WO2017142424A1

WO2017142424A1 - Treatment of neurodegenerative disorders

Info

Publication number: WO2017142424A1
Application number: PCT/NZ2017/050013
Authority: WO
Inventors: Garth Cooper
Original assignee: Garth Cooper
Priority date: 2016-02-18
Filing date: 2017-02-09
Publication date: 2017-08-24

Abstract

Pharmaceutical compositions and methods for the treatment of subjects, including humans, who have, or are at risk for, Alzheimer's disease comprising tetramine copper chelators in combination with an agent effective to reduce one or more of encephalic glucose, encephalic sorbitol or encephalic fructose, such as amylin, GLP-1 agonists and DPP-IV inhibitors.

Description

TREATMENT OF NEURODEGENERATIVE DISORDERS

FIELD OF THE INVENTION

[0001] The present i nvention relates generally to compounds, compositions and methods of treatment, and include compounds, compositions and methods for treating Alzheimer's disease, for improving physiological and/or neurological deficits associated with Alzheimer's disease.

BACKGROUND TO THE INVENTION

[0002] The following incl udes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed invention, or that any publication or document that is specifical ly or implicitly referenced is prior art.

[0003] Alzheimer's disease (AD) is an age-related neurodegenerative disease associated with pathological characteristics of senile plaques and neurofibrillary tangles. It is the most common form of dementia in humans, with a progression from episodic memory problems to a general decline in cognitive function. In 2013, approximately 44 million people were estimated to be affected by dementia .

[0004] A number of mechanisms have been proposed as causative of AD. As yet, however, no curative treatment with proven disease modifying effects exists. [0005] Current treatments for AD are directed to treating cognitive and behavioural symptoms. Two types of drugs are currently used to treat cognitive symptoms of AD; cholinesterase inhibitors, which provide the neurotransmitter acetylcholine that is commonly depleted in the AD brain and thereby purportedly improve cel l-to-cell communication; and memantine hydrochloride (Namenda™), which is believed to inhibit signal transduction of N-methyl-d-aspartate (N MDA) receptors, and reportedly slows the progression of symptoms i n moderate to severe AD.

[0006] Other medications, such as antidepressants, are sometimes used to hel p control the behavioral symptoms associated with AD. [0007] There are no known approved treatments that are directed to the cause of AD, the underlying dysfunction or the resulting cell and tissue damage. Clearly there is a need for compounds and methods that limit or prevent damage associated with or causative of AD, as well as damage to organelles, cells and tissues that occurs as a consequence of AD.

[0008] There is also a need for compounds and methods that limit or prevent damage to cells and tissues that occurs directly or indirectly as a result of necrosis and/or inappropriate apoptosis associated with or causative of AD. Agents and methods that maintain neuronal and glial cell integrity represent novel protective agents with utility in limiting AD and AD-related dysfunction.

[0009] Copper chelation in combination with treatment to lower encephalic glucose, encephalic sorbitol, and/or encephalic fructose is proposed herei n to treat AD. Triethylenetetramine dihydrochloride, a chelating compound for removal of excess copper from the body, is prescribed for Wilson's disease patients who cannot tolerate penicillami ne. Triethylenetetramine dihydrochloride is /V,/V'-bis(2- aminoethyl)-l,2-ethanediamine dihydrochloride. It is a white to pale yellow crystalline hygroscopic powder. Syprine^® (triethylenetetramine dihydrochloride) is available as 250 mg capsules for oral admi nistration. See Siegemund R, et al., "Mode of action of triethylenetetramine dihydrochloride on copper metabolism in Wilson's disease," Acta Neurol. Scand. 83(6) : 364-6 (June 1991).

[0010] U.S. Patent Nos. 6,610,693, 6,348,465 and 6,951,890 provide copper chelators and other agents (e.g. , zinc which prevents copper absorption) to decrease copper values for the benefit of subjects suffering from diabetes and its

complications. See also, Cooper, G.J ., et al., "Treatment of diabetes with copper bindi ng compounds," U .S. Pat. App. No. 2005/0159489, published July 21, 2005;

Cooper, G.J ., et al., "Copper antagonist compounds," U .S. Pat. App. No.

2005/0159364, published July 21, 2005; Cooper, G.J., et a/., "Preventing and/or treating cardiovascular disease and/or associated heart failure," U .S. Pat. App. No. 2003/0203973, published October 30, 2003. These also relate to therapies usi ng copper antagonists, including triethylenetetramine, for example. Various experimental and clinical results are described in Cooper, G.J., et al., "Regeneration of the heart in diabetes mellitus by selective copper chelation," Diabetes 53: 2501- 2508 (2004) . See also Cooper. G.J., et al., "Demonstration of a Hyperglycemia- Driven Pathogenic Abnormality of Copper Homeostasis in Diabetes and Its

Reversability by Selective Chelation : Quantitative Comparisons Between the Biology of Copper and Eight Other N utritionally Essential Elements in Normal and Diabetic Subjects," Diabetes 54: 1468-1476 (2005) .

