synthesizing the polymer can result in the coupling of two or more polymer chains and can increase the weight average molecular weight of the polymer complex. When this coupling occurs, it is further understood that the bond is biodegradable.

[0016] The active moiety can be any compound or molecule that provides a therapeutic effect in a subject. In certain embodiments, the compound or molecule has a molecular weight of 2000 Daltons or less, 1500 Daltons or less, 1000 Daltons or less, 500 Daltons or less, or 250 Daltons or less. In certain embodiments, the compound or molecule is a MetAP2 inhibitor. In certain embodiments, the compound or molecule is fumagillin, fumagillol, or an analog, derivative, salt, or ester thereof. The compound or molecule selected depends on the condition or disease being treated. In certain embodiments, two or more active moieties can be used. In certain embodiments, an active moiety and an inert “capping” moiety can be used. In certain embodiments, the condition being treated is obesity. In the compositions of the present invention, the conjugate moiety is linked to the active moiety via a linker. Any linker structure known in the art can be used to link the modified active moiety to the conjugate moiety. The linker used depends on the physiological state of the target tissue, the properties of the active moiety being optimized, and the cleavage mechanism. D’Souza et al. review various linkers, including those that function via protein cleavage: “Release from Polymeric Prodrugs: Linkages and Their Degradation” J. Pharm. Sci., 93, 1962 - 1979 (2004). Blencoe et al. describe various self-immolative linkers: “Self-immolative linkers in polymeric delivery systems” Polym. Chem. 2, 773 - 790 (2011). Ducry et al. review linkers in “Antibody-Drug Conjugates: Linking Cytotoxic Payloads to Monoclonal Antibodies” Bioconj. Chem. 21, 5 - 13 (2010).Peptide linkers suitable for cleavage by matrix metalloproteinase (MMP) are described in Chau et al., "Antitumor efficacy of a novel polymer-peptide-drug conjugate in human tumor xenograft models", Int. J. Cancer 118, 1519-1526 (2006), and Chau et al., US Patent Application Publication No. 2004 / 0116348. Chemical properties of other linkers suitable for the compositions of the present invention are shown in Shiose et al., Biol. Pharm. Bull. 30(12) 2365-2370 (2007); Shiose et al., Bioconjugate Chem. 20(1) 60-70 (2009); US Patent No. 7,553,816 to Senter; US Patent No. 7,223,837 to De Groot; US Patent No. 6,759,509 to King; US Patent No. 6,835,807 to Susaki; US Patent No. 6,436,912 to Susaki; and US Patent No. 7,943,569 to Gemeinhart;

[0017] In certain embodiments, the linker is a peptide linker. Preferred peptide linkers are described in U.S. Patent No. 6,835,807 to Susaki et al., U.S. Patent No. 6,291,671 to Inoue et al., U.S. Patent No. 6,811,996 to Inoue et al., U.S. Patent No. 7,041,818 to Susaki et al., U.S. Patent No. 7,091,186 to Senter et al., and U.S. Patent No. 7,553,816 to Senter et al., the entireties of each of which are incorporated by reference. Additional preferred peptides and their cleavage are described in Shiose et al., Biol. Pharm. Bull. 30(12)2365 - 2370(2007) and Shiose et al., Bioconjugate Chem. 20(1)60 - 70(2009). Peptide linkers suitable for cleavage by matrix metalloproteinase (MMP) are described in Chau et al., “Antitumor efficacy of a novel polymer - peptide - drug conjugate in human tumor xenograft models” Int. J. Cancer 118,1519 - 1526(2006), and Chau et al., U.S. Patent Application Publication No. 2004 / 0116348.

[0018] The linker may be cleaved by any mechanism known in the art. For example, the linker may be designed for protein cleavage or intracellular protein cleavage. In certain embodiments, the linker is designed such that there is no cleavage of the linker in plasma, or the cleavage rate in plasma is very low. Preferred linker structures are described in further detail below.

[0019] In certain embodiments, the linker has a structure that is preferentially cleaved in diseased tissue. Since hydrolases are present in both normal and diseased tissue, the linker should be cleaved by hydrolases that are more active and / or more present in diseased tissue. For example, tumors generally have an upregulated metabolic rate and, in particular, overexpress proteases including cathepsin. The upregulation and role of proteases in cancer are described in Mason et al., Trends in Cell Biology 21, 228-237 (2011).

[0020] In certain embodiments, the class of modified active moieties are those that bind irreversibly to their target; i.e., after dissociation from the complex, the active moiety covalently binds to the biochemical target. Once bound, the active moiety does not diffuse or transport extracellularly. In the case of irreversible binding, for target-directedness to occur, the rate at which the small molecule binds to the target, K rev1 should be significant relative to the rate of small molecule efflux, k sm-1 . If the rate of efflux is high relative to small molecule binding, an equilibrium of the small molecule is established between plasma and the intracellular compartment, and the advantage of intracellular delivery over extracellular delivery is lost.

[0021] In other embodiments, the class of modified active moieties are those that bind reversibly to their target. In the case of reversible binding, for target-directedness to occur, the equilibrium constant for small molecule binding to the target, K = k rev1 / k rev-1 should be large, and the "on-rate", k rev1 should be greater than the rate of small molecule efflux, k sm-1should be high relative to. If the rate of efflux is high relative to the rate of small molecule binding, an equilibrium of the small molecule is established between plasma and the intracellular compartment, and the advantage of intracellular delivery over extracellular delivery is lost. Such relationships are schematically shown below. In the formula: [PC] is the concentration of the polymer complex; [SM] is the concentration of the free small molecule; plasma is the plasma concentration; intracellular (icell) is the intracellular concentration; intracellular (icell)-target is the small molecule reversibly bound to the intracellular target; and inert is the inert metabolite of the small molecule. In certain embodiments, k rev-1 When = 0, that moiety binds irreversibly to the target.

[0022] [Chemical formula]

[0023] In other embodiments, the class of modified active moieties are moieties having a very high equilibrium constant and a high "on-rate" for efflux. In other embodiments, the class of modified active moieties are moieties that undergo intracellular metabolism at a high rate relative to efflux.

[0024] In certain embodiments, modification of the active moiety is achieved by using a linker having a structure such that upon cleavage, a fragment of the linker remains attached to the active moiety. The fragment can alter any of the molecular weight, hydrophobicity, polar surface area, or charge of the active moiety, thereby generating a modified active moiety with reduced efflux from the target cell compared to the unmodified active moiety. For example, by coupling a MetAP2 inhibitory active moiety via a linker described herein, a complex is obtained that generates an active moiety (modified active moiety) to which a fragment of the linker is attached upon cleavage of the linker. The modified active moieties described herein can have reduced efflux from the cell compared to the unmodified active moiety, resulting in a modified active moiety with better efficacy relative to the parent small molecule, and better pharmacokinetic profiles relative to the parent small molecule.

[0025] The present invention is as follows:

[0026] [Chemical formula]

[0027] (In each occurrence, independently, R4 is hydrogen or C1-C6 alkyl; R5 is hydrogen or C1-C6 alkyl; R6 is C2-C6 hydroxyalkyl; Z is -NH-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-L or -NH-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-Q-X-Y-C(O)-W; AA1 is glycine, alanine, or H2N(CH2)mCO2H, where m is 2, 3, 4, or 5; AA2 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA3 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA4 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA5 is a bond, or glycine, valine, tyrosine, tryptophan, phenylalanine, methionine, leucine, isoleucine, or asparagine; AA6 is a bond, or alanine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine, or H2N(CH2)mCO2H, where m is 2, 3, 4, or 5; L is -OH, -O-succinimide, -O-sulfosuccinimide, alkoxy, aryloxy, acyloxy, aroyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, -NH2, -NH(C2-C6 hydroxyalkyl), halide, or perfluoroalkyloxy; Q is NR, O, or S; X is M-(C(R)2) p-M-J-M-(C(R)2) p -M-V, where M is a bond or C(O); J is a bond or ((CH2) q Q) r , C5-C8 cycloalkyl, aryl, heteroaryl, NR, O, or S; Y is NR, O, or S; R is hydrogen or alkyl; V is a bond or:

[0028]

Chemical formula

[0029] ; R 9 is alkyl, aryl, aralkyl, or a bond; or R 9 together with Y forms a heterocycle; R 10 is an amide or a bond; R 11 is hydrogen or alkyl; W is a MetAP2 inhibitor moiety or alkyl; x ranges from 1 to about 450; y ranges from 1 to about 30; n ranges from 1 to about 50; p ranges from 0 to 20; q is 2 or 3; and r is 1, 2, 3, 4, 5, or 6) Provide a complex having a linker with the structure of .

[0030] In certain embodiments, R4 is C1-C6 alkyl. In certain embodiments, R4 is methyl. In certain embodiments, R5 is C1-C6 alkyl. In certain embodiments, R5 is methyl. In certain embodiments, R6 is 2-hydroxyethyl, 2-hydroxypropyl, or 3-hydroxypropyl. In certain embodiments, R6 is 2-hydroxypropyl.

[0031] In certain embodiments, the compound has a molecular weight of less than about 60 kDa. In other embodiments, the molecular weight is less than about 45 kDa. In other embodiments, the molecular weight is less than about 35 kDa.

[0032] In certain embodiments, the ratio of x to y ranges from about 30:1 to about 3:1. In other embodiments, the ratio of x to y ranges from about 19:2 to about 7:2. In certain embodiments, the ratio of x to y ranges from about 9:1 to about 4:1. In certain embodiments, the ratio of x to y is about 11:1. In certain embodiments, the ratio of x to y is about 9:1. In certain embodiments, the ratio of x to y is about 4:1.

[0033] In certain embodiments, Z is -hydrogen-AAi-AA2-AA3-AA4-AA5-AA6-C(O)-L. In certain embodiments, L is methoxy, ethoxy, pentafluorophenyloxy, phenyloxy, acetoxy, fluoride, chloride, methoxycarbonyloxy; ethoxycarbonyloxy, phenyloxycarbonyloxy, 4-nitrophenyloxy, trifluoromethoxy, pentafluoroethoxy, or trifluoroethoxy. In certain embodiments, L is 4-nitrophenyloxy.

[0034] In certain embodiments, Z is -hydrogen-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-Q-X-Y-C(O)-W. In certain embodiments, AA1 is glycine. In certain embodiments, AA2 is glycine. In certain embodiments, AA3 is glycine. In certain embodiments, AA4 is glycine or phenylalanine. In certain embodiments, AA5 is leucine, phenylalanine, valine or tyrosine. In certain embodiments, AA6 is asparagine, citrulline, glutamine, glycine, leucine, methionine, threonine or tyrosine. In certain embodiments, AA5-AA6 is Leu-Cit, Leu-Gln, Leu-Gly, Leu-Leu, Leu-Met, Leu-Thr, Phe-Cit, Phe-Gln, Phe-Leu, Phe-Met, Phe-Thr, Val-Asn, Val-Cit, Val-Gln, Val-Leu, Val-Met, Val-Thr, Tyr-Cit, Tyr-Leu, or Tyr-Met. In certain embodiments, AA1, AA3 and AA5 are glycine, valine, tyrosine, tryptophan, phenylalanine, methionine, leucine, isoleucine, or asparagine. In certain embodiments, AA2, AA4 and AA6 are glycine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, threonine or tyrosine. In certain embodiments, AA2 is a bond; and AA3 is a bond. In certain embodiments, AA1 is glycine; AA4 is phenylalanine; AA5 is leucine; and AA6 is glycine.

[0035] In certain embodiments, W is:

[0036]

Chemical formula

[0037]

Chemical formula

[0038] [Chemistry]

[0039] [Chemistry]

[0040] [Chemistry]

[0041] and

[0042] wherein R2 is -OH or methoxy; and R3 is hydrogen, -OH or methoxy.

[0043] In certain embodiments, W is:

[0044] [Chemistry]

[0045] is.

[0046] In certain embodiments, W is:

[0047] [Chemistry]

[0048] is.

[0049] In certain embodiments, Q is NR. In other embodiments, Q is S.

[0050] In certain embodiments, J is NR. In other embodiments, J is ((CH2) q Q) r is. In other embodiments, J is C5-C8 cycloalkyl. In certain embodiments, J is aryl.

[0051] In certain embodiments, Y is NR. In other embodiments, Y is S.

[0052] In certain embodiments, -Q-X-Y is:

[0053]

Chemical formula

[0054]

Chemical formula

[0055] wherein;

[0056] V is:

[0057]

Chemical formula

[0058] , or a bond; R 12 is hydrogen or Me; R 12 is R 14 together with forms a piperidine ring; R 11 is hydrogen or Me; and R 13 is R 12 together with forms a piperidine ring.

[0059] In certain embodiments, -Q-X-Y- is:

[0060]

Chemical formula

[0061] wherein.

[0062] In certain embodiments, -Q-X-Y- is:

[0063] [Chemical formula]

[0064] is as follows.

[0065] In certain embodiments, -Q-X-Y- is:

[0066] [Chemical formula]

[0067] is as follows.

[0068] In certain embodiments, -Q-X-Y- is:

[0069] [Chemical formula]

[0070] is as follows. In certain embodiments, -Q-X-Y- is:

[0071] [Chemical formula]

[0072] is as follows.

[0073] In certain embodiments, R4 and R5 are methyl; R6 is 2-hydroxypropyl; Z is -NH-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-Q-X-Y-C(O)-W; AA1 is glycine; AA2 is a bond; AA3 is a bond; AA4 is phenylalanine; AA5 is leucine; AA6 is glycine; -Q-X-Y- is:

[0074] [Chemical formula]

[0075] is as follows, and W is:

[0076]

Chem.

[0077] is as follows.

[0078] In certain embodiments, R4 and R5 are methyl; R6 is 2-hydroxypropyl; Z is -NH-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-Q-X-Y-C(O)-W; AA1 is glycine; AA2 is a bond; AA3 is a bond; AA4 is phenylalanine, AA5 is leucine, AA6 is glycine; -Q-X-Y- is:

[0079]

Chem.

[0080] and W is:

[0081]

Chem.

[0082] is as follows.

[0083] In certain embodiments, R4 and R5 are methyl; R6 is 2-hydroxypropyl; Z is -NH-AA1-AA2-AA3-AA4-AA5-AA6-C(O)-Q-X-Y-C(O)-W; AA1 is glycine; AA2 is a bond; AA3 is a bond; AA4 is phenylalanine; AA5 is leucine; AA6 is glycine; -Q-X-Y- is:

[0084]

Chem.

[0085] and W is:

[0086]

Chem.

[0087] is.

[0088] In certain embodiments, -Q-X-Y- is the following scheme:

[0089]

Chem.

[0090] as shown, a self-immolative linker that releases a MetAP2 inhibitor in the form of a carbamate derivative.

[0091] Another aspect of the present invention provides a complex having a linker having the structure of Z-Q-X-Y-C(O)-W, wherein, in each occurrence independently, Z is H2N-AA2-AA3-AA4-AA5-AA6-C(O)- or hydrogen; AA2 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA3 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA4 is a bond, or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine; AA5 is a bond, alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, valine, tryptophan, or ; AA6 is alanine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine or H2N(CH2)mCO2H, where m is 2, 3, 4 or 5, Q is NR, O, or S; X is M-(C(R)2) P -M-J-M-(C(R)2) P -M-V, where M is a bond, or C(O); J is a bond, or ((CH2) q Q) r , C5-C8 cycloalkyl, aryl, heteroaryl, NR, O, or S; Y is NR, O, or S; R is hydrogen or alkyl; V is a bond or:

[0092]

Chemical formula

[0093] and; R 9 is alkyl, aryl, aralkyl, or a bond; or R 9 combines with Y to form a heterocyclic ring; R 10 is an amide or a bond, R 11 is hydrogen or alkyl, W is a MetAP2 inhibitor moiety, p is from 0 to 20; q is 2 or 3; and r is 1, 2, 3, 4, 5, or 6.

[0094] In certain embodiments, Z is H2N-AA5-AA6-C(O)-. In certain embodiments, AA5 is alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, valine, tryptophan, or tyrosine, and AA6 is glycine. In certain embodiments, AA5 is leucine and AA6 is glycine. In certain embodiments, AA5 is valine and AA6 is glycine. In certain embodiments, AA5 is phenylalanine and AA6 is glycine. In certain embodiments, AA5 is glycine and AA6 is glycine. In certain embodiments, AA5 is not valine.

[0095] In other embodiments, Z is Η2Ν-ΑΑ3-ΑΑ4-ΑΑ5-ΑΑ6-C(O)-. In certain embodiments, AA5 is alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, valine, tryptophan, or tyrosine, and each of AA3, AA4, or AA6 is glycine. In certain embodiments, AA5 is leucine and each of AA3, AA4, or AA6 is glycine. In certain embodiments, AA5 is valine and each of AA3, AA4, or AA6 is glycine. In certain embodiments, AA5 is phenylalanine and each of AA3, AA4, or AA6 is glycine. In certain embodiments, AA3 is glycine, AA4 is phenylalanine, AA5 is leucine, and AA6 is glycine. In certain embodiments, each of AA3, AA4, AA5, and AA6 is glycine. In certain embodiments, AA5 is not valine.

[0096] In certain embodiments, Z is hydrogen. In other embodiments, Z is H2N-AA6-C(O)-. In certain embodiments, AA6 is glycine.

[0097] In certain embodiments, Q is NR. In certain embodiments, M is a bond. In certain embodiments, J is a bond. In certain embodiments, Y is NR.

[0098] In certain embodiments, W is:

[0099]

Chemical formula

[0100]

Chemical formula

[0101]

Chemical formula

[0102]

Chem.

[0103]

Chem.

[0104] being;

[0105] wherein, R2 is -OH or methoxy; and R3 is hydrogen, -OH or methoxy.

[0106] In certain embodiments, W is:

[0107]

Chem.

[0108]

Chem.

[0109] being.

[0110] In certain embodiments, W is:

[0111]

Chem.

[0112] being.

[0113] In certain embodiments, -Q-X-Y- is:

[0114]

Chem.

[0115]

Chem.

[0116] and;

[0117] V is:

[0118] [Chem.]

[0119] or a bond; R 12 is hydrogen or Me; R 12 is R 14 together with R 11 forms a piperidine ring; and R 12 together with R

[0120] In certain embodiments, Z is H2N-AA5-AA6-C(O)-; AA5 is leucine, and AA6 is glycine; Q-X-Y is:

[0121] [Chem.]

[0122] and; and W is:

[0123] [Chem.]

[0124] is.

[0125] In certain embodiments, Z is H2N-AA5-AA6-C(O)-, AA5 is valine, and AA6 is glycine; Q-X-Y is:

[0126] [Chem.]

[0127] and; and W is:

[0128]

Chem.

[0129] is as follows.

[0130] In certain embodiments, Z is H2N-AA5-AA6-C(O)-; AA5 is phenylalanine, and AA6 is glycine; Q-X-Y is:

[0131]

Chem.

[0132] and W is:

[0133]

Chem.

[0134] is as follows.

[0135] In certain embodiments, Z is H2N-AA5-AA6-C(O)-; AA5 is glycine, and AA6 is glycine; Q-X-Y is:

[0136]

Chem.

[0137] and W is:

[0138]

Chem.

[0139] is as follows.

[0140] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is leucine, and each of AA3, AA4, or AA6 is glycine; Q-X-Y is:

[0141]

Chemical formula

[0142] and W is:

[0143]

Chemical formula

[0144] is.

[0145] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is valine, and each of AA3, AA 4、 AA6 is glycine; Q-X-Y is:

[0146]

Chemical formula

[0147] and W is:

[0148]

Chemical formula

[0149] is.

[0150] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is phenylalanine, and each of AA3, AA 4、 AA6 is glycine; Q-X-Y is:

[0151]

Chemical formula

[0152] and W is:

[0153] [Chem.]

[0154] is.

[0155] In certain embodiments, Z is H2N-AA3-AA4-AA5-A-C(O)-; AA3 is glycine, AA4 is phenylalanine, AA5 is leucine, and AA6 is glycine; and Q-X-Y is:

[0156] [Chem.]

[0157] and W is:

[0158] [Chem.]

[0159] is.

[0160] In certain embodiments, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; each of AA3, AA4, AA5, and AA6 is glycine; and Q-X-Y is:

[0161] [Chem.]

[0162] and W is:

[0163] [Chem.]

[0164] is.

[0165] In one embodiment, Z is H2N-AA6-C(O)-; AA6 is glycine; Q-X-Y is:

[0166] [Chemical formula]

[0167] and W is

[0168] [Chemical formula]

[0169] is.

[0170] In one embodiment, Z is hydrogen; Q-X-Y is:

[0171] [Chemical formula]

[0172] and W is:

[0173] [Chemical formula]

[0174] is.

[0175] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is leucine, and AA6 is glycine; Q-X-Y is:

[0176] [Chemical formula]

[0177] and W is:

[0178]

Chem.

[0179] is.

[0180] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is valine, and AA6 is glycine; Q-X-Y is:

[0181]

Chem.

[0182] and W is:

[0183]

Chem.

[0184] is.

[0185] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is phenylalanine, and AA6 is glycine; Q-X-Y is:

[0186]

Chem.

[0187] and W is:

[0188]

Chem.

[0189] is.

[0190] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is glycine, and AA6 is glycine; Q-X-Y is:

[0191]

Chemical formula

[0192] and W is:

[0193]

Chemical formula

[0194] is.

[0195] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is leucine, and each of AA3, AA4 or AA6 is glycine; Q-X-Y is:

[0196]

Chemical formula

[0197] and W is:

[0198]

Chemical formula

[0199] is.

[0200] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is valine, and each of AA3, AA4, or AA6 is glycine; Q-X-Y is:

[0201]

Chemical formula

[0202] and; and W is:

[0203]

Chem.

[0204] is.

[0205] In certain embodiments, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is phenylalanine, and each of AA3, AA4, or AA6 is glycine; Q-X-Y is:

[0206]

Chem.

[0207] and; and W is:

[0208]

Chem.

[0209] is.

[0210] In certain embodiments, Z is H2N-AA3-AA-AA5-AA6-C(O)-; AA3 is glycine, AA4 is phenylalanine, AA5 is leucine, and AA6 is glycine; Q-X-Y is:

[0211]

Chem.

[0212] and; and W is:

[0213]

Chem.

[0214] is.

[0215] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; each of AA3, AA4, AA5 and AA6 is glycine; Q-X-Y is:

[0216]

Chemical formula

[0217] and W is:

[0218]

Chemical formula

[0219] is.

[0220] In one embodiment, Z is H2N-AA6-C(O)-; AA6 is glycine; Q-X-Y is:

[0221]

Chemical formula

[0222] and W is:

[0223]

Chemical formula

[0224] is.

[0225] In one embodiment, Z is hydrogen; Q-X-Y is:

[0226]

Chemical formula

[0227] and W is:

[0228] [Chemical formula]

[0229] is as follows.

[0230] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is leucine, and AA6 is glycine; Q-X-Y is:

[0231] [Chemical formula]

[0232] and W is:

[0233] [Chemical formula]

[0234] is as follows.

[0235] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is valine, and AA6 is glycine; Q-X-Y is:

[0236] [Chemical formula]

[0237] and W is:

[0238] [Chemical formula]

[0239] is as follows.

[0240] In one embodiment, Z is H2N-AA5-AA6-C(O)-; AA5 is phenylalanine, and AA6 is glycine; Q-X-Y is:

[0241] [Chemical formula]

[0242] and W is:

[0243] [Chemical formula]

[0244] is.

[0245] In certain embodiments, Z is H2N-AA5-AA6-C(O)-; AA5 is glycine, and AA6 is glycine; Q-X-Y is:

[0246] [Chemical formula]

[0247] and W is:

[0248] [Chemical formula]

[0249] is.

[0250] In certain embodiments, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is leucine, and each of AA3, AA4, or AA6 is glycine; Q-X-Y is:

[0251] [Chemical formula]

[0252] and W is:

[0253] [Chemical formula]

[0254] is.

[0255] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is valine, and each of AA3, AA4, or AA6 is glycine; Q-X-Y is:

[0256]

Chemical formula

[0257] and; and W is:

[0258]

Chemical formula

[0259] is.

[0260] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA5 is phenylalanine, and each of AA3, AA4 or AA6 is glycine; Q-X-Y is:

[0261]

Chemical formula

[0262] and; and W is:

[0263]

Chemical formula

[0264] is.

[0265] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; AA3 is glycine, AA4 is phenylalanine, AA5 is leucine, and AA6 is glycine; Q-X-Y is:

[0266]

Chemical formula

[0267] and W is:

[0268]

Chemical formula

[0269] is.

[0270] In one embodiment, Z is H2N-AA3-AA4-AA5-AA6-C(O)-; each of AA3, AA4, AA5 and AA6 is glycine; Q-X-Y is:

[0271]

Chemical formula

[0272] and W is:

[0273]

Chemical formula

[0274] is.

[0275] In one embodiment, Z is H2N-AA6-C(O)-; AA6 is glycine; Q-X-Y is:

[0276]

Chemical formula

[0277] and; and W is:

[0278]

Chem.

[0279] is.

[0280] In certain embodiments, Z is hydrogen; Q-X-Y is:

[0281]

Chem.

[0282] and; and W is:

[0283]

Chem.

[0284] is.

[0285] Other active moieties that can be modified for use in the complexes of the present invention include the following structures:

[0286]

Chem.

[0287] In certain embodiments, the active moiety is an anti-obesity compound. In other embodiments, the active moiety is a molecule that inhibits methionine aminopeptidase-2 (MetAP2), such as fumagillin, fumagillol, or an analogue, derivative, salt, or ester thereof. Further, preferred MetAP2 inhibitors are described in U.S. Patent No. 6,242,494 to Craig et al., U.S. Patent No. 6,063,812 to Hong et al., U.S. Patent No. 6,887,863 to Craig et al., U.S. Patent No. 7,030,262 to BaMaung et al., U.S. Patent No. 7,491,718 to Comess et al., the entireties of each of which are incorporated by reference. Additional preferred MetAP2 inhibitors are described in Wang et al., “Correlation of tumor growth suppression and methionine aminopeptidase-2 activity blockade using an orally active inhibitor,” PNAS 105(6)1838-1843 (2008); Lee et al., “Design, Synthesis, and Antiangiogenic Effects of a Series of Potent Novel Fumagillin Analogues,” Chem. Pharm. Bull. 55(7)1024-1029 (2007); Jeong et al., “Total synthesis and antiangiogenic activity of cyclopentane analogues of fumagillol,” Bioorganic and Medicinal Chemistry Letters 15,3580-3583 (2005); Arico-Muendel et al., “Carbamate Analogues of Fumagillin as Potent, Targeted Inhibitors of Methionine Aminopeptidase-2,” J. Med. Chem. 52, 8047-8056 (2009); and International Publication No. WO 2010 / 003475 to Heinrich et al.

[0288] Fumagillin is a small molecule used as an antibacterial and an antiprotozoal agent. Its physicochemical properties and manufacturing methods are known (see U.S. Patent No. 2,803,586 and Turner, J.R. et al., The Stereochemistry of Fumagillin, Proc. Natl. Acad. Sci. 48, 733-735 (1962)). Fumagillin, a fermentation product, may be hydrolyzed to obtain alcohol fumagillol, which may be converted to various derivatives including carbamoyl fumagillol, MW 325. The synthesis and preparation of carbamoyl fumagillol and some small molecule derivatives are described in U.S. Patent No. 5,166,172.

[0289] Fumagillin and related compounds are believed to exert their biological effects through inhibition of MetAP2. This enzyme removes the N-terminal methionine from nascent cell proteins (see Tucker, L.A et al., “Ectopic Expression of Methionine Aminopeptidase-2 Causes Cell Transformation and Stimulates Proliferation”, Oncogene 27, 3967 (2008)).

[0290] Carbamoyl fumagillol and derivatives, as well as other MetAP2 inhibitors, have shown therapeutic effects in preclinical and clinical studies. These compounds inhibit cell proliferation and angiogenesis as described in U.S. Patent No. 5,166,172. Fumagillin analogs or derivatives, such as CKD-732 and PI-2458 etc., have been well studied in various systems as described in Bernier et al., “Fumagillin class inhibitors of methionine aminopeptidase-2” Drugs of the Future 30(5):497-508, 2005.

[0291] The anti-obesity effects of fumagillin and its analogs are known. Rupnick et al., "Adipose tissue mass can be regulated through the vasculature," PNAS 99, 10730-10735, 2002, described weight loss in ob / ob mice with TNP-470 in the range of 2.5 mg / kg to 10 mg / kg per day. Brakenhielm described the prevention of obesity with a dose of 15 or 20 mg / kg of TNP-470 every other day: "The Angiogenesis Inhibitor, TNP-470, Prevents Diet-Induced and Genetic Obesity in Mice," Circulation Research 94:1579-1588, 2004. Kim et al., "Assessment of the anti-obesity effects of the TNP-470 analog, CKD-732," J Molecular Endocrinology 38, 455-465, 2007, described weight loss in C57BL / 6J mice and SD rats at a dose of 5 mg / kg / day. Lijnen et al., "Fumagillin reduces adipose tissue formation in murine models of nutritionally induced obesity," Obesity 12, 2241-2246, 2010, described that oral delivery of 1 mg / kg of fumagillin daily resulted in weight loss in C57BL / 6 mice.

[0292] One of these derivatives, chloroacetyl carbamoyl fumagillol (TNP-470), has been widely studied (see H. Mann-Steinberg et al., “TNP-470: The Resurrection of the First Synthetic Angiogenesis Inhibitor”, Chapter 35 in Folkman and Figg, Angiogenesis: An Integrative Approach from Science to Medicine, Springer NY (2008)). TNP-470 has shown activity against a number of cancers, including lung cancer, cervical cancer, ovarian cancer, breast cancer and colon cancer. Due to dose-limiting neurotoxicity, TNP-470 has been tested using multiple dosing regimens, but these attempts to limit its toxicity have not been successful. Thus, TNP-470 has been found to be too toxic for use in humans. TNP-470 has a short half-life and requires long-term intravenous administration for therapeutic use. Carbamoyl fumagillol, a metabolite of TNP-470, has a half-life of 12 minutes in humans (see Herbst 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”, Journal of Clinical Oncology 20(22)4440-4447(2002)). Furthermore, fumagillin and its derivatives are hydrophobic and difficult to formulate.

[0293] Despite the known usefulness of fumagillin derivatives, their use in therapy has not been successful because the problems of their low water solubility, short half-life values, and neurotoxic side effects cannot be overcome. Based on the previously observed dose-limiting neurotoxicity, the MTD of TNP-470 in combination with paclitaxel has been determined to be 60 mg / m2 administered three times a week: Herbst 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” Journal of Clinical Oncology 20, 4440-4447, 2002. Similarly, Shin et al., “A Phase 1 pharmacokinetic and pharmacodynamics study of CKD-732, an antiangiogenic agent, in patients with refractory solid cancer“ Investigational New Drugs 28, 650-658, 2010” reported that the MTD of CKD-732 is 15 mg / m2 / day administered on a schedule every four days due to confusion and insomnia. Thus, the compounds of the present invention are more potent, exhibit reduced toxicity (lower neurotoxicity), have improved water solubility, are more stable, and / or have a longer half-life (serum half-life) compared to currently known fumagillin derivatives.

[0294] As used herein, the expression “reduced toxicity” has its ordinary meaning as understood by one of ordinary skill in the art. Merely by way of example and in no way limiting the meaning of this term, administration of a fumagillin analog complex results in fewer side effects in an open-field test using mice compared to administration of the fumagillin analog alone.

[0295] The expression "improved water solubility" has its ordinary meaning as understood by a person skilled in the art. Merely an example, and in no way limiting the meaning of this term, the following description regarding this term is helpful: the fumagillin analog is incorporated covalently in a complex and thus dissolves more in water than the amount in which the unbound fumagillin analog dissolves alone in water.

[0296] The expression "longer half-life" has its ordinary meaning as understood by a person skilled in the art. Merely an example, and in no way limiting the meaning of this term, the following description regarding this term is helpful: any obvious increase in the length of time required to inactivate the fumagillin complex in either in vivo or in vitro as compared to the half-life of the fumagillin analog alone in either in vivo or in vitro.

[0297] Not bound by any theory, the non-enzymatic action of MetAP2 that suppresses the activity of extracellular signal-regulated kinases 1 and 2 (ERK1 / 2) may be as important as the binding of MetAP2 to eukaryotic translation initiation factor, elF. Cellular responses to MetAP2 inhibition that reflect potential ERK-related processes may include suppression of sterol regulatory element-binding protein (SREBP) activity, leading to reduced lipid and cholesterol biosynthesis. Interestingly, changes in the expression patterns of liver and adipose tissue genes after long-term (about 9 months) fumagillin exposure suggest that inhibition of MetAP2 can alter the relative abundance of factors involved in inflammation, consistent with reduced ERK-dependent cellular processes. The putative mechanism of MetAP2 inhibition leading to mobilization of stored fat as an energy source in the body and catabolism of free fatty acids is supported by changes in plasma β-hydroxybutyrate, adiponectin, leptin, and FGF21 observed in previous studies. The increase in the levels of adiponectin and FGF21, major catabolic hormones, accompanied by the appearance of ketone bodies (β-hydroxybutyrate), suggests that inhibition of MetAP2 by the compounds of the present invention, complex or modified fumagillin, fumagillol, or their analogs, derivatives, salts or esters, promotes energy consumption, fat utilization, and lipid excretion. The decrease in leptin observed in previous studies and in the studies provided herein is also consistent with a decrease in total adipose tissue and a negative energy balance. There is also a possibility that the compounds of the present invention, complex or modified fumagillin, fumagillol, or their analogs, derivatives, salts or esters, form a covalent bond with MetAP2, thereby irreversibly inhibiting and inactivating existing enzymes until a newly generated pool of MetAP2 is produced in target tissues (e.g., liver and adipose tissue).

[0298] In certain embodiments, the compounds of the present invention, complex or modified fumagillin, fumagillol, or their analogs, derivatives, salts or esters have the structure of the following formula, for example, as shown in Table 1:

[0299]

Table 1A

[0300]

Table 1B

[0301]

Table 1C

[0302]

Table 1D

[0303]

Table 1E

[0304]

Table 1F

[0305]

Table 1G

[0306] * In the formula, the polymer is as follows:

[0307]

Chemical formula

[0308] has the structure of, and preferably the following:

[0309]

Chemical formula

[0310] has the structure of.

[0311] For the purposes of the present invention, chemical elements are specified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986 - 87, inside cover.

[0312] The term "alkyl" refers to a fully saturated branched or unbranched carbon chain radical having a specified number of carbon atoms, or, when not specified, up to 30 carbon atoms. For example, "lower alkyl" refers to an alkyl having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and their alkyl positional isomers. Alkyls having 10 to 30 carbon atoms include decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, and tetracosyl. In certain embodiments, a straight or branched chain alkyl has 30 or fewer carbon atoms in its main chain (e.g., C1 - C 30 , C3 - C 30 ) in the case of a branched chain), more preferably 20 or fewer carbon atoms. Similarly, certain cycloalkyls have 3 to 10 carbon atoms in their ring structure, and the ring structure may have 5, 6, or 7 carbons.

[0313] Unless otherwise specified, "lower alkyl" as used herein refers to an alkyl group as defined above, but having 1 to 10 carbon atoms, or 1 to 6 carbon atoms in its main chain structure, such as methyl, ethyl, n - propyl, isopropyl, n - butyl, isobutyl, sec - butyl, and tert - butyl, etc. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this specification, an alkyl group is a lower alkyl. In certain embodiments, a substituent referred to as alkyl in this specification is a lower alkyl.

[0314] As used herein, the term "carbocyclic ring" refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

[0315] As used herein, the term "aryl" includes 5-, 6- and 7-membered monocyclic aromatic groups which may contain from 0 to 4 heteroatoms, such as benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. An aryl group having a heteroatom in the ring structure may also be referred to as an "aryl heterocycle" or "heterocyclic aromatic". The aromatic ring may be substituted at one or more ring positions with substituents such as those described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heterocyclic aromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring structures having two or more rings in which two or more carbons are shared by two adjacent rings (these rings are "fused rings"), where at least one ring is aromatic, and for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl.