[0011] It is an object of the invention to go at least some way to fulfilling these needs and provide other related advantages, and/or to at least provide the publ ic with a useful choice. Those skilled in the art wil l recognize further advantages and benefits of the invention after readi ng the disclosure.

SUMMARY OF THE INVENTION

[0012] The invention descri bed and clai med herein have many attributes and embodiments including, but not l imited to, those set forth or described or referenced in this Brief Summary. It is not i ntended to be all-inclusive and the invention described and claimed herein are not limited to or by the features or embodiments identified in this Brief Summary, which is included for purposes of ill ustration only and not restriction.

[0013] The present i nvention relates generally to compounds, compositions and methods for treating Alzheimer's disease (AD).

[0014] The present invention is directed in part to the treatment of AD by administration to a mammalian subject in need thereof an effective amount of a copper binding tetramine compound and an effective amount of an agent effective to reduce the amount or concentration of one or more of encephalic gl ucose, encephalic sorbitol, or encephalic fructose in the mammalian subject.

[0015] The present i nvention is also directed in part to the treatment of AD by administration to a mammalian subject i n need thereof an effective amount of a) a compound according to Formula (I) or Formula (II), and

b) an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject.

[0016] In various embodiments, the copper binding tetramine compound binds Cu(II), for example, is a tetramine compound that is specific for Cu(II) over Cu(I) . Particularly contemplated tetramine compounds include triethylenetetramine (2,2,2 tetrami ne), 2,3,2 tetramine and 3,3,3 tetramine as well as salts, active metabolites, derivatives, and prodrugs thereof. [0017] In one embodiment, the tetramine compound is triethylenetetramine disuccinate.

[0018] In various embodiments, the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

[0019] In one example, the amyli n analogue is Symlin .

[0020] In various examples, the GLP-1 agonist is exenatide, liraglutide, lixisenatide, albiglutide, or dulaglutide, or any combination of two or more thereof.

[0021] In various examples, the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagliptin, Vildagli ptin, Saxagliptin,

Linagliptin, Anagliptin, Teneligliptin, Aloglipti n, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin.

[0022] In one example, the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linaglipti n, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia .

[0023] Administration of the copper binding tetramine compound and of the agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject may be si multaneous, sequential, or separate.

[0024] In one embodiment, administration is of triethylenetetramine disuccinate and amyl in or Syml in, and may be simultaneous, sequential, or separate

administration.

[0025] In one embodiment, administration is of triethylenetetramine disuccinate and one or more agents selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagli ptin, Anagliptin, Teneligliptin, Alogl iptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin, and may be simultaneous, sequential, or separate administration.

[0026] In one embodiment, administration is of triethylenetetramine disuccinate and one or more agents selected from the group comprising alogliptin, linaglipti n, saxagl iptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia, and may be si multaneous, sequential, or separate admi nistration.

[0027] Si milarly, administration of the one or more compounds of Formula (I), and/or the one or more compounds of Formula (II), and of the agent effective to reduce the amount or concentration of one or more of encephalic gl ucose, encephalic sorbitol, or encephalic fructose in the mammalian subject, may be simultaneous, sequential, or separate

[0028] In sti ll further embodiments, methods are provided for treating AD by administering

a) one or more copper binding tetramine compounds, one or more compounds of Formula (I), and/or one or more compounds of Formula (II), and

b) one or more agents effective to reduce the amount or concentration of one or more of encephal ic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject,

in the form of one or more pharmaceutical compositions.

[0029] Thus, pharmaceutical compositions are also provided comprising one or more copper binding tetramine compounds, compounds of Formula (I), or compounds of Formula (II), additionally comprising or formulated to be admi nistered in conjunction in with an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject, wherein the one or more pharmaceutical compositions comprises a pharmaceutically acceptable carrier or diluent.

[0030] In the context of the invention, AD includes tissue damage or degeneration associated with, caused by, or causative of AD, including damage or degeneration in which free radical mediated oxidative injury is involved, and damage or degeneration associated with AD i n which cells inappropriately undergo apoptosis. Thus, treatment or amel ioration of symptoms of AD, including cognitive and behavioural symptoms, are particularly contemplated .

[0031] In various embodiments, the mammalian subject is selected from the group consisti ng of a human, a domestic and farm ani mal, and zoo, sports, or pet animal, such as a dog, a horse, a cat, a sheep, a pig, a cow, or a deer. In a particularly contemplated example, the mammalian subject is a human .

[0032] In various embodiments, the mammalian subject is a non-diabetic subject, for example, a non-diabetic human subject, or a human subject who is not undergoing treatment for diabetes. In various embodiments, the mammalian subject does not suffer from Wilson's disease.