[0316] "Alkenyl" refers to any branched or unbranched unsaturated carbon chain radical having a specific number of carbon atoms, or up to 26 carbon atoms when no limit on the number of carbon atoms is specified; and having one or more double bonds in the radical. Alkenyls having 6 to 26 carbon atoms are exemplified by the various isomeric forms of hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl, where the unsaturated bond may be located at any position of the radical, and the double bond may have either a (Z) structure or an (E) structure.

[0317] The term "alkynyl" refers to a hydrocarbyl radical within the scope of alkenyl but having one or more triple bonds.

[0318] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group bonded to an oxygen radical, as defined below. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by oxygen. Thus, an alkyl substituent that makes an alkyl into an ether can be -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2) m -R1 (where m and R1 are described below), and can be represented by one of them, is alkoxyl or similar thereto.

[0319] The term "heterocyclyl" or "heterocyclic group" refers to a 3- to 10-membered ring structure, more preferably a 3- to 7-membered ring, whose ring structure contains 1 to 4 heteroatoms. The heterocycle can be a polycycle. Examples of heterocyclyl groups include thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolidine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, phrazine, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinone and pyrrolidinone, sultams, sultones, and the like. The heterocycle can be substituted at one or more positions with the above-described substituents such as halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfamil, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like.

[0320] The term "alkylthio" refers to a sulfur radical bonded to an alkyl group as defined above. In certain embodiments, the "alkylthio" moiety is represented by one of -(S)-alkyl, -(S)-alkenyl, -(S)-alkynyl, and -(S)-(CH2) m -R1 (where m and R1 are defined below). Representative alkylthio groups include methylthio, ethylthio, and the like.

[0321] As used herein, the term "nitro" means -NO2; the term "halogen" refers to F, Cl, Br, or I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.

[0322] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines, for example, of the general formula:

[0323]

Chemical formula

[0324] refer to moieties that can be represented by

[0325] wherein R3, R5, and R6 are each independently hydrogen, alkyl, alkenyl, -(CH2) m -R1, or R3 and R5 together with the N atom to which they are attached form a heterocyclic ring having 4 to 8 atoms in the ring structure; R1 represents alkenyl, aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclic; and m is an integer in the range of 0 or 1 to 8. In certain embodiments, only one of R3 or R5 may be carbonyl, for example, R3, R5, and nitrogen do not together form an imide. In certain embodiments, R3 and R5 (and optionally R6) each independently represent hydrogen, alkyl, alkenyl, or -(CH2) m -R1. Thus, the term "alkylamine" as used herein means one in which a substituted or unsubstituted alkyl is attached to an amine group as defined above, i.e., at least one of R3 and R5 is an alkyl group. In certain embodiments, the amino group or alkylamine is basic, i.e., pK a satisfies pK a ≧7.00. The protonated forms of these functional groups have a pK a higher than 7.00 for water.

[0326] The term "carbonyl" (C(O)) is recognized in the art and has the general formula:

[0327] [Chemical formula]

[0328] and includes moieties that can be represented by:

[0329] wherein X represents a bond, or oxygen or sulfur, and R7 represents hydrogen, alkyl, alkenyl, -(CH2) m -R1 or a pharmaceutically acceptable salt, and R8 represents hydrogen, alkyl, alkenyl or -(CH2) m -R1 (where m and R1 are as defined above). When X is oxygen and R7 or R8 is not hydrogen, the above formula represents an "ester". When X is oxygen and R7 is as defined above, the above moiety is referred to herein as a carboxyl group, and in particular when R7 is hydrogen, the above formula represents "carboxylic acid". When X is oxygen and R8 is hydrogen, the above formula represents "formate". Generally, when the oxygen atom in the above formula is replaced by sulfur, the above formula represents a "thiocarbonyl" group. When X is sulfur and R7 or R8 is not hydrogen, the above formula represents a "thioester" group. When X is sulfur and R7 is hydrogen, the above formula represents a "thiocarboxylic acid" group. When X is sulfur and R8 is hydrogen, the above formula represents a "thioformate" group. On the other hand, when X is a bond and R7 is not hydrogen, the above formula represents a "ketone" group. When X is a bond and R7 is hydrogen, the above formula represents an "aldehyde" group.

[0330] As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In broad aspects, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, those described above. Permissible substituents can be one or more, and in suitable organic compounds, can be the same or different. For the purposes of the present invention, heteroatoms such as nitrogen can have hydrogen substituents and / or permissible substituents of any of the organic compounds described herein that satisfy the valence of the heteroatom. The present invention is not intended to be limited in any way by the permissible substituents of an organic compound. The expressions "substituted" or "substituted with" are understood to include the implicit condition that such substitution is based on the permissible valences of the substituted atoms and substituents, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo conversions such as rearrangement, cyclization, elimination, etc.

[0331] The term "sulfamoyl" is recognized in the art and has the general formula:

[0332]

Chemical formula

[0333] and includes moieties that can be represented by

[0334] wherein R3 and R5 are as defined above.

[0335] The term "sulfate" is recognized in the art and has the general formula:

[0336]

Chemical formula

[0337] and includes moieties that can be represented by

[0338] In the formula, R7 is as defined above.

[0339] The term "sulfamide" is recognized in the art and has the general formula:

[0340]

Chem.

[0341] and includes a moiety that can be represented by:

[0342] In the formula, R2 and R4 are as defined above.

[0343] The term "sulfonate" is recognized in the art and has the general formula:

[0344]

Chem.

[0345] and includes a moiety that can be represented by:

[0346] In the formula, R7 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

[0347] As used herein, the terms "sulfoxide" or "sulfinyl" have the general formula:

[0348]

Chem.

[0349] and refer to a moiety that can be represented by:

[0350] In the formula, R 12 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

[0351] Similar substitutions for alkenyl and alkynyl groups can form, for example, aminoalkenyl, aminoalkynyl, amidoalkenyl, amidoalkynyl, iminoalkenyl, iminoalkynyl, thioalkenyl, thioalkynyl, carbonyl-substituted alkenyl or alkynyl.

[0352] As used herein, when any structure is described one or more times, for example, the definitions of each expression such as alkyl, m, n, etc. are intended to be independent of the definitions in other places in the same structure.

[0353] The term "amino acid" is intended to encompass all compounds, whether natural or synthetic, that contain both an amino functional group and an acid functional group, including amino acid analogs and derivatives. In certain embodiments, the amino acids contemplated in the present invention are natural amino acids present in proteins, or natural assimilation or catabolism products of such amino acids that contain an amino group and a carboxyl group. Natural amino acids are identified throughout this specification by the conventional three-letter and / or one-letter abbreviations corresponding to the common names of the amino acids, according to the following table. The abbreviations are acceptable in the field of peptides and are recommended by the IUPAC-IUB Commission on Biochemical Nomenclature.

[0354] The term "amino acid residue" means an amino acid. Generally, the abbreviations used herein to refer to natural amino acids are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (Biochemistry (1972) 11:1726-1732). For example, Met, He, Leu, Ala, and Gly represent the "residues" of methionine, isoleucine, leucine, alanine, and glycine, respectively. A residue means a radical obtained by removing the OH portion of the carboxyl group and the hydrogen portion of the α-amino group from the corresponding α-amino acid.

[0355] The term "amino acid side chain" is, as defined in K.D. Kopple's "Peptides and Amino Acids", W.A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33, the portion of the amino acid residue excluding the main chain; examples of such side chains of common amino acids are -CH2CH2SCH3 (side chain of methionine), -CH2(CH3)-CH2CH3 (side chain of isoleucine), -CH2CH(CH3)2 (side chain of leucine) or H- (side chain of glycine). These side chains hang down from the main chain Cα carbon.

[0356] As used herein, the term "peptide" refers to a sequence of amino acid residues linked by peptide bonds or modified peptide bonds. The term "peptide" is intended to encompass peptide mimetics, peptide derivatives, peptidomimetics and peptide variants. The term "peptide" is understood to include peptides of any length. Peptide sequences described herein are generally represented according to accepted conventions, with the N-terminal amino acid on the left and the C-terminal amino acid on the right (e.g., H2N-AA1-AA2-AA3-AA4-AA5-AA6-CO2H).

[0357] Certain compounds of the invention may exist as specific geometric or stereoisomeric forms. The invention contemplates all such compounds, including cis and trans isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, which are included within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, and mixtures thereof, are intended to be included in the invention. Any presentation of a particular isomer is merely illustrative (e.g., an illustration of a trans isomer also encompasses a cis isomer).

[0358] For example, if a specific enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or induction with a chiral auxiliary group. In this case, the resulting mixture of diastereomers can be separated, and the auxiliary group can be cleaved to obtain the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as an amino group or an acidic functional group such as a carboxyl group, a diastereomeric salt can be formed with a suitable optically active acid or base, and then the diastereomers thus formed can be resolved by fractional recrystallization or chromatographic means known in the art, and then the pure enantiomer can be recovered.

[0359] Synthesis of the compound of the present invention In the synthesis method of the present invention, a wide range of functional groups are tolerated; thus, various substituted starting materials can be used. In this method, generally, the desired final compound is provided at or near the end of the overall process. However, in some cases, it may be desirable to further convert the compound into its pharmaceutically acceptable salt, ester, or prodrug.

[0360] The compounds of the present invention can be prepared in various ways using standard synthetic methods and procedures known to those skilled in the art or apparent to those skilled in the art in light of the teachings herein, using commercially available starting materials, compounds known in the literature, or intermediates prepared for immediate use. Standard synthetic methods, as well as procedures for the preparation of organic molecules and the transformation and manipulation of functional groups, can be obtained from relevant scientific literature or standard textbooks in the field. Without being limited to any one or several sources, classical literature, for example, Smith, M.B., March, J., March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5, which is incorporated herein by reference. thedition, John Wiley & Sons: New York, 2001; and Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999 are well-known, useful, and recognized references for organic synthesis to those skilled in the art. The following description of synthetic methods is for illustrative purposes of the basic procedures for the preparation of the compounds of the present invention and is not intended to be limiting.

[0361] The compounds of the present invention can be conveniently prepared by various methods known to those skilled in the art. The compounds of the present invention can be prepared from commercially available starting materials or starting materials that can be prepared using procedures described in the literature according to the schemes and examples provided herein. The compounds of the present invention, and their syntheses, are further described in International Publication No. WO 2011 / 150088 pamphlet and International Publication No. WO 2011 / 150022 pamphlet. Each of these publications is hereby incorporated by reference in its entirety for all purposes.

[0362] Pharmaceutical composition The present invention also provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof, and a pharmaceutically acceptable carrier or excipient.

[0363] As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences, which is a standard reference in the art. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, glucose solution, and 5% human serum albumin.

[0364] Pharmaceutically acceptable carriers include solid carriers such as lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Preferred liquid carriers include syrup, peanut oil, olive oil, water, and the like. Similarly, the carrier or diluent may include retardant materials known in the art, for example, glyceryl monostearate or glyceryl distearate including wax alone, ethyl cellulose, hydroxypropyl methyl cellulose, methyl methacrylate, or the like. Other fillers, excipients, flavoring agents, and other additives such as those known in the art may also be included in the pharmaceutical compositions of the present invention. Non-aqueous excipients such as liposomes and non-volatile oils may also be used. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the compositions is contemplated, except where any conventional medium or agent is incompatible with the active compound. Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, the pharmaceutical composition includes DMSO.

[0365] The term "pharmaceutically acceptable salt" refers to relatively non-toxic, inorganic and organic acid addition salts of a compound. These salts can be prepared in situ during the final isolation and purification of the compound, or by reacting the purified compound in its free base form separately with a suitable organic or inorganic acid and isolating the salt so formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate, and the like. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful in the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0366] As used herein, the expression "pharmaceutically acceptable" refers to ligands, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment that exhibits a reasonable benefit / risk ratio without causing excessive toxicity, irritation, allergic response, or other problems or complications, and are substantially non-pyrogenic.

[0367] As used herein, the term "metabolite" means a metabolite of a compound of the present invention, or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof, that exhibits activity similar to that of the compound of the present invention, or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof, in vivo.

[0368] As used herein, the term "prodrug" means a compound of the present invention, or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof, covalently linked to one or more pro-moieties, such as an amino acid moiety or other water-soluble moiety. The compound of the present invention, or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof, can be released from the pro-moiety via hydrolytic, oxidative, and / or enzymatic release mechanisms. In one embodiment, the prodrug compositions of the present invention exhibit the additional benefits of increased water solubility, improved stability, and improved pharmacokinetic profiles. The pro-moiety can be selected to obtain the desired prodrug properties. For example, the pro-moiety, such as an amino acid moiety or other water-soluble moiety such as a phosphate within R4, can be selected based on solubility, stability, bioavailability, and / or delivery or uptake in vivo. Examples of prodrugs include, but are not limited to, esters of hydroxy functional groups (e.g., acetate esters, dialkylaminoacetate esters, formate esters, phosphate esters, sulfate esters, and benzoate ester derivatives) and carbamate esters (e.g., Ν,Ν-dimethylaminocarbonyl), esters of carboxyl functional groups (e.g., ethyl esters, morpholinoethanol esters), N-acyl derivatives of amino functional groups (e.g., N-acetyl), N-Mannich bases, Schiff bases, and enamines, and oximes, acetals, ketals, enol esters, and the like of ketone and aldehyde functional groups: see Bundegaard, H., Design of Prodrugs, pi-92, Elesevier, New York-Oxford (1985).

[0369] Treatment method The present invention provides a method of inducing or causing weight loss in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to induce or cause weight loss. In certain embodiments, the subject is overweight or obese. In certain embodiments, inducing or causing weight loss is increasing weight loss.

[0370] The present invention also provides a method of preventing or delaying weight gain in a subject at risk thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to prevent or delay weight gain. In certain embodiments, the subject is at risk of becoming overweight or obese.

[0371] The present invention provides a method of treating obesity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to treat or ameliorate obesity.

[0372] The present invention also provides a method of preventing or delaying the onset of obesity in a subject at risk thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to prevent or delay the onset of obesity.

[0373] The present invention provides a method of treating metabolic syndrome or one or more components thereof in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to treat or ameliorate metabolic syndrome or one or more components thereof.

[0374] The present invention also provides a method for preventing or delaying the onset of metabolic syndrome or one or more of its components in a subject at risk thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention for preventing or delaying the onset of metabolic syndrome or one or more of its components.

[0375] The present invention also provides a method for reducing body weight in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention for reducing body weight. In certain embodiments, the subject is overweight or obese. In certain embodiments, the subject needs to reduce excessive adipose tissue.

[0376] Obesity and overweight refer to a subject having excessive fat relative to lean body mass. Excessive fat accumulation is associated with an increase in the size (hypertrophy or adiposity) and number (hyperplasia) of adipose tissue cells. Obesity can be caused by any factor, regardless of genetic (e.g., Prader-Willi syndrome) or environmental factors. Obesity is measured in various ways, from the perspective of absolute body weight, weight elongation ratio, degree of excess body fat, visceral or subcutaneous fat distribution, and social and aesthetic norms. A common criterion for body fat is the Body Mass Index (BMI). BMI represents the ratio of body weight (expressed in kilograms) to the square of height (expressed in meters). The obesity index can be accurately calculated using the following formula: SI unit: BMI = body weight (kg) / (height 2 (m 2 ), or US unit: BMI = (body weight (pounds) * 703) / (height 2 (inches 2 ).

[0377] As used herein, "overweight" refers to a state in which a healthy adult otherwise has a BMI of 25 kg / m 2 ~29.9 kg / m 2 . As used herein, "obesity" or "obese" refers to a state in which a healthy adult otherwise has a BMI of 30 kg / m 2Refers to the state of having the above BMI. Obesity has several subcategories. Adults with a BMI of 35 kg / m 2 or higher are called "severe obesity" or "morbid obesity". Adults with a BMI of 40 - 44.9 kg / m 2 or higher, or adults with a BMI of 35 kg / m 2 or higher and having at least one obesity - related health condition are called "pathological obesity" or "morbid obesity". Adults with a BMI of 45 kg / m 2 or higher are called "super - obesity" or "super - obesity disorder". In children, the definitions of overweight and obesity take into account the effect of age and gender on body fat.

[0378] Different countries may define obesity and overweight by different BMIs. The term "obesity" is intended to encompass the definitions in all countries. For example, in Asians, an increased risk associated with obesity occurs at a lower body mass index (BMI). In Asian countries including Japan, "obesity" refers to a subject having at least one obesity - induced or obesity - related co - morbidity that would require weight loss or would be improved by weight loss and having a BMI of 25.0 kg / m 2 or higher. People in South and Central America tend to be classified as closer to Asians than Europeans or North Americans.

[0379] The fact that excessive adipose tissue occurs selectively in different parts of the body and that the development of adipose tissue can be more dangerous in some parts of the body than in others cannot be explained by BMI. For example, "central obesity," which is typically associated with an "apple-shaped" body type, is due to excessive fat accumulation in the abdominal region, including abdominal and visceral fat, and has a higher risk of comorbidities than "peripheral obesity," which is typically associated with a "pear-shaped" body type and is due to excessive fat accumulation, particularly in the waist. The measurement of waist-to-hip ratio (WHR) can be used as an index of central obesity. The minimum WHR indicating central obesity is variously set, and adults with central obesity typically have a WHR of about 0.85 or more for women and about 0.9 or more for men.

[0380] The determination of a disease is carried out by standard methods known in the art, for example, by monitoring appropriate markers. For example, with respect to obesity, the following markers can be monitored: weight, BMI, body composition surveys, body fat distribution, central fat distribution, food or calorie intake, behavioral measurements of hunger and satiety, metabolic rate, and obesity-related comorbidities.

[0381] Methods for determining whether a subject is overweight or obese, which explain the ratio of excessive adipose tissue to fat-free mass, include obtaining information on the subject's body composition. Body composition can be obtained by measuring the thickness of subcutaneous fat at multiple locations on the body, such as the abdomen, subscapular region, arms, buttocks, and thighs. These measurements are then used to estimate total body fat with an acceptable error of about 4 percentage points. Another method is bioelectrical impedance analysis (BIA), which estimates body fat using the resistance of an electric current passing through the body. Another method is to use a large tank of water to measure body buoyancy. More body fat results in greater buoyancy, and more muscle mass tends to sink. Another method is dual-energy X-ray absorptiometry (DEXA). DEXA can non-invasively measure body composition, particularly total body fat and / or regional body fat mass. Magnetic resonance imaging (MRI) can also be used to non-invasively measure body composition.

[0382] For all methods described herein, references to the compounds of the invention include compositions such as pharmaceutical compositions described herein that include one or more of these compounds. These compositions may further include suitable excipients, such as pharmaceutically acceptable excipients including buffers known in the art. The invention can be used alone or in combination with other conventional methods of treatment.

[0383] Subjects in need of treatment provided by the invention can have at least one obesity-induced or obesity-related co-morbidity, i.e., a disease and other adverse health conditions that are related to, exacerbated by, or induced by being overweight or obese (i.e., diagnosed with or suffering from a co-morbidity). In other embodiments, the subject can have at least two obesity-induced or obesity-related co-morbidities.

[0384] Obesity-induced or obesity-related co-morbidities include, but are not limited to, diabetes, non-insulin-dependent type II diabetes, impaired glucose tolerance, impaired fasting glucose, dysglycaemia, elevated plasma insulin concentration, insulin resistance syndrome, hyperlipidaemia, lipid disorder, elevated free fatty acids, hypertension, hyperuricaemia, gout, coronary artery disease, heart disease, myocardial infarction, angina pectoris, microvascular damage, sleep apnoea, obstructive sleep apnoea, Pickwickian syndrome, fatty liver; cerebral infarction, stroke, cerebral thrombosis, respiratory complications, cholelithiasis, gallbladder disease, kidney disease, gastro-oesophageal reflux, stress urinary incontinence, arteriosclerosis, heart disease, abnormal heart rhythm, arrhythmia, transient ischaemic attack, orthopaedic disorder, osteoarthritis, degenerative arthritis, lumbodynia, menstrual disorder, hormonal imbalance, endocrine disease and infertility. In particular, co-morbidities include: hypertension, hyperlipidaemia, lipid disorder, impaired glucose tolerance, cardiovascular disease, sleep apnoea, diabetes, and other obesity-related conditions.

[0385] The present invention provides a method for treating obesity or inducing, causing or increasing (reducing body weight) weight loss in a subject in need thereof, and for treating one or more of these obesity-induced or obesity-related co-morbidities in a subject suffering from such co-morbidities, the method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention for treating or alleviating obesity or reducing body weight and for treating or alleviating one or more of the obesity-induced or obesity-related co-morbidities.

[0386] The present invention provides a method for treating metabolic disorders or metabolic syndrome in a subject in need thereof, wherein the syndrome is characterized by a group of metabolic risk factors including: 1) abdominal obesity (excessive adipose tissue inside and around the abdomen); 2) atherogenic dyslipidemia (high triglycerides; low HDL cholesterol and high LDL cholesterol, or low HDL:LDL ratio); 3) elevated blood pressure; 4) insulin resistance or impaired glucose tolerance; 5) a prothrombotic state (e.g., high fibrinogen or high plasminogen activator inhibitor-1 in the blood); 6) a proinflammatory state (e.g., elevated CRP in the blood); and 7) prediabetes or type 2 diabetes. The present invention can treat metabolic diseases alone, or in combination with treating obesity or inducing, causing, or increasing weight loss.

[0387] The present invention also provides a method for treating, reducing, or improving one or more cardiometabolic risk factors selected from the group consisting of plasma triglyceride levels, LDL-cholesterol levels, C-reactive protein (CRP) levels, and blood pressure (systolic blood pressure and / or diastolic blood pressure) in a subject suffering from said risk factors, in addition to treating obesity or inducing, causing, or increasing weight loss in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention for treating or ameliorating obesity or reducing weight and treating or ameliorating one or more risk factors.

[0388] The compounds of the present invention, or pharmaceutically acceptable salts, prodrugs, metabolites, analogs, or derivatives thereof, can also be administered in combination with a second active agent. The second active agent may be complexed with a polymer.

[0389] As the intended second active agent, those administered for treating type 2 diabetes: for example, sulfonylureas (e.g., chlorpropamide, glibide, glibenclamide, glimepiride); meglitinides (e.g., repaglinide and nateglinide); biguanides (e.g., metformin); thiazolidinediones (rosiglitazone, troglitazone, and pioglitazone); glucagon-like peptide 1 mimetics (e.g., exenatide and liraglutide); sodium-glucose cotransporter inhibitors (e.g., dapagliflozin), dipeptidyl peptidase 4 inhibitors (e.g., gliptin), sodium-glucose linked transporter inhibitors, renin inhibitors, and alpha-glucosidase inhibitors (e.g., acarbose and meglitol); and / or those administered for treating heart disorders and conditions associated with overweight or obesity such as hypertension, dyslipidemia, ischemic heart disease, cardiomyopathy, myocardial infarction, stroke, venous thromboembolic disease, and pulmonary hypertension, for example, chlorthalidone; hydrochlorothiazide; indapamide, metolazone; loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide, lasix, torsemide); potassium-sparing agents (e.g., amiloride hydrochloride, spironolactone, and triamterene); peripheral agents (e.g., reserpine); central alpha-agonists (e.g., clonidine hydrochloride, guanabenz acetate, guanfacine hydrochloride, and methyldopa); alpha-blockers (e.g., doxazosin mesylate, prazosin hydrochloride, and terazosin hydrochloride); beta-blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol fumarate, carteolol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, penbutolol sulfate, pindolol, propranolol hydrochloride, and pyrilamine maleate); combination of alpha-blockers and beta-blockers (e.g., carvedilol and labetalol hydrochloride); direct vasodilators (e.g., hydralazine hydrochloride and minoxidil); calcium antagonists (e.g., diltiazem hydrochloride and verapamil hydrochloride); dihydropyridines (e.g., amlodipine besylate, felodipine, isradipine, nicardipine, nifedipine, and nisoldipine);ACE inhibitors (benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, quinapril hydrochloride, ramipril,trandolapril); angiotensin II receptor blockers (e.g., losartan potassium, valsartan, and irbesartan); and combinations thereof; and statins such as mevastatin, lovastatin, pravastatin, simvastatin, velostatin, dihydrocompactin, fluvastatin, atorvastatin, darvastatin, carvastatin, cerivastatin, beva statin, cefvastatin, rosuvastatin, pitavastatin, and glenvastatin, typically for the treatment of dyslipidemia.;

[0390] Other second active agents that can be co-administered (e.g., sequentially or simultaneously) include: agents administered for treating ischemic heart disease, including statins, nitrates (e.g., isosorbide dinitrate and isosorbide mononitrate), β-blockers, and calcium channel antagonists; agents administered for treating cardiomyopathy, including positive inotropes (e.g., digoxin), diuretics (e.g., furosemide), ACE inhibitors, calcium antagonists, antiarrhythmics (e.g., sotolol, amiodarone, and disopyramide), and β-blockers; agents administered for treating myocardial infarction, including ACE inhibitors, angiotensin II receptor blockers, direct vasodilators, β-blockers, antiarrhythmics, and thrombolytics (e.g., alteplase, reteplase, tenecteplase, anisoylated plasminogen streptokinase activator complex, and urokinase); agents administered for treating stroke, including antiplatelet agents (e.g., aspirin, clopidogrel, dipyridamole, and ticlopidine), anticoagulants (e.g., heparin), and thrombolytics; agents administered for treating venous thromboembolic disease, including antiplatelet agents, anticoagulants, and thrombolytics; agents administered for treating pulmonary hypertension, including positive inotropes, anticoagulants, diuretics, potassium (e.g., K-dur), vasodilators (e.g., nifedipine and diltiazem), bosentan, epoprostenol, and sildenafil; agents administered for treating asthma, including bronchodilators, anti-inflammatory agents, leukotriene blockers, and anti-Ige agents. Specific asthma medications include zafirlukast, flunisolide, triamcinolone, beclomethasone, terbutaline, fluticasone, formoterol, beclomethasone, salmeterol, theophylline, and xopenex; agents administered for treating sleep apnea include modafinil and amphetamine; agents administered for treating non-alcoholic fatty liver disease include antioxidants (e.g., vitamins E and C), insulin sensitizers (metformin, pioglitazone, rosiglitazone, and betaine), hepatoprotectants, and antihyperlipidemic agents;Agents administered for treating osteoarthritis of the load-bearing joint include acetaminophen, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, etodolac, oxaprozin, naproxen, diclofenac, and nabumetone), COX-2 inhibitors (e.g., celecoxib), steroids, supplements (e.g., glucosamine and chondroitin sulfate), and artificial joint fluid; agents administered for treating Prader-Willi syndrome include human growth hormone (HGH), somatropin, and anti-obesity drugs (e.g., orlistat, sibutramine, methamphetamine, ionamin, phentermine, bupropion, diethylpropion, fenproporex, benzphetamine, and topiramate); agents administered for treating polycystic ovary syndrome include insulin-sensitizing agents, combinations of synthetic estrogen and progesterone, spironolactone, eflornithine, and clomiphene; agents administered for treating erectile dysfunction include phosphodiesterase inhibitors (e.g., tadalafil, sildenafil citrate, and vardenafil), prostaglandin E analogs (e.g., alprostadil), alkaloids (e.g., yohimbine), and testosterone; agents administered for treating infertility include clomiphene, clomiphene citrate, bromocriptine, gonadotropin-releasing hormone (GnRH), GnRH agonists, GnRH antagonists, tamoxifen / nolvadex, gonadotropins, human chorionic gonadotropin (HCG), human menopausal gonadotropin (HmG), progesterone, recombinant follicle-stimulating hormone (FSH), urofollitropin, heparin, follitropin alpha, and follitropin beta; agents administered for treating childbirth complications include bupivacaine hydrochloride, dinoprostone PGE2, meperidine HC1, Ferro-folic-500 / iberet-folic-500, meperidine, methylergonovine maleate, ropivacaine HC1, nalbuphine HC1, oxymorphone HC1, oxytocin, dinoprostone, ritodrine, scopolamine hydrobromide, sufentanil citrate, and labor-inducing agents;Examples of drugs administered for treating depression include serotonin reuptake inhibitors (e.g., fluoxetine, escitalopram, citalopram, paroxetine, sertraline, and venlafaxine); tricyclic antidepressants (e.g., amitriptyline, amoxapine, clomipramine, desipramine, doxepin hydrochloride, doxepin, imipramine, iprindole, lofepramine, nortriptyline, opipramol, protriptyline, and trimipramine); monoamine oxidase inhibitors (e.g., isocarboxazid, moclobemide, phenelzine, tranylcypromine, selegiline, rasagiline, nialamide, iproniazid, iproclozide, troxatone, linezolid, dienolide kavapyrone desmethoxyyangonin, and dextroamphetamine); stimulants (e.g., amphetamine, methamphetamine, methylphenidate, and arecoline); antipsychotics (e.g., butyrophenone, phenothiazine, thioxanthene, clozapine, olanzapine, risperidone, quetiapine, diprasidone, amisulpride, paliperidone, symbiax, tetrabenazine, and cannabidiol); and mood stabilizers (e.g., lithium carbonate, valproic acid, divalproex sodium, sodium valproate, lamotrigine, carbamazepine, gabapentin, oxcarbazepine, and topiramate). Examples of drugs administered for treating anxiety include serotonin reuptake inhibitors, mood stabilizers, benzodiazepines (e.g., alprazolam, clonazepam, diazepam, and lorazepam), tricyclic antidepressants, monoamine oxidase inhibitors, and β-blockers; and other anti-obesity drugs including serotonin and norepinephrine reuptake inhibitors; norepinephrine reuptake inhibitors; selective serotonin reuptake inhibitors; and intestinal lipase inhibitors. Specific anti-obesity drugs include orlistat, sibutramine, methamphetamine, ionamin, phentermine, bupropion, diethylpropion, fenfluramine, benzphetermine, and topamax.;

[0391] The present invention also provides a method for reducing adipocytes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to reduce adipocytes or adipose tissue. The present invention also provides a method for preventing an increase in adipocytes in a subject at risk thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to prevent an increase in adipocytes. Reducing adipocytes means decreasing the number of adipocytes or the size (fat mass). Preventing an increase in adipocytes means decreasing or maintaining the number of adipocytes, or decreasing or maintaining the size of adipocytes. In certain embodiments, administration of the compounds of the present invention causes adipocytes to contract in a subject in need thereof. The adipose tissue can be white adipose tissue or brown adipose tissue.

[0392] The present invention also provides a method for reducing food intake in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to reduce food intake.

[0393] A decrease in food intake means a decrease in the daily food intake. The decrease in the daily food intake can be from about 5% to about 50% (e.g., about 5%, about 10%, about 20%, about 30%, about 40%, or about 50%). Based on a daily diet of 2000 kcal, the decrease is from about 100 kcal to about 1000 kcal per day (e.g., about 100 kcal, about 200 kcal, about 400 kcal, about 600 kcal, about 800 kcal, or about 1000 kcal).

[0394] The present invention also provides a method for reducing hunger in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of the present invention to reduce hunger. The subject may also have a decrease in food intake.

[0395] Hunger can be evaluated in a fasting state using a 10 - point visual analog scale (VAS) that is widely used in research on appetite. See Flint et al., Int. J. Obes. Relat. Metab. Disord. 24(1):38 - 48, 2000. Specifically, on a scale from 1 to 10, where 10 indicates extreme hunger and 1 indicates no hunger, the subject is asked to evaluate their overall hunger over the previous two days.

[0396] The method of the present invention can also reduce abdominal circumference in a subject in need thereof. Abdominal circumference is evaluated using a tape measure placed at the abdomen 1 cm above the iliac crest. The subject of the present invention can have a reduction in abdominal circumference of about 1 inch (2.54 cm) to about 20 inches (50.8 cm) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 inches (2.54 cm, 5.08 cm, 7.62 cm, 10.16 cm, 12.7 cm, 15.24 cm, 17.78 cm, 20.32 cm, 22.86 cm, 25.4 cm, 27.94 cm, 30.48 cm, 33.02 cm, 35.56 cm, 38.1 cm, 40.64 cm, 43.18 cm, 45.72 cm, 48.26 cm or 50.8 cm)).

[0397] In the method of the present invention, administration of the compound results in a reduction in body fat and maintenance of substantial muscle mass in the patient. In certain embodiments, compared to patients undergoing only caloric restriction therapy, administration of the compound promotes fat oxidation in the patient. For example, provided herein is a method for reducing body fat in a patient in need thereof. Such a patient can maintain a substantially greater muscle mass compared to the reduction in body fat in patients undergoing only energy - restricted diet therapy.

[0398] The present invention also provides a method of improving surgical outcomes in a subject in need thereof, the method comprising administering, prior to surgery, a therapeutically effective amount of at least one compound of the present invention to the subject to improve surgical outcomes. In certain embodiments, the administration reduces liver and / or abdominal fat in the patient and improves surgical outcomes. In certain embodiments, the surgery is not an emergency surgery. Such surgery can include bariatric surgery, cardiovascular surgery, abdominal surgery, or plastic surgery.

[0399] As used herein, the term "patient" or "subject" can mean a human or non-human subject. In certain embodiments, the subject is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, livestock, sport animals, pets, primates (including humans), horses, dogs, cats, mice, and rats. In certain embodiments, the mammal is a human.

[0400] As used herein, a "subject in need thereof" is a subject that is overweight or obese (with or without one or more co-existing conditions), or a subject that has a high risk of becoming overweight or developing obesity relative to the general population. In certain aspects, the subject in need thereof is an obese subject having a BMI of 30 kg / m 2 or greater. In certain aspects, the subject in need thereof is a subject that is overweight or obese, or has a high risk of becoming overweight or developing obesity relative to the general population, and is not afflicted with or diagnosed with a disorder selected from the group consisting of cancer, hyperproliferative disorders, retinal angiogenesis due to macular degeneration, psoriasis and pyogenic granuloma, rheumatic, immune and degenerative joint diseases.

[0401] The term "preventive or therapeutic" treatment is recognized in the art and includes administering a composition of one or more agents to a host. When the treatment is carried out before the clinical signs of an undesirable condition (e.g., a disease or other undesirable condition in a host animal), the treatment is preventive (i.e., protects the host from developing the undesirable condition), while when the treatment is carried out after the signs of an undesirable condition, the treatment is therapeutic (i.e., is intended to reduce, alleviate, or stabilize an existing undesirable condition or its side effects).

[0402] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to: ameliorating one or more symptoms associated with a disease; reducing its severity; or alleviating it; including one or more of these. With regard to obesity, beneficial or desired clinical results include any one or more of the following: reducing or maintaining body weight; controlling food intake or calorie intake (including reducing it); increasing the metabolic rate or inhibiting a decrease in the metabolic rate; and ameliorating, reducing the severity of, and / or alleviating any of the disorders associated with obesity, such as diabetes, non-insulin-dependent diabetes, hyperglycemia, impaired glucose tolerance, insulin resistance, dyslipidemia, lipid abnormalities, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, abdominal obesity, eating disorders, metabolic syndrome, hypertension, osteoarthritis, myocardial infarction, fatty liver disease, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), stroke, and other related diseases; improving the quality of life of a patient suffering from obesity, and / or extending lifespan.

[0403] As used herein, "delaying" the onset of obesity means deferring, preventing, decelerating, retarding, stabilizing, and / or postponing the onset of the disease. This delay can be of various lengths depending on the disease history and / or the medical history of the subject being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention in that the individual does not develop the disease. For example, one result of delaying the onset can be to reduce the weight of a subject at risk of obesity compared to the subject's weight immediately prior to administration of the composition described herein. Another result of delaying the onset can be to prevent the weight that has previously decreased as a result of diet, exercise, or drug therapy from returning. Another result of delaying the onset can be to prevent the onset of obesity in a subject at risk of obesity if treatment is implemented prior to the onset of obesity. Another result of delaying the onset can be to reduce the occurrence and / or severity of obesity-related disorders in a subject at risk of obesity if the treatment is implemented prior to the onset of obesity.

[0404] An individual "at risk" of obesity may or may not have a detectable disease and may or may not exhibit a detectable disease prior to the implementation of the treatment methods described herein. "At risk" means that the individual has one or more so-called risk factors that are measurable parameters correlated with the onset of obesity. An individual having one or more of these risk factors has a higher probability of being obese than an individual not having these risk factors. These risk factors include, but are not limited to, age, diet, physical inactivity, metabolic syndrome, family history of obesity, ethnicity, genetic syndromes, previous medical history (e.g., eating disorders, metabolic syndrome, and obesity), presence of a precursor disease (e.g., overweight). For example, a healthy individual with a BMI less than 25.0 - 30.0 kg / m 2 or an individual with at least one co-existing condition and a BMI less than 25.0 kg / m 2 - 27.0 kg / m 2 is at risk of obesity.

[0405] The "onset" of obesity means the onset and / or progression of the disease in an individual (which may be different embodiments of the present invention). The onset of obesity can be detected using standard clinical techniques described herein. However, the onset can also represent the progression of the disease, which may not be detectable initially. For the purposes of the present invention, progression refers to the biological process of the disease state, in which case progression is measured by the assessment of height and weight for estimating BMI, measurement of abdominal circumference, assessment of co-existing conditions, and the onset and / or worsening of obesity complications such as atherosclerosis, type II diabetes, polycystic ovary disease, cardiovascular disease, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia, and cholelithiasis. These various diagnostic tests are known in the art. "Onset" includes occurrence, recurrence, and onset. As used herein, the "onset" or "occurrence" of obesity includes initial onset and / or recurrence.

[0406] As used herein, "weight control" or "weight improvement" refers to reducing or maintaining weight in an individual (compared to the pre-treatment level). In some embodiments, the weight is maintained generally within the normal range. Weight can be reduced by decreasing calorie intake and / or reducing body fat accumulation. In some embodiments, the weight is reduced in an individual by at least about 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% compared to the pre-treatment level.

[0407] As used herein, "controlling food intake" refers to reducing or maintaining food intake in an individual (compared to the pre-treatment level). In some embodiments, the food intake is maintained generally within the normal range. In some embodiments, the food intake is reduced in an individual by about 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% compared to the pre-treatment level.

[0408] The "therapeutically effective amount" of a compound for use in therapy refers to the amount of the compound in a formulation that, when administered as part of a desired dosage regimen, alleviates symptoms, causes remission of a condition, or delays or prevents the onset of a disease state, according to clinically acceptable criteria, such as a reasonable benefit / risk ratio applicable to any medical treatment, when administered (to a mammal, preferably a human) for the disorder or condition to be treated or for cosmetic purposes. "Therapeutically effective amount" is synonymous with "effective dosage".

[0409] As used herein, the "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce a beneficial or desired result. In prophylactic use, beneficial or desired results include removing or reducing the risk of a disease, reducing its severity, or delaying its onset, including biochemical, histological, and / or behavioral signs of the disease, complications of the disease, and intermediate pathological phenotypes that appear during the development of the disease. In therapeutic use, beneficial or desired results include, for example, reducing the intensity, duration, or frequency of an episode of a disease; and reducing one or more signs (biochemical, histological, and / or behavioral) resulting from the disease, including complications of the disease and intermediate pathological phenotypes that appear during the development of the disease; increasing the quality of life of a patient suffering from the disease; reducing the dosage of other agents required to treat the disease; enhancing the effect of another drug therapy; and / or delaying the progression of the disease in the patient; and other clinical results. The effective dosage can be administered in one or more administrations. For the purposes of the present invention, the effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood in the clinical situation, the effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, in combination with one or more other agents, when a desired result can be achieved or is achieved, the "effective dosage" may be considered in the context of administering one or more therapeutic agents, and the administration of a single agent in an effective amount may also be considered. For example, the effective amount of a compound of the present invention for treating obesity is an amount sufficient to treat or alleviate one or more symptoms associated with obesity."Effective amount" means an amount sufficient to bring about one or more of the following, which may also correspond to various embodiments of the present invention: reducing, decreasing or controlling body weight, reducing, decreasing or controlling food intake, increasing the metabolic rate, reducing one or more symptoms resulting from diseases associated with obesity, increasing the quality of life of a patient suffering from obesity, and / or extending lifespan.

[0410] When providing one or more of the compounds described herein to a subject, the dosage of the compound administered will vary depending on factors such as the subject's age, weight, height, gender, general medical condition, previous medical history, disease progression, route of administration, formulation, and factors of the same kind.

[0411] The dosage of the compounds of the present invention can be determined empirically in an individual who has received one or more administrations. The individual is administered the compounds of the present invention at gradually increasing dosages. To evaluate the effectiveness of the compounds of the present invention, markers of the disease state can be monitored. It will be apparent to those skilled in the art that the dosage will vary depending on the individual, the stage of the disease (e.g., the stage of obesity), as well as past treatments and co-administered treatments.

[0412] The toxicity and therapeutic effectiveness of the compounds of the present invention can be determined by standard pharmaceutical procedures in experimental animals. The toxic dose can be determined as the maximum tolerated dose (MTD), or the LD50 (the dose lethal to 50% of the population). The effective dose can be determined as the ED50 (the dose therapeutically effective in 50% of the population), or the dose required to produce some average change in the animal (e.g., the dose required to produce an average decrease in systolic blood pressure of 10 mmHg in the subject group).

[0413] It is ideal for the effective dose and the toxic dose to be determined in the same species. However, if determined in different species, allometric scaling can be used to convert to the effective or toxic dose for another species. The dose ratio of the toxic effect to the therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. When comparing mice to rats, the generally accepted magnification factor is 2, and the rat dose is estimated to be one-half of the dose in mice. Thus, if the toxic dose in rats is 100 mg / kg and the effective dose in mice is 1 mg / kg, the therapeutic index in rats can be calculated by taking the effective dose in rats as 1 mg / kg / 2, i.e., 0.5 mg / kg, and the therapeutic index is 200. The FDA defines that a drug has a narrow therapeutic range if (a) the difference between the median lethal dose and the median effective dose is less than 2-fold; or (b) the difference between the minimum toxic concentration and the minimum effective concentration in the blood is less than 2-fold.

[0414] Compounds of the present invention showing a high therapeutic index are preferred. Although compounds of the present invention showing toxic side effects can also be used, care should be taken in the design of the delivery system that delivers such compounds of the present invention to the site of the diseased tissue in order to minimize the potential for damage to uninfected cells and thereby reduce side effects.

[0415] To formulate the dosage range for use in humans, data obtained from animal experiments can be used. The dosage of such compounds of the present invention is preferably in the range of blood concentration that includes an effective dosage with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. In the compounds of the present invention having an MW of less than 1000, initially, a therapeutically effective amount can be estimated from cell culture assays. On the other hand, for dosages of complexes that require cleavage of the linker to release the active moiety, better estimation is possible with animal models. Using such information, useful dosages in humans can be determined more accurately. It is known in the art that complexing with a polymer weakens (dilutes) the active moiety activity (the polymer is a diluent). This is illustrated in the mouse dosing model of the anticancer agents shown in the following table.

[0416]

Table 2

[0417] Therefore, when the therapeutic index is not improved, it is well understood that complexing with a polymer increases the clinical dosage. This is illustrated in the human dosing model of the anticancer agents shown in the following table.

[0418]

Table 3

[0419] The polymer complexed and modified compounds of the present invention surprisingly provide greater efficacy and lower toxicity compared to the uncomplexed and / or unmodified parent drug / active moiety.

[0420] For example, the fumagillol complex and modified fumagillol compounds of the present invention are surprisingly superior to the fumagillol small molecule and, at equimolar doses, resulted in increased weight loss in DIO mice. The compounds of the present invention can be used at lower molar doses and less frequent dosing to result in equivalent weight loss. The lower molar doses and reduced dosing frequency reduce systemic drug exposure and systemic drug toxicity. Further, the fumagillol complex and modified fumagillol compounds of the present invention provide an action similar to that of the fumagillol small molecule, i.e., a favorable fat loss and reduction in food intake in DIO mice.

[0421] Conventional polymer complexes dilute the activity, increase the dose 5 - 20 fold, and result in only a small change (less than 2 fold) in the therapeutic index. In contrast, the polymer composite compounds of the present invention surprisingly and unexpectedly increase the therapeutic index (substantial improvement) and exhibit increased activity at lower doses.

[0422] In the method of the present invention, the polymer composite compounds of the present invention exhibit less frequent dosing (e.g., q4d: dosing every 4 days, q7d: dosing every 7 days, q8d: dosing every 8 days), a dose reduced to at least 84 mol% of the fumagillol equivalent, and a reduced AUC in non-target compartments, while the therapeutic index increases (more than 10 fold).

[0423] In another embodiment, an effective dose, e.g., the daily dose of the compound of the present invention, is provided herein. For example, a method is provided herein that includes administering a dose of the compound of the present invention effective for weight loss. For example, in the methods described herein, the intended dosing of the compound of the present invention can include administering a dose of about 200 mg / day, about 80 mg / day, about 40 mg / day, about 20 mg / day, about 10 mg / day, about 5 mg / day, about 3 mg / day, about 2 mg / day, about 1 mg / day, about 0.5 mg / day, about 0.2 mg / day, about 0.05 mg / day, about 0.01 mg / day, or about 0.001 mg / day, regardless of body weight.

[0424] The effective amount of the drug for weight loss in a patient may be administered based on body weight or body surface area and can be from about 0.0001 mg / kg to about 5 mg / kg body weight per day. For example, the intended dosage can be from about 0.001 to 5 mg / kg body weight per day, from about 0.001 mg / kg to 1 mg / kg body weight per day, from about 0.001 mg / kg to 0.1 mg / kg body weight per day, from about 0.001 to about 0.010 mg / kg body weight per day, or about 0.007 mg / kg body weight per day.

[0425] The compounds of the present invention can be administered in an amount sufficient to reduce the patient's body weight by about 0.5 kg / week to about 1 kg / week (or about 0.5% to about 1% of body weight per week). In certain embodiments, the weekly weight loss occurs during the duration of treatment.

[0426] Administration of the compounds of the present invention according to the methods of the present invention may be continuous or intermittent, depending, for example, on the physiological state of the patient receiving the administration, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to a skilled practitioner. Administration of the compounds of the present invention may be essentially continuous over a preselected period or may be a series of spaced dosings.

[0427] For repeated administration over several days or longer periods, the treatment is continued, depending on the condition, until a desirable suppression of symptoms or a sufficient therapeutic level is achieved. For example, dosing once to five times a week is contemplated. In certain embodiments, the compounds of the invention are administered about every four days. Other dosing regimens include once to five times a week, every three to four days, or less frequent regimens. In some embodiments, the compounds of the invention are administered about once a week, once every two weeks, or about one to four times a month, depending on the duration of the response to drug administration. Intermittent dosing regimens with staggered dosages, separated by two days to a maximum of seven days, or 14 days, may be used. In some embodiments, treatment may be initiated with daily dosing and then changed to weekly to monthly dosing. The progress of this treatment can be readily monitored by conventional techniques and assays, or by measuring MetAP2 as described in U.S. Patent No. 6,548,477.

[0428] The frequency of administration can be determined and adjusted over the course of treatment. For example, the frequency of administration can be determined or adjusted based on the type and severity of the disease being treated, whether the purpose of drug administration is prophylactic or therapeutic, previous treatment history, the patient's clinical history, and response to the drug, as well as the instructions of the attending physician. Typically, the clinician administers the compounds of the invention until a dosage that achieves a desirable result is reached.

[0429] Treatment can be continued for as long as desired or limited to a short period. Suitable treatment periods can be, for example, at least about 1 week, at least about 4 weeks, at least about 1 month, at least about 6 months, at least about 1 year, at least about 2 years, or indefinite. The treatment period can end when a desired result, such as a targeted weight loss, is achieved, for example, when a reduction of about 5% of body weight, about 10% of body weight, about 20% of body weight, about 30% or more of body weight is achieved. The treatment regimen can include: a corrective phase in which the compound of the invention is administered at a dosage, or dosing frequency, sufficient to effect a reduction in excess fat accumulation; and then a maintenance phase in which the compound is administered at a lower dosage, or lower dosing frequency, sufficient to prevent recurrence of excess fat accumulation.

[0430] The compound, or a pharmaceutically acceptable salt, ester or prodrug thereof (or a pharmaceutical composition thereof) can be administered by any means known in the art. For example, the compounds or compositions of the invention can be administered orally, nasally, transdermally, topically, by pulmonary administration, by inhalation, buccally, sublingually, intraperintoneally, subcutaneously, intramuscularly, intravenously, rectally, intrathoracically, intrathecally and parenterally. Administration can be systemic administration such as intravenous administration or topical administration. In certain embodiments, the route of administration can be intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrathoracic, intrauterine, rectal, vaginal, topical routes, and the like. In certain embodiments, the compound is administered subcutaneously.

[0431] In one aspect, the compounds of the present invention, or pharmaceutically acceptable salts, solvates, diastereomers, and polymorphs thereof, can be administered in a suitable dosage form or formulation prepared by combining (i.e., by making a pharmaceutical composition of the present invention) a therapeutically effective amount (e.g., an effective level sufficient to achieve the desired therapeutic effect) of the compound of the present invention or a pharmaceutically acceptable salt, solvate, diastereomer, and polymorph thereof (as the active ingredient) with a standard pharmaceutical carrier or diluent, according to conventional procedures. These procedures can include appropriately mixing, granulating, and compressing or dissolving the ingredients to achieve the desired formulation.

[0432] Parenteral dosage forms can be prepared by any means known in the art. For example, sterile aqueous or oily suspensions for injection can be formulated according to known techniques, using suitable dispersing or wetting agents and suspending agents.

[0433] Oral dosage forms, such as capsules, tablets, pills, powders, and granules, can be prepared using any suitable method known in the art. For example, the compounds of the present invention can be mixed with enteric materials and compressed into tablets. Alternatively, the formulations of the present invention can be incorporated into chewable tablets, crushable tablets, tablets that rapidly dissolve in the mouth, or gargles.

[0434] For pulmonary (e.g., intratracheal) administration, the compounds of the present invention can be formulated with conventional excipients into an inhalable composition in the form of a fine powder or a nebulizable liquid. For ocular administration, the compounds of the present invention can be formulated with conventional excipients into, for example, the form of eye drops or eye implants. Excipients useful in eye drops include, for example, thickening agents or gelling agents to minimize loss due to tearing by improving retention in the eye.

[0435] Liquid dosage forms for oral or other administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active agent, the liquid dosage forms can include inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, dimethylformamide, oils (more particularly, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, compositions for ocular delivery, oral delivery, or other systemic delivery can include adjuvants such as wetting agents, emulsifying agents, and suspending agents.

[0436] Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. The liquid formulation can be sprayed directly, or the lyophilized powder can be sprayed after reconstitution. Alternatively, the compounds of the present invention can be aerosolized using a fluorocarbon formulation and a metered - dose inhaler, or inhaled as a lyophilized powder and a micronized powder.

[0437] Dosage forms for topical or transdermal administration of the pharmaceutical compositions of the present invention can include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is commingled, under sterile conditions, with a pharmaceutically acceptable carrier and any required preservative or buffer, as needed. For example, administration by the dermal route is achieved with aqueous drops, sprays, emulsions, or creams.

[0438] Transdermal patches can have the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable vehicle. Absorption enhancers may be used to increase the flux of the compound across the skin. The rate can also be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0439] Compositions for rectal or vaginal administration may be suppositories, prepared by mixing the compounds of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or suppository wax, which is solid at ambient temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity to release the active agent. Alternatively, administration can also be effected by inserting an endoscope into the rectum of the subject and then releasing the intended formulation from the lumen of the endoscope.

[0440] One of ordinary skill in the art may refer to general references for a detailed description of the known or equivalent techniques described herein. These references include Ausubel et al., Current Protocols in Molecular Biology; John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed.), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990). Of course, these references can also be referred to in the preparation and use of aspects of the present invention.

Examples

[0441] Examples are provided below to further illustrate various features of the present invention. The examples also illustrate useful methodologies for practicing the present invention. These examples do not limit the invention claimed in the claims.

[0442] Basic procedures The polymer product of the present invention was purified using tangential flow filtration (TFF). The Pall Minimate™ Capsule and Minimate™ TFF System were used, and TFF was carried out according to the manufacturer's instructions. A Minimate TFF Capsule equipped with a 5 kDa Omega membrane (5K) or a Minimate TFF Capsule equipped with a 10 kDa Omega membrane (10K) cartridge was used for purification. In all cases, the permeate water was discarded and the retentate was lyophilized to obtain the polymer product. The structure of the product was 1 confirmed by 1H NMR and small molecules were also characterized by mass spectrometry (MS). The polymer weights reported in the examples were not corrected for moisture content.

[0443] Carbamoyl fumagillol and chloroacetyl carbamoyl fumagillol can be prepared according to the methods disclosed in U.S. Patent No. 5,166,172 (Kishimoto et al., which is hereby incorporated by reference). p-Nitrophenyl fumagyl-6-yl carbonate can be prepared according to the published procedure. (See Han, C. et al., Biorg. Med. Chem. Lett. 2000, 10, 39-43.) MA-GFLG-ONp can be prepared according to the methods disclosed in U.S. Patent No. 5,258,453 (Kopecek et al., which is hereby incorporated by reference).

[0444] Synthesis of poly(HPMA-co-MA-GFLG-ONp)

[0445]

Chemical formula

[0446] A mixture of hydroxypropyl methacrylamide (HPMA, 22.16 g, 155 mmol), N-methacrylgly-phe-leu-gly p-nitrophenyl ester (MA-GFLG-ONp, 10.00 g, 17.19 mmol), AIBN (1.484 g, 9.037 mmol) and acetone (225 g) was degassed (freezing, applying pump, thawing for 4 cycles). The resulting reaction mixture was stirred at 50 °C for 48 h and then cooled to room temperature. The desired product was purified by trituration with acetone and then dried in vacuo to give 17.6 g of poly(HPMA-co-MA-GFLG-ONp) as a white solid. The structure was 1 confirmed by 1H NMR, indicating that the product was substantially free of impurities (e.g., p-nitrophenol). According to ultraviolet absorbance, the copolymer contained 0.47 mmol of p-nitrophenyl ester per gram of polymer. The copolymer of this example was used in most of the subsequent examples. A wide variety of copolymers based on various monomers and / or monomer ratios may be prepared according to this procedure by adjusting the stoichiometric ratio and / or using different monomers.

[0447] Synthesis of poly(HPMA-co-MA-GFLG-OH)

[0448] Poly(HPMA-co-MA-GFLG-ONp) (700 mg) was added portionwise to a solution of 0.1 M NaOH (11.3 mL) at 0 °C. The yellow reaction mixture was stirred at 0 °C for 0.5 h and then at room temperature for 4 h. Half of the solution was acidified to pH = 6 with 0.1 M HCl. The aqueous phase was extracted with ethyl acetate to remove any excess p-nitrophenol. Lyophilization of the aqueous phase gave poly(HPMA-co-MA-GFLG-OH) as a colorless solid (360 mg).

[0449] Synthesis of poly(HPMA-co-MA-GG-ONp)

[0450] [Chemical formula]

[0451] A mixture of hydroxypropyl methacrylamide (HPMA, 82.5 g), N-methacrylgly-gly p-nitrophenyl ester (MA-GG-ONp, 16.8 g), AIBN (5.7 g), and acetone (875 g) was purged with argon for 90 minutes. The resulting reaction mixture was stirred at 50 °C for 48 hours and then cooled to room temperature. The desired product was purified by trituration with acetone and then dried in vacuo to give 69.3 g of poly(HPMA-co-MA-GG-ONp) as a white solid. The structure was 1 confirmed by 1H NMR, indicating that the product was substantially free of impurities (e.g., p-nitrophenol). The amount of p-nitrophenyl ester per gram of polymer may be determined by ultraviolet absorbance. A wide variety of copolymers based on various monomers and / or monomer ratios may be prepared according to this procedure by adjustment of the stoichiometric ratio and / or use of different monomers.

[0452] Synthesis of poly(HPMA-co-MA-GFLG-NHCH2CH2N(Me)BOC) and basic procedure A

[0453]

Chemical formula

[0454] A solution of poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 mL) and H2O (10 mL) was added dropwise to a solution of tert-butyl N-(2-aminoethyl)-N-methylcarbamate (0.20 g, 1.15 mmol) in water (20 mL) at 0 °C over 15 minutes. The reaction mixture was stirred at 0 °C for 15 minutes, then warmed to room temperature and stirred for 12 hours. The solvent was evaporated under reduced pressure. The resulting residue was dissolved in water (50 mL) and the pH was adjusted to approximately 8.0 with 0.1 M NaOH. The solution was filtered through a VacuCap filter and then purified using TFF (10K). The polymer-containing solution was washed with 25 mM NaCl solution (800 mL) (as part of the TFF process) to remove p-nitrophenol, the pH of the solution was adjusted to approximately 4 with 0.1 M HCl, and then washed with water (400 mL) (as part of the TFF process). Freeze-drying the polymer solution isolated the compound poly(HPMA-co-MA-GFLG-NHCH2CH2N(Me)BOC) as a pale yellow solid (720 mg, 71%).

[0455] Synthesis of Fmoc-Phe-Gly-NH-(CH2)6NH-Boc: To a solution of Fmoc-Phe-Gly-OH (0.66 g) in anhydrous THF (20 mL) under N2 at 0 °C were added N,N'-dicyclohexylcarbodiimide (0.307 g) and 1-hydroxybenzotriazole hydrate (0.201 g). After stirring for 15 minutes, N-Boc-1,6-diaminohexane (0.322 g) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solids were filtered off and washed with EtOAc. The filtrate and washings were then concentrated under reduced pressure. Purification of the resulting residue by flash column chromatography (0 - 10% MeOH in CH2Cl2) gave Fmoc-Phe-Gly-NH-(CH2)6NH-Boc as a white solid (0.9 g).

[0456] Synthesis of Fmoc-Phe-Gly-NH-(CH2)6NH2-TFA: Fmoc-Phe-Gly-NH-(CH2)6NH-Boc (0.7 g) was dissolved in CH2Cl2 (4 mL) at 0 °C under N2, and then trifluoroacetic acid (TFA) (4 mL) was added. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours under N2. The solvent was removed under reduced pressure, and the residue was dried under high vacuum to obtain 0.71 g of Fmoc-Phe-Gly-NH(CH2)6-NH2 TFA. This crude material was used for the preparation without further purification.

[0457] Synthesis of Fmoc-Phe-Gly-NH(CH2)6NH-CO-fumagillol:

[0458]

Chemical formula

[0459] To a 0 °C solution of the compound Fmoc-Phe-Gly-NH(CH2)6-NH2 TFA (0.71 g) in anhydrous CH2Cl2 (20 mL) and DMF (1 mL) under N2, nitrophenyl fumagil-6-yl carbonate (0.536 g) was added. Then diisopropylethylamine (DIPEA) (0.74 mL) was added. The reaction mixture was allowed to warm to room temperature and then stirred at the same temperature overnight. The solvent was removed under reduced pressure, and the resulting residue was dissolved in EtOAc (70 mL). The EtOAc was washed with water and brine. Then the ethyl acetate solution was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0 - 10% MeOH in CH2Cl2) to obtain Fmoc-Phe-Gly-NH-(CH2)6NH-CO-fumagillol as an off-white solid (0.81 g).

[0460] Synthesis of H-Phe-Gly-NH(CH2)6NH-CO-fumagillol

[0461]

Chemical formula

[0462] Under N2, DBU (0.15 g) was added to a 0 °C solution of compound Fmoc-Phe-Gly-NH-(CH2)6NH-CO-fumagillol (0.80 g) in anhydrous CH2Cl2 (20 mL). The reaction mixture was allowed to warm to room temperature. The solvent was removed under reduced pressure, and the resulting residue was purified by flash column chromatography (0 - 10% MeOH in CH2C12) to give H-Phe-Gly-NH-(CH2)6H-CO-fumagillol as a pale yellow rubber (0.45 g, 76%).

[0463] Synthesis of poly[HPMA-co-MA-GGFG-N-(6-aminohexyl)carbamoyl fumagillol] and basic procedure B

[0464] [Chemical formula]

[0465] At 0 °C under N2, H-Phe-Gly-NH(CH2)6NHCO-fumagillol (0.45 g) in anhydrous DMF (5 mL) was added to a solution of poly(HPMA-co-MA-GG-ONp) (0.68 g) in anhydrous DMF (12 mL), followed by diisopropylethylamine (DIPEA) (0.25 mL). The reaction mixture was allowed to warm to room temperature and stirred overnight under N2, then 3-amino-1-propanol (0.032 g) was added. The mixture was stirred for an additional 1 hour. The solvent was removed under reduced pressure, and the resulting residue was dissolved in 300 mL of distilled water and extracted with EtOAc (4 times). Saturated aqueous NaCl solution (50 mL) was used to facilitate phase separation. Trace amounts of EtOAc were removed from the polymer solution by stirring under a stream of nitrogen gas. The polymer solution was filtered through a vacu cap filter (pH = 5.56), concentrated to 30 mL by TFF with a 10K capsule, and washed with water (700 mL) by TFF. The polymer was then lyophilized to give the desired polymer complex poly[HPMA-co-MA-GGFG-N-(6-aminohexyl)carbamoyl fumagillol] as a pale pink foamy solid (0.685 g). The spiroepoxide content was determined by reaction with 2-mercaptopyrimidine and found to be 0.4 mmol / g.

[0466] Synthesis of Poly[HPMA-co-MA-GGLG-N-(6-aminohexyl)carbamoyl fumarigirole]

[0467]

Chem.

[0468] The dipeptide H-Leu-Gly-NH(CH2)6NHCO-Fum was prepared using standard techniques and coupled to poly(HPMA-co-MA-GG-ONp) using basic procedure B.

[0469] Synthesis of Poly[HPMA-co-MA-GGVG-N-(6-aminohexyl)carbamoyl fumarigirole]

[0470]

Chem.

[0471] The dipeptide H-Val-Gly-NH(CH2)6NHCO-Fum was prepared using standard techniques and coupled to poly(HPMA-co-MA-GG-ONp) using basic procedure B.

[0472] Synthesis of Poly[HPMA-co-MA-GGGG-N-(6-aminohexyl)carbamoyl fumarigirole]:

[0473]

Chem.

[0474] The dipeptide H-Gly-Gly-NH(CH2)6NHCO-Fum was prepared using standard techniques and coupled to poly(HPMA-co-MA-GG-ONp) using basic procedure B.

[0475] Synthesis of poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoyl fumagillol] via poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexylamine-HCl)]:

[0476] [Chemical formula]

[0477] Using ds-1,4-diaminocyclohexane (0.914 g) and poly(HPMA-co-MA-GFLG-ONp) (1.5 g), following the basic procedure C, poly[HPMA-co-MA-GFLG-N-(ds-4-aminocyclohexylamine-HCl)] was obtained as an off-white solid (1.08 g).

[0478] Using poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexylamine-HCl)] (0.98 g), p-nitrophenyl fumagill-6-yl carbonate (0.465 g), and DIEA (0.268 g) in 16 mL of DMF, the basic procedure F was followed. The solvent was evaporated and the solution was diluted with water. The aqueous phase (total 500 mL) was extracted with ethyl acetate (total 80 mL) and purified by TFF using an additional 350 mL of water. The concentrated water was diluted with water, extracted with ethyl acetate, and lyophilized to obtain poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoyl fumagillol] as a pale pink solid (0.79 g).

[0479] 11H NMR (DMSO-d6): δ 7.90 - 8.35 (m, 4H, amide - NH), 7.0 - 7.70 (m, 25H, phenylalanine and amide - NH), 5.26 (m, H - 5 - Fum), 5.18 (bt, alkene - Fum), 4.60 - 4.90 (m, 14H), 4.50 - 4.60 (m, 1H, α - proton of phenylalanine), 4.10 - 4.30 (m, 1H, α - proton of leucine), 3.40 - 3.80 (m, 21H), 3.26 (s, 3H, OMe - Fum), 2.80 - 3.10 (m, 31H), 2.17 (m, 2H, allyl - Fum), 0.37 - 2.0 [m, 166H {1.69 (s, 3H, Fum - Me), 1.59 (s, 3H, Fum - Me), 1.07 (s, 3H, Fum - Me)}].

[0480] Synthesis of poly(HPMA - co - MA - GFLG - NHCH2CH2NH2HCl) and basic procedure C or reaction of diamine with poly(HPMA - co - MA - GFLG - ONp):

[0481]

Chem.

[0482] A solution of ethylenediamine (0.33 g, 5.49 mmol) in water (20 mL), pH 11.7, was adjusted to pH 9.1 by the addition of 37% aqueous HCl (17 - 18 drops). The solution was cooled in an ice bath, and poly(HPMA - co - MA - GFLG - ONp) (1.03 g) in DMF (6 mL) was added dropwise over 20 minutes while maintaining the temperature below 4 °C. The solution was stirred at 4 °C for 20 minutes and at room temperature for 50 minutes to obtain a lemon - yellow solution, pH 8.1. The solution was evaporated at 40 °C. H2O (10 mL, three times) was added and evaporated. The product was diluted with water (60 mL), and the solution was adjusted to pH 8.0 with NaOH. The solution was filtered through a VacuCap filter and purified by TFF as follows. First, the polymer solution was washed with 25 mM NaCl solution (800 mL) to remove p - nitrophenol. The solution was washed with water (400 mL) and then adjusted to pH 4 with 0.1 M HCl. The TFF - concentrated water was collected, and the filter was washed twice with 10 mL of water. Combining the concentrated water and the washings gave a polymer solution, which was lyophilized to isolate the compound poly(HPMA - co - MA - GFLG - NHCH2CH2NH2HCl) as a pale - yellow solid (0.71 g, 72%).

[0483] Synthesis of N - [(2R)1 - hydroxy - 2 - methylbutan - 2 - yl]carbamoyl fumariglone and basic procedure D:

[0484]

Chemical formula

[0485] A solution of p - nitrophenyl fumarigl - 6 - yl carbonate (400 mg, 0.89 mmol) and (R) - 2 - amino - 3 - methyl - 1 - butanol (280 mg, 2.71 mmol) in ethanol (10 mL) was stirred at room temperature for 12 hours. The yellow solution was concentrated, and the residue was purified by flash chromatography (methanol / methylene chloride) to obtain N - [(2R)1 - hydroxy - 2 - methylbutan - 2 - yl]carbamoyl fumariglone (340 mg, 0.83 mmol) as a colorless oil.

[0486] Synthesis of N-(6-hydroxyhexyl)carbamoyl fumarigirole:

[0487]

Chemical formula

[0488] Using p-nitrophenyl fumarigyl-6-yl carbonate (150 mg) and 6-aminohexanol (48 mg) in ethanol (10 mL), the basic procedure D was followed. The product was isolated as a colorless oil (110 mg, 78%).

[0489] Synthesis of N-[1-(hydroxymethyl)cyclopentyl]carbamoyl fumarigirole:

[0490]

Chemical formula

[0491] Using p-nitrophenyl fumarigyl-6-yl carbonate (100 mg) and cycloleucinol (52 mg) in ethanol (3 mL) and THF (1 mL), following the basic procedure D, N-[1-(hydroxymethyl)cyclopentyl]carbamoyl fumarigirole was obtained as an oil (50 mg).

[0492] Synthesis of N-(1-hydroxy-2-methylpropan-2-yl)carbamoyl fumarigirole:

[0493]

Chemical formula

[0494] Using p-nitrophenyl fumarigl-6-yl carbonate (100 mg) and 2-amino-2-methylpropanol (40 mg) in ethanol (3 mL) and THF (2 mL), following the basic procedure D, N-(1-hydroxy-2-methylpropan-2-yl)carbamoyl fumariglol was obtained as an oil (37 mg).

[0495] Synthesis of fumarigl-6-yl (2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate:

[0496]

Chemical formula

[0497] Following the basic procedure D. S-Prolinol (68 mg, 0.67 mmol) was reacted with p-nitrophenyl fumarigl-6-yl carbonate (150 mg, 0.335 mmol) in ethanol (4 mL). The product was purified by flash chromatography (methanol / methylene chloride) to give fumarigl-6-yl (2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate as a white foam (81 mg, 63%).

[0498] Synthesis of N-(6-aminohexyl)carbamoyl fumariglol:

[0499]

Chemical formula

[0500] A solution of 1,6-diaminohexane (0.13 g) in methanol (8 mL) was cooled to 0 °C, and a solution of p-nitrophenyl fumaroyl-6-yl carbonate (0.13 g) in methanol (2 mL) was added dropwise. The solvent was reduced to 2 mL by rotary evaporation. Ethyl acetate was added, and the organic phase was washed with water, 0.1 N NaOH, water, and brine, and dried over sodium sulfate. The solvent was evaporated, and the residue was dissolved in ethanol (15 mL). DL-Tartaric acid (16 mg) was added, and the solution was stored overnight and then evaporated to approximately 0.5 mL. Addition of ether formed a white solid. The solid was collected by filtration, washed with ether, and dried to give the tartrate salt (74 mg) of N-(6-aminohexyl)carbamoyl fumarogirole.

[0501] Synthesis of fumaroyl-6-yl [trans-(4-aminocyclohexyl)]carbamate:

[0502]

Chemical formula

[0503] To a solution of trans-1,4-diaminocyclohexane (1.3 g) in methanol (80 ml) at 0 - 5 °C, a solution of fumaroyl-6-yl 4-nitrophenyl carbonate (1.0 g) in methanol (20 ml) and CH2Cl2 (20 ml) was added over 30 minutes, and then stirred for 30 minutes. After concentration to 20 ml on a rotary evaporator and dilution with ethyl acetate (75 ml), the organic layer was washed with water (30 ml), 0.1 N NaOH (30 ml), water, and brine (30 ml), dried (MgSO4), and concentrated under reduced pressure to give 0.78 g of a solid. This was dissolved in ethanol (80 ml), and DL-tartaric acid (127 mg) was added. After 1 hour, a solution was formed, which was left overnight and then concentrated under reduced pressure to remove substantially all of the ethanol. MTBE (100 ml) was added, concentrated, and MTBE (30 ml) was added. The solid was collected by filtration, washed with MTBE (twice with 10 ml), and dried in vacuo to give fumaroyl-6-yl [trans-(4-aminocyclohexyl)]carbamate hemitartrate (0.73 g), melting point 180 - 185 °C.

[0504] Synthesis of poly[HPMA-co-MA-GFLG-NH(CH2)6NH2-HCl]: Using 1,6-diaminohexane (621 mg, 5.36 mmol) and poly(HPMA-co-MA-GFLG-ONp) (1.0 g), the basic procedure C was followed. The crude product was purified by TFF (5K) using aqueous NaCl (25 mM), then acidified to pH 4.0 with 0.1 M HCl and further purified by TFF using water, and poly[HPMA-co-MA-GFLG-NH(CH2)6NH2-HCl] was obtained as an off-white solid (860 mg).

[0505] Synthesis of p-nitrophenyl N-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoyl fumaroyl-6-yl carbonate and basic procedure E:

[0506]

Chemical formula

[0507] Under N2 at 0 °C, DMAP (660 mg, 5.40 mmol) was added to a solution of alcohol N-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoyl fumaroyl alcohol (1.11 g) in methylene chloride, and p-nitrophenyl chloroformate (810 mg) was added little by little. The reaction mixture was stirred at 0 °C for 1 hour. The solvent was evaporated, the obtained residue was dissolved in EtOAc, washed with water and brine, and dried (Na2SO4). Evaporation of EtOAc gave a crude product, which was purified by flash chromatography (silica type, elution with 100% hexane, then 2 - 30% EtOAc). The fractions containing the pure product were combined and evaporated, and N-[(2R)1-(p-nitrophenylcarbonylhydroxy-2-methylbutan-2-yl]carbamoyl fumaroyl alcohol (1.25 g, 80%) was isolated as a white solid.

[0508] Synthesis of N-[1-(p-Nitrophenoxycarbonylhydroxymethyl)-2-methylpropan-2-yl]carbamoyl fumagillol:

[0509]

Chem.

[0510] According to basic procedure E, dimethyl alcohol (60 mg), p-nitrophenyl fumagill-6-yl carbonate (46 mg), and DMAP (37 mg) were reacted in methylene chloride (8 mL). The reaction mixture was diluted with ethyl acetate and washed with water (3 times) and then brine. The organic phase was dried (Na2SO4) and evaporated to give a yellow foamy solid (87 mg), which was used without further purification.

[0511] Synthesis of N-[1-(p-Nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoyl fumagillol:

[0512]

Chem.

[0513] According to basic procedure E, N-[1-(Hydroxymethyl)cyclopentyl]carbamoyl fumagillol (the product of Example 14, 74 mg), p-nitrophenyl chloroformate (53 mg), and DMAP (43 mg) were reacted in methylene chloride (5 mL). After extraction, N-[1-(p-Nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoyl fumagillol (100 mg) was used without further purification.

[0514] Synthesis of poly[HPMA-co-MA-GFLG-NH(CH2)6NH carbamoyl-[1-hydroxy-3-methylbutan-2-yl]carbamoyl fumagillol] and basic procedure F:

[0515]

Chem.

[0516] To a solution of polymer (400 mg) and p-nitrophenyl N-[(2R)1-hydroxy-3-methylbutan-2-yl]carbamoyl fumaroyl-6-yl carbonate (240 mg) in DMF (8 mL) at 0 °C, DIEA (0.11 g) was added dropwise. The solution was stirred at 0 °C for 1 hour and then allowed to warm to room temperature. After 3 days, the solvent was evaporated and water (80 mL) was added. The aqueous phase was extracted with ethyl acetate (total 500 mL) until the starting carbonate could no longer be detected by mass spectrometry. The aqueous phase was purified by TFF (10K) and the concentrated water was lyophilized to obtain the complex as a white solid (380 mg, 77%).

[0517] 1 H NMR (DMSO-d6): δ 8.25 (bs, 2H, amide-NH), 8.0 (bs, 1H, amide-NH), 7.70 (bs, 2H, amide-NH), 7.10 - 7.30 (m, 15H, phenylalanine and amide-NH), 7.10 (bt, 1H, NH-Fum), 6.92 (bd, 1H, NH-Fum), 5.26 (m, H-5-Fum), 5.18 (bt, alkene-Fum), 4.50 - 4.80 (m, 1H, α-proton of phenylalanine), 4.0 - 4.21 (m, 1H, α-proton of leucine), 3.50 - 3.84 (m, 19H), 3.29 (s, 3H, OMe-Fum), 2.80 - 3.10 (m, 28H), 2.51 (d, 1H, J = 4.4 Hz, H-2-Fum), 2.19 (m, 2H, allyl-Fum), 0.82 - 1.92 [m, 131H{1.84 (m, 2H, Fum), 1.72 (s, 3H, Fum-Me), 1.60 (s, 3H, Fum-Me), 1.09 (s, 3H, Fum-Me), 0.84 (dd, 6H, Fum-isopropyl}]。

[0518] Synthesis of poly[HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoylfumagirole]:

[0519]

Chemical Structure

[0520] In DMF (10 mL), poly(HPMA-co-MA-GFLG-NHCH2CH2NH2-HCl) (200 mg), p-nitrophenyl fumaroyl-6-yl carbonate (100 mg), and DIEA (57 mg) were used, and the basic procedure F was followed. The product was purified by TFF (10K) using water and freeze-dried, and the complex was obtained as a pale yellow solid (160 mg).

[0521] Synthesis of poly[HPMA-co-MA-GFLG-N(Me)-(2-methylaminoethyl)carbamoyl fumarogirole]:

[0522]

Chemical formula

[0523] In DMF (5 mL), poly(HPMA-co-MA-GFLG-N(Me)CH2CH2NHMe-HCl) (200 mg), p-nitrophenyl fumaroyl-6-yl carbonate (100 mg), and DIEA (57 mg) were used, and the basic procedure F was followed. The product was purified by using TFF (10K) with water and freeze-dried, and the complex was obtained as an off-white solid (180 mg).

[0524] Synthesis of poly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumarogirole):

[0525]

Chemical formula

[0526] Using poly(HPMA-co-MA-GFLG-NHCH2CH2NH2-HCl) (200 mg), p-nitrophenyldihydrofumagyl-6-yl carbonate (200 mg), and DIEA (57 mg) in DMF (10 mL), the basic procedure F was followed. The product was purified by TFF (10K) using water (150 mL) and freeze-dried to obtain poly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagylol as a pale yellow solid (160 mg).

[0527] Synthesis of poly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagylol]:

[0528]

Chemical formula

[0529] Using poly(HPMA-co-MA-GFLG-NHCH2CH2CH2NH2-HCl) (220 mg), p-nitrophenylfumagyl-6-yl carbonate (110 mg), and DIEA (63 mg) in DMF (6 mL), the basic procedure F was followed. The solvent was evaporated and the resulting solution was diluted with water. The aqueous phase was extracted with ethyl acetate and purified by TFF using 350 mL of water. The concentrated water was freeze-dried to obtain poly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagylol] as a light pink powder (200 mg).

[0530] Synthesis of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagylol]:

[0531]

Chemical formula

[0532] Using poly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexylamine-HCl)] (1.0 g), p-nitrophenyl fumaroyl-6-yl carbonate (0.48 g), and DIEA (0.27 g) in DMF (25 mL), the basic procedure F was followed. The solvent was evaporated and the solution was diluted with water. The aqueous phase (300 mL) was extracted with ethyl acetate (total 700 mL) and purified by TFF using an additional 350 mL of water. Freeze-drying the concentrated water gave poly[HPMA-co-MA-GFLG-N-(4-aminocyclohexyl)carbamoyl fumaroylol] as a pale pink solid (0.9 g).

[0533] 1 H NMR (DMSO-d6): δ 8.10 - 8.35 (m, 3H, amide-NH), 7.90 - 8.10 (m, amide-NH), 7.05 - 7.32 (m, 22H, amide-NH) 5.27 (m, H-5-Fum), 5.18 (bt, alkene-Fum), 4.60 - 4.90 (m, 14H), 4.50 - 4.60 (m, 1H, α-proton of phenylalanine), 4.10 - 4.30 (m, 1H, α-proton of leucine), 3.40 - 3.80 (m, 21H), 3.27 (s, 3H, OMe-Fum), 2.80 - 3.20 (m, 33H), 2.56 (d, 1H, H = 3.90 Hz, H-2-Fum), 2.18 (m, 2H, allyl-Fum), 0.37 - 2.0 [m, 147H{1.70 (s, 3H, Fum-Me), 1.60 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum-Me)}].

[0534] Synthesis of poly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexyl)carbamoyl fumaroylol]:

[0535]

Chemical Structure

[0536] In 25 mL of DMF, poly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexylamine-HCl)] (1.0 g), p-nitrophenyl fumaroyl-6-yl carbonate (0.48 g), and DIEA (0.27 g) were used and the basic procedure F was followed. The solvent was evaporated and the solution was diluted with water. The aqueous phase (300 mL) was extracted with ethyl acetate (total 700 mL) and purified by TFF using an additional 350 mL of water. Freeze-drying the concentrated water gave poly[HPMA-co-MA-GFLG-N-(3-aminohexyl)carbamoyl fumaroylol] as a pale pink solid (0.9 g).

[0537] 1 1H NMR (DMSO-d6): δ 7.90 - 8.35 (m, 4H, amide - NH), 7.0 - 7.70 (m, 25H, phenylalanine and amide - NH), 5.26 (m, H - 5 - Fum), 5.18 (bt, alkene - Fum), 4.60 - 4.90 (m, 14H), 4.50 - 4.60 (m, 1H, α - proton of phenylalanine), 4.10 - 4.30 (m, 1H, α - proton of leucine), 3.40 - 3.80 (m, 21H), 3.26 (s, 3H, OMe - Fum), 2.80 - 3.10 (m, 31H), 2.17 (m, 2H, allyl - Fum), 0.37 - 2.0 [m, 166H {1.69 (s, 3H, Fum - Me), 1.59 (s, 3H, Fum - Me), 1.07 (s, 3H, Fum - Me)}].

[0538] Synthesis of poly[HPMA-co-MA-GFLG-N-[2-(4-aminophenyl)ethyl]carbamoyl fumaroylol]:

[0539]

Chemical Structure

[0540] A suspension of poly[HPMA-co-MA-GFLG-OH] (200 mg), N-[2-(4-aminophenyl)ethyl]carbamoyl fumagillol] (100 mg), and DIEA (75 mg) in DMF (6 mL) at 0 °C was added EDCI (total 44 mg) little by little. The solution was left to warm at room temperature and stirred overnight. The solvent was evaporated, the residue was suspended in water, and the suspension was extracted with EtOAc (7 times, total 250 mL). The aqueous phase was purified by TFF (10K) using water (350 mL). Freeze-drying the concentrated water gave the polymer as a white fluffy solid (170 mg).

[0541] Synthesis of poly[HPMA-co-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoyl fumagillol]:

[0542]

Chemical formula

[0543] To a solution of 2,2'-(ethylenedioxy)bis(ethylamine) (0.79 g, 5.34 mmol) in distilled water (20 mL) at 0 °C (pH = 11.56), concentrated HCl was added until the pH of the solution became 9.01 (measured with a pH meter). Poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 mL) and H2O (10 mL) was added dropwise to the amine-containing solution over 15 minutes, and the reaction mixture was stirred at 0 °C for 15 minutes. Then, the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. The pH of the solution was measured to be 8.15. The reaction mixture was diluted with distilled water (300 mL), filtered through a VacuCap filter, and the reaction flask was washed with water (100 mL). The polymer solution was concentrated to 40 mL by TFF (10K), washed with 25 mM NaCl (800 mL) to remove p-nitrophenol, then the pH was adjusted to 4 with 0.1 M HCl, and then washed with water (400 mL). Freeze-drying the pure polymer solution isolated poly[HPMA-co-MA-GFLG-NH-2-[2-(2-aminoethoxy)ethoxy]ethylamine-HCl] as a pink solid (800 mg, 78%).

[0544] A mixture of p-nitrophenyl fumaroyl-6-yl carbonate (93 mg, 0.208 mmol) and poly[HPMA-co-MA-GFLG-N-2-[(2-(2-aminoethoxy)]ethoxy)ethylamine-HCl] (200 mg, 0.104 mmol) in anhydrous DMF (5 mL) at 0 °C under N2 was added with DIEA (57 mg, 0.416 mmol). The reaction mixture was left to warm to room temperature and stirred for 12 h. The solvent was removed under reduced pressure, and the resulting residue was suspended in water (30 mL) and extracted with EtOAc (the aqueous and organic phases formed from the resulting emulsion were separated using centrifugation), and excess p-nitrophenyl fumaroyl-6-yl carbonate and p-nitrophenol were removed. Nitrogen was bubbled through the aqueous solution to remove traces of EtOAc, and the solution was purified by washing with water (150 mL) using TFF (5K) to remove DIEA hydrochloride. Freeze-drying the polymer solution gave the desired polymer complex poly[HPMA-co-MA-GFLG-N-2-[2-(2-aminoethoxy)ethoxyethyl]carbamoyl fumaroylrol] (220 mg, 95%) as an off-white solid.

[0545] Synthesis of poly[HPMA-co-MA-GFLG-NH-(6-aminodecyl)carbamoyl fumaroylrol]:

[0546]

Chemical formula

[0547] Under N₂ at 0 °C, to a mixture of p-nitrophenyl fumaroyl-6-yl carbonate (300 mg, 0.67 mmol) and poly[HPMA-co-MA-GFLG-N-10-[decylamine-HCl] (300 mg, 0.15 mmol; prepared in the same manner as in Example 33 except that 1,10-diaminodecane was used as the amine) in anhydrous DMF (6 mL) was added DIEA (83 mg, 0.64 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours. The solvent was removed under reduced pressure, and the resulting residue was suspended in water (30 mL) and extracted with EtOAc (the aqueous and organic phases formed from the resulting emulsion were separated using centrifugation), and excess p-nitrophenyl fumaroyl-6-yl carbonate and p-nitrophenol were removed. Nitrogen was passed through the aqueous solution to remove trace amounts of EtOAc. The crude aqueous solution was purified by washing with water (150 mL) using TFF (10K) to remove DIEA hydrochloride. Lyophilization of the polymer solution gave the desired polymer complex poly[HPMA-co-MA-GFLG-NH-(10-aminodecyl)carbamoyl fumaroylrol] (300 mg, 87%) as an off-white solid.

[0548] Synthesis of N-(2-acetamidoethyl)carbamoyl fumaroylrol:

[0549]

Chemical formula

[0550] To a solution of p-nitrophenyl fumaroyl-6-yl carbonate (200 mg) in ethanol (5 mL) at 0 °C was added N-(2-aminoethyl)acetamide (0.132 mL). The solution was stirred at 0 °C for 1 hour and at room temperature overnight. The reaction was diluted with ethyl acetate and washed with water. The aqueous phase was back-extracted with ethyl acetate, and the organic phases were combined and dried (MgSO₄). The crude product was purified by flash chromatography. The product was a yellow solid (120 mg).

[0551] Synthesis of the following compound:

[0552]

Chem.

[0553] To a solution of poly(HPMA-co-MA-GFLG-NHCH2CH2NH2-HCl) (200 mg) and N-(5-carboxypentyl)carbamoyl fumariglomycin (96 mg) in DMF (6 mL) at 0 °C was added DIEA (104 mg), followed by the addition of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (42 mg). The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated, and the residue was dissolved in water (50 mL) and extracted with ethyl acetate (200 mL). The aqueous phase was purified by TFF using water (450 mL). Lyophilization of the concentrated water gave the polymer (200 mg) as a pale yellow solid.

[0554] Synthesis of the following compound:

[0555]

Chem.

[0556] To a solution of poly[HPMA-co-MA-GFLG-N(CH2)6H2-HCl] (216 mg) and 2-carboxyethylcarbamoyl fumariglomycin (91 mg) in DMF (8 mL) at 0 °C was added DIEA (118 mg), followed by the addition of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (88 mg). The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated, and the residue was dissolved in water (50 mL) and extracted with ethyl acetate (200 mL). The aqueous phase was purified by TFF (10K) using water (1 L). Lyophilization of the concentrated water gave the polymer (170 mg) as a pale yellow solid.

[0557] Synthesis of the following compound:

[0558]

Chem.

[0559] Using poly(HPMA-co-MA-GFLG-NHCH2CH2CH2NH2-HCl) (220 mg) and carbonate (Example 24, 100 mg) in DMF (6 mL) together with DIEA (63 mg), the basic procedure F was followed. The reaction product was extracted with ethyl acetate. Following purification by TFF (10K) using water, and then lyophilization, the product was isolated as a pale pink powder (140 mg).

[0560] BocNHCH2CH2N(Me)CH2C(O)NHC(O)2-fumagillol-6-yl (alkylation of N-BOC, N'-methylethylenediamine with chloroacetylcarbamoyl fumagillol):

[0561]

Chemical Structure

[0562] A solution of TNP-470 (0.2 g) and DIEA (0.105 g) in DMF (3 mL) was cooled to 0 °C. A solution of tert-butyl N-[2-(methylamino)ethyl]carbamate (0.105 g) in DMF (3 mL) was added and the mixture was stirred at 0 °C for 3 hours and then overnight. The reaction product was diluted with ethyl acetate and extracted with water. The aqueous phase was back-extracted with ethyl acetate, the organic phases were combined and extracted with brine, dried (MgSO4), and evaporated to give an oil. The product fraction was purified by silica gel chromatography (methanol / methylene chloride) and evaporated to give BocNHCH2CH2N(Me)CH2C(O)NHC(O)2-fumagillol-6-yl as a white foam (0.16 g, 60%).

[0563] Reaction of tert-butyl N-[2-aminoethyl]carbamate with chloroacetylcarbamoyl fumagillol

[0564]

Chemical Structure

[0565] 30 μL aliquots of a 1 M solution of Boc-ethylenediamine in DMF were added to DMF (270 μL). The solution was cooled to 0 °C and a solution of TNP-470 (48 mg) in DMF (600 μL) was added dropwise over 2 minutes. The reaction was monitored by LC / MS. The maximum amount of the desired alkylated product observed was 34%. Carbamoyl fumagillol was also produced. The ratio of the desired product to carbamoyl fumagillol was 1.0 to 0.4. Attempts to isolate the desired product resulted in the isolation of hydantoin and fumagillol. Thus, the desired product could not be isolated due to its rate of decomposition. Thus, TNP-470 could not be alkylated according to the described method.

[0566] In vivo study in DIO C57B16 mice - body weight change, food intake, body composition Thirteen-week-old C57B16 male mice (N = 6) with an average body weight of 34 g were allowed free access to a high-fat diet (Harlan diet) TD.06414, which consists of 60% of Kcal from fat. On the first day of the study, the animals were randomly divided into groups such that the average body weight of each group was 33.9 g. The mice were treated with either phosphate-buffered saline (vehicle), TNP-470, or Compound 16 (subcutaneous administration on the back). Treatment was continued for 31 days at the doses and schedule shown in the table below. The animals were weighed every other day. Food intake was measured once a week. On day 33, gross observations were made and body composition was determined.

[0567] Figure 1 compares the weight loss in obese DIO mice after treatment with the fumagillol-compound (Compound 16) of the invention or TNP-470 (synthetic fumagillin analog) at different doses / regimens listed in Table 2.

[0568] [Table 4]

[0569] The results in Figure 1 show that the weight gain in the excipient control group was 16%, and the weight loss when compound 16 was administered in the q4d dosing regimen was 19%. Administration of compound 16 provides both therapeutic and preventive effects. Specifically, compound 16 induces or promotes weight loss and also prevents weight gain. Compound 16 is superior to TNP-470 in terms of the degree of weight loss. Compound 16 is superior to TNP-470 in that the dose of fumagillol is reduced.

[0570] Table 3 compares the body fat composition in obese DIO mice after treatment with compound 16 or TNP-470 at different doses / regimens described herein. Analyses were performed on day 33 for gross findings. The total fat in the excipient group was 13.2% when expressed as a percentage of body weight, while the total fat in the groups treated with compound 16 was 8.2% (1 mg / kg, qod) or 5.6% (6 mg / kg, q4d).

[0571]

Table 5

[0572] Figure 2 compares the average daily food intake in obese DIO mice after treatment with compound 16 or TNP-470 at different doses / regimens. The results in Figure 2 show a decreased food intake after treatment with compound 16, and compound 16 results in a greater decrease in food intake than TNP-470.

[0573] Figure 3 compares the body composition (fat vs. body weight) in obese DIO mice after treatment with compound 16 or TNP-470 at different doses / regimens. The results in Figure 3 show that the decreased body weight is directly correlated with fat loss.

[0574] In vivo study of DIO C57B16 mice - weight change, food intake, glucose tolerance, body composition dose response Fifteen-week-old male C57B16 mice (N = 6) with an average body weight of 42 g were allowed to freely consume TD.06414, a high-fat diet (Harlan diet) composed of 60% Kcal from fat. On the first day of the test, the animals were treated at different doses with either phosphate-buffered saline (vehicle) or Compound 16 (administered subcutaneously on the back). The treatment was continued for 29 days at the doses and schedule shown in the following table. The animals' body weights were measured every other day. The food intake was measured once a week. On the 24th day (the mice were most recently treated with Compound 16 on the 21st day), an intraperitoneal (IP) glucose tolerance test (GTT) was performed on the vehicle group and four groups treated with Compound 16 after an overnight fast. The body weights of each animal were measured, and baseline fasting glucose measurements were collected. Each animal was given a dose of 1 gram per kilogram of glucose as a 25% solution by intraperitoneal injection. Blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after intraperitoneal administration of glucose (via tail vein blood samples using the AlphaTRAK blood glucose monitoring system (including a glucose meter and test strips) commercially available from Abbott Laboratories (North Chicago, Illinois, USA)). The AlphaTRAK meter displays results from 20 to 750 mg / dL (1.1 to 41.7 mmol / L). On the 32nd day (the mice were most recently administered on the 29th day), the animals were fasted for 3 hours, their body weights were measured, blood was collected by cardiac puncture, gross findings were obtained, and body composition was determined. Blood analysis was performed by Idexx laboratories. Blood glucose was 278, 290, 265, 259, and 227 mg / dL for doses 0, 0.2, 0.6, 2.0, and 6.0, respectively. Blood urea nitrogen (BUN) was 21.8, 22.0, 19.7, 15.3, and 16.5 for doses 0, 0.2, 0.6, 2.0, and 6.0, respectively.

[0575] Table 4 shows blood glucose levels as a function of time and Compound 16 dose. Table 4 shows that as the dose of Compound 16 increases, the resulting blood glucose level decreases, and this is true even at the lowest dose of 0.2 mg / kg. These results are shown in Figure 4.

[0576] [Table 6]

[0577] Table 5 shows that when the dose of Compound 16 was increased, the result was a significant improvement in weight loss at a dose of more than 0.2 mg / kg q4d. Table 6 shows that the doses of 2 mg / kg q4d and 6 mg / kg q4d led to a significant decrease in food intake compared to the vehicle control, and that the food intake was dose-responsive. The one-week food intake of the 2 mg / kg group from Day 9 to 29 was 90% of that of the vehicle, while the food intake of the 6 mg / kg group was 75% of that of the vehicle.

[0578]

Table 7

[0579]

Table 8

[0580] Table 7 shows that adipose tissue was lost earlier than other tissues, as the mice in the control group had approximately 13% fat, while the mice in the 2 mg / kg·q4d group and the 6 mg / kg·q4d group had 11% and 10% fat, respectively.

[0581]

Table 9

[0582] The results in Figure 5 show that weight loss improved after treatment with Compound 16 at a dose of 0.6 mg / kg or more using the q4d schedule. The weight loss was dose-responsive, increasing as the dose increased.

[0583] The results in Table 8 show a decrease in cholesterol, triglyceride, HDL, LDL, and HDL / LDL ratio associated with an increase in the dose of Compound 16. These results are shown in Figure 6.

[0584]

Table 10

[0585] The results in Table 9 show favorable changes in alkaline phosphatase, SGPT, SGOT, and CPK accompanying the increased dosage of Compound 16.

[0586]

Table 11

[0587] Example Fifteen-week-old male C57B16 mice (N = 6) with an average body weight of 42 g were allowed free access to TD.06414, a high-fat diet (Harlan diet) in which 60% of the Kcal is composed of fat. On the first day of the test, the animals were treated with either phosphate-buffered saline (vehicle), or Compound 16, 28, 29, or 30 at a dosage of 2 mg / kg, or Compound 31 at a dosage of 6 mg / kg, based on a q4d schedule (administered subcutaneously in the back). The animals were weighed every other day. Figure 10 compares the weight loss in obese DIO mice after treatment with various conjugates of the present invention for 23 days. The results in Figure 10 show that as a result of only the change in the linker, there is a change in the degree of weight loss.

[0588] Example Male Sprague Dawley rats (N = 3) at 9 - 10 weeks of age with an average body weight of 300 g were allowed free access to a standard rodent diet (PharmaServ lab diet 5001). The rats were treated with compound 16 at either 100 mg / kg or 200 mg / kg (intravenously, via the tail vein) on days 1, 8, 15, 22, and 29. The body weights of the rats were measured regularly, and blood samples were collected on days 10, 17, and 24. For blood collection during survival, the rats were anesthetized with an inhalation mixture of 4% isoflurane and 1.5% oxygen, and then at least 1 mL of blood was collected by puncture of the retro - orbital plexus. On day 31, the animals were weighed, blood was collected by cardiac puncture, gross findings were obtained, and body composition was determined. There were no remarkable clinical findings regarding albumin, albumin / globulin ratio, alkaline phosphatase, ALT (SGPT), AST (SGOT), bicarbonate, direct bilirubin, indirect bilirubin, total bilirubin, BUN, BUN / creatinine ratio, calcium, chloride, cholesterol, CK, creatinine, globulin, glucose, phosphorus, potassium, sodium, sodium / potassium ratio, and total protein, as long as they were compared with the normal range and pre - dosing data. Apart from weight loss and other findings reported herein, the animals appeared generally normal and showed no signs of neurotoxicity such as ataxia, disorientation, tremors, or convulsions. The results in Figure 7 indicate that compound 16 is tolerated at high doses based on a q7d dosing schedule.

[0589] Example Male Sprague Dawley rats (N = 3, average body weight 350 g) were administered either vehicle, compound 1 (30 mg / kg), or compound 16 (200 mg / kg) by a single intravenous bolus. Blood samples were collected by submandibular venipuncture at 0, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 hours. Aliquots of each sample were diluted with methanol containing propranolol as an internal standard and analyzed by LC / MS / MS with a lower limit of quantification of 2.5 nM. In both cases of administration of compound 1 or compound 16, the analyte was compound 1. The half-life of the small molecule compound 1 was in the range of 10 - 15 minutes, the maximum plasma concentration (Cmax) was approximately 15 μM, and it occurred at T0. For the polymer complex of compound 16, the released small molecule exhibited a maximum plasma concentration of approximately 0.3 μM at about 3 hours and a terminal elimination half-life of 10 hours. These results are shown in Figure 9.

[0590] In vivo test of Example DIO Levin rats - body weight change, food intake, body composition, schedule - dose response, leptin levels: A study was conducted to evaluate the relative efficacy of compound 16, which is a fumagillol polymer complex, and compound 1, which is a small molecule fumagillol derivative, and CKD - 732 (also known as beloranib and ZGN - 433). CKD - 732 as referred to herein has the following structure:

[0591] [Chemical formula]

[0592] is the hemicitrate salt, and compound 1 was also tested in the form of the hemicitrate salt.

[0593] The test article was administered subcutaneously to diet-induced obesity (DIO) Levin-DS rat models based on a schedule of every four days (q4d). Also, the efficacy of Compound 16 was evaluated based on a once-weekly (q7d) dosing schedule. Diet intervention (standard Chow, Labdiet 5001; 3.4 kcal / g) was included for comparison with drug intervention. Male rats, just 3 weeks old, were allowed free access to Harlan TD.06414 pellets (5.1 kcal / g) in which 60% of the calories were from fat and 21% from carbohydrates. Prior to dosing, the rats were randomly divided into groups of 3 animals with an average body weight of 595 gm. The rats were treated with either phosphate-buffered saline (vehicle), Compound 16 or Compound 1, or CKD-732 (administered subcutaneously on the back). Compound 16 was dissolved in the vehicle. Compound 1 in the form of hemitartrate and CKD-732 in the form of hemitartrate were dissolved in ethanol and then diluted with the vehicle. All doses were at a volume of 5.0 ml / kg. Treatment was continued for 68 days at the doses and schedules shown in Table 10 below. Since the molecular weight of CKD-732 is 15% greater than that of Compound 1, CKD-732 was administered at 1.15 mg / kg, while Compound 1 was administered at 1 mg / kg for the purpose of comparison on a molar basis. On Day 1 at the first administration, the rats were 14 weeks old. Also, on Day 1, Group 2 was switched from a high-fat diet to a standard chow diet, while the other groups maintained a high-fat diet throughout the test period.

[0594]

Table 12

[0595] The animals had their body weights measured every other day. The food intake was measured once a week. Blood samples were taken approximately once a week throughout the test for the evaluation of serum chemistry including glucose and insulin. On Day 48, all rats were subjected to a 4-hour fasting oral glucose tolerance test (OGTT). The animals were administered 8 mL / kg of 25% glucose (2 g / kg) per os (PO). On Day 68, gross observations were made to determine the body composition.

[0596] Figure 11 shows the change in body weight with respect to the test day for each group. A significant decrease in body weight was observed in both the polymer complex administration groups of Q4D and Q7D. Treatment with Compound 16 at 3 mg / kg (Q4D) or 6 mg / kg (Q7D) showed a greater weight loss than the change to standard chow. At the end of the test, Compound 16 at 3 mg / kg based on the Q4D schedule showed a body weight 22.1% lower than the vehicle control and 6.2% lower than the rats based on the standard chow diet. Approximately 10 weeks later, treatment with Compound 16 at 6 mg / kg based on the Q7D schedule showed a body weight similar to that of the treatment with a 3 mg / kg dose based on the Q4D schedule. Compound 1 administered at 1 mg / kg and CDK-732 administered at 1.15 mg / kg based on the Q4D schedule showed body weights 3.9% or 3.2% lower than the vehicle. Compound 1 administered at 3 mg / kg based on the Q4D schedule showed a body weight 8.9% lower than the vehicle. The polymer complex is approximately 1 / 6 by weight of the active fumarigirole derivative.

[0597] Figure 12 shows the final body weight on day 68 for all groups as a function of the average daily fumarigirole exposure. There was no fumarigirole exposure in either the vehicle or the standard diet. Compound 16 showed a greater weight loss with a significantly lower fumarigirole exposure compared to Compound 1 or CKD-732 based on the same schedule as this polymer complex. All groups were administered based on the Q4D schedule, except for 6 mg / kg of Compound 16 administered at Q7D.

[0598]

Table 13

[0599] Figure 13 shows the decrease in serum insulin levels in male Levin DIO rats maintained on a 60% fat diet and administered the compounds of the present invention on the q4d (3 mg / kg) and q7d (6 mg / kg) schedules, compared to the cases of the standard diet intervention and the vehicle group.

[0600]

Table 14

[0601] Table 12 shows the change in fasting insulin levels in each group. All groups (except the vehicle control group) showed a decrease in insulin levels, illustrating that the compounds of the present invention can reduce insulin levels based on a less frequent dosing schedule.

[0602] Figure 14 shows the results of an oral glucose tolerance test (OGTT) on insulin levels in rats treated with the compounds of the present invention based on the q4d and q7d schedules, compared with the cases of standard diet intervention and the vehicle group. The standard diet intervention also resulted in lower insulin levels. Compared with the vehicle, the insulin levels remained decreased in the presence of abnormally high glucose levels, indicating that the level of insulin required to reduce blood glucose was lower (see Figure 15), suggesting an improvement / recovery of insulin sensitivity.

[0603] Figure 15 shows the decreased glucose levels over time with different treatments following an oral glucose challenge.

[0604]

Table 15

[0605] Figure 16 shows the product of glucose (mM / L) × insulin (uU / ml) / 22.5, which is an index commonly recognized for insulin sensitivity, in male Levin DIO rats (Matthews et al., Diabetologia (1985) 28, 412±419; Pickavance et al., British Journal of Pharmacology (1999) 128, 1570±1576).

[0606]

Table 16

[0607] Leptin, an adipocyte hormone, is a known appetite suppressant. Leptin resistance (abnormally high levels unrelated to food intake) is known to occur in patients and animals with diet-induced obesity (Levin et al., Am J Physiol Regul Integr Comp Physiol. 2002 Oct;283(4):R941-8). Low levels of leptin have been associated with hyperphagia (Sindelar et al., 1999, Enriori et al., 2006). Food intake was measured once a week. Serum leptin levels were measured on day 29 and plotted against food intake for the week including day 29. Animals on a standard chow diet were hyperphagic and showed significantly greater food intake relative to leptin levels than those shown by the compounds of the present invention. Treatment with Compound 1 did not result in a significant decrease in leptin levels. Figure 17 shows food intake in grams for each group for one week. The standard chow group showed a significant increase in food intake after switching from a high-fat diet to a standard diet. Hyperphagia is known to occur to maintain calorie intake.

[0608]

Table 17

[0609] Figure 18 shows the change from baseline in leptin levels in male Levin DIO rats maintained on a high-fat diet and treated with the complex of the present invention or a standard chow intervention. For Compound 16, a dose-dependent response was observed in the change from baseline in leptin levels.

[0610]

Table 18

[0611] Example In Vivo Tests in DIO Mice - Body Weight Change, Food Intake, Schedule - Dose Response Twenty-one-week-old male C57B1 / 6 mice with an average body weight of 46.8 g (N = 9 / group) were allowed free access to a high-fat diet consisting of 60% of Kcal from fat. The animals were administered according to the schedule in Table 17 below.

[0612]

Table 19

[0613] Compound administration was carried out at 9 - 10 am on the day of administration. Groups 6 and 7 received a total of 17 administrations. The q4d groups (1, 2, 3, 4, 8, 9) received a total of 9 administrations. The q8d group (5) received a total of 5 drug administrations. Body weight and food intake were measured every other day. Blood glucose was measured in the fed state at 9:00 am on days - 7, 0, 7, 14, 21, and 28 (blood glucose was measured before administration on the administration day). Blood glucose was measured with a glucometer. An intraperitoneal glucose tolerance test (ipGTT, 6 - hour fasting) was performed.

[0614] The test ended on the 34th day. The liver and epididymal white adipose tissue (eWAT) were collected, weighed, and stored at - 80°C. Serum was collected, and AST, ALT, ALP, CK, BUN, creatinine, calcium, potassium, sodium, chloride, total protein, albumin, total bilirubin, glucose, triglyceride, and cholesterol were determined. Insulin samples were measured using a commercially available kit.

[0615] What is called a polymer in this example is poly[HPMA - co - MA - GFLG - N-(6 - aminohexyl)acetamide], which is a polymer without fumarigirole. The synthesis of poly[HPMA - co - MA - GFLG - N-(6 - aminohexyl)acetamide] is described in WO 2011 / 150022 pamphlet, which is incorporated herein by reference in its entirety.

[0616] Structure of poly[HPMA - co - MA - GFLG - N-(6 - aminohexyl)acetamide]

[0617]

Chemical formula

[0618] Figure 19 shows the surprising and unexpected finding that the 12 mg / kg dose based on the Q8D schedule results in a greater initial weight loss than the 6 mg / kg dose based on the Q4D schedule. By the end of the study, the weight loss in the Q8D group had stopped, while the weight loss in the 6 mg / kg group appeared to continue. The polymer without fumagillol shows a weight change similar to that of the excipient.

[0619] Figure 20 shows that the small molecule CKD-732 (Compound B) administered based on the Q2D (QOD) schedule showed a better response than the same average daily dose administered based on the Q4D schedule. As expected, the small molecule showed a better response with more frequent dosing.

[0620] Figure 21 shows a decrease in food intake with the compounds of the present invention. Note the initial significant decrease in food intake, subsequent recovery, and then a periodic decrease-recovery pattern in the 12 mg / kg group based on the Q8D schedule.

[0621] Figure 22 shows significantly decreased insulin levels during ipGTT in male C57B16 mice maintained on a high-fat diet. The compounds of the present invention greatly reduce the amount of insulin secreted by β-cells in the presence of hyperglycemia, showing a decrease in resistance and an improvement in insulin sensitivity. Also note that fasting insulin decreased in all compound 16 groups of mice.

[0622] Figure 23 shows the change in total insulin AUC in male C57B16 mice maintained on a high-fat diet during a glucose challenge as a function of the treatment group.

[0623] Figure 24 shows that blood glucose decreased throughout the treatment period compared to the excipient and polymer groups.

[0624] Figure 25 shows the product of glucose (mg / dl) and insulin (μU / ml) / 405 (Akagiri et al., A Mouse Model of Metabolic Syndrome, J. Clin. Biochem. Nutr., 42, 150-157, March 2008), that is, HOMA-ir measurement (Bonora et al., Diabetes Care. 2002, 25, 1135-1141), which is an established measurement method for insulin resistance and a method for predicting cardiovascular disease.

[0625]

Table 20

[0626] Effectiveness of Various Compounds in the DIO Mouse Model C57B16 male mice (N = 6) were allowed free access to TD.06414, a high-fat diet (Harlan diet) in which 60% of the Kcal is composed of fat. On the first day of the test, the animals were randomly divided into groups so that the average body weight of the mice in each group was 47 g. The mice were treated with either phosphate-buffered saline (vehicle) or the compounds listed in Table 19 dissolved in the vehicle (administered subcutaneously in the back). The treatment was continued for 26 days at the doses and schedule shown in Table 19 below. The polymer referred to in this example is poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide], a polymer that does not contain fumagillol.

[0627]

Chem.

[0628] Compound cis-16 is Compound 16 in which 1,4-diaminocyclohexane is in the cis configuration rather than the trans configuration as shown in the illustration of Compound 16.

[0629] Compound aa is the reaction product of 2KDa MW methoxy-terminated PEG amine and p-nitrophenyl fumagyl-6-yl carbonate.

[0630]

Chem.

[0631] Compound bb is:

[0632]

Chem.

[0633] It is. The synthesis of poly[HPMA-co-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoyl fumagillol] is described in WO 2011 / 150022 pamphlet, and this document is incorporated herein by reference in its entirety.

[0634]

Table 21

Claims

Claim 1 A pharmaceutical composition for improving or restoring insulin sensitivity, wherein the subject is overweight or obese, and the pharmaceutical composition comprises a compound selected from the group consisting of the following formula: 【Chemical 1】 [Chemical Formula 2] A pharmaceutical composition comprising a compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

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