[0033] In one embodiment, the reduction in the amount or concentration of encephalic glucose, encephalic sorbitol, or encephal ic fructose is by a statistically significant amount. In one example, the reduction in the amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose is to an amount or concentration substantially equivalent to the amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose, respectively, found in a mammalian subject of the same species not suffering from AD. [0034] In one embodiment, the reduction in the amount or concentration of encephalic glucose is to an amount or concentration substantially equivalent to the amount or concentration of encephalic glucose found in a mammalian subject of the same species not suffering from AD.

[0035] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose in the hippocampus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the hippocampus of a mammalian subject of the same species not suffering from AD. [0036] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose i n the mammalian subject reduces glucose i n the entorhi nal cortex. In one embodiment, the reduction is to an amount or

concentration substantially equivalent to that found in the entorhinal cortex of a mammalian subject of the same species not suffering from AD.

[0037] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose in the middle- temporal gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the middle-temporal gyrus of a mammalian subject of the same species not suffering from AD.

[0038] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose in the sensory cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the sensory cortex of a mammalian subject of the same species not sufferi ng from AD.

[0039] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose i n the motor cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the motor cortex of a mammalian subject of the same species not suffering from AD. [0040] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose in the ci ngulate gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the cingulate gyrus of a mammalian subject of the same species not sufferi ng from AD.

[0041] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces glucose in the cerebel lum. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the cerebel lum of a mammalian subject of the same species not suffering from AD.

[0042] In one embodiment, the reduction in the amount or concentration of encephalic sorbitol is to an amount or concentration substantially equivalent to the amount or concentration of encephalic sorbitol found in a mammalian subject of the same species not suffering from AD.

[0043] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the hippocampus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the hippocampus of a mammalian subject of the same species not suffering from AD.

[0044] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces sorbitol in the entorhi nal cortex. In one embodiment, the reduction is to an amount or

[0045] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the middle- temporal gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the middle-temporal gyrus of a mammalian subject of the same species not suffering from AD. [0046] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the sensory cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the sensory cortex of a mammalian subject of the same species not sufferi ng from AD.

[0047] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the motor cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the motor cortex of a mammalian subject of the same species not suffering from AD.

[0048] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the ci ngulate gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the ci ngulate gyrus of a mammalian subject of the same species not sufferi ng from AD.

[0049] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces sorbitol in the cerebel lum. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the cerebel lum of a mammalian subject of the same species not suffering from AD. [0050] In one embodiment, the reduction in the amount or concentration of encephalic fructose is to an amount or concentration substantially equivalent to the amount or concentration of encephalic fructose found in a mammalian subject of the same species not suffering from AD. [0051] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces fructose in the hippocampus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the hippocampus of a mammalian subject of the same species not suffering from AD.

[0052] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces fructose in the entorhi nal cortex. In one embodiment, the reduction is to an amount or

[0053] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces fructose in the middle- temporal gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the middle-temporal gyrus of a mammalian subject of the same species not suffering from AD.

[0054] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces fructose in the sensory cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the sensory cortex of a mammalian subject of the same species not sufferi ng from AD.

[0055] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces fructose i n the motor cortex. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the motor cortex of a mammalian subject of the same species not suffering from AD. [0056] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephal ic fructose in the mammalian subject reduces fructose in the ci ngulate gyrus. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the cingulate gyrus of a mammalian subject of the same species not sufferi ng from AD.

[0057] In one embodiment, administration of the one or more agents effective to reduce the amount or concentration of encephalic glucose, encephalic sorbitol, and/or encephalic fructose in the mammalian subject reduces fructose in the cerebel lum. In one embodiment, the reduction is to an amount or concentration substantially equivalent to that found in the cerebel lum of a mammalian subject of the same species not suffering from AD.

[0058] Diagnostic or prognostic methods for determining the presence or likel ihood of developing AD are also provided.

[0059] In one embodiment, the invention relates to a method of assessing a mammalian subject's risk of developing AD which comprises:

analysing a encephalic sample from said subject to determine the amount or concentration of glucose, fructose, or sorbitol in the sample, and

comparing the amount or concentration of glucose, fructose, or sorbitol in the sample to the amount or concentration of glucose, fructose, or sorbitol found i n a mammalian subject of the same species not suffering from AD,

wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose in the sample is indicative of an increased risk of developi ng AD.

[0060] In one embodiment, an elevated concentration of encephalic glucose in a sample from a human subject i ndicative of an increased risk of developing AD is above about 10 μιτιοΙ glucose/g wet weight.

[0061] In one embodiment, the i nvention relates to a method of diagnosing AD in a mammalian subject, the method comprising

comparing the amount or concentration of glucose, fructose, or sorbitol in the sample to the amount or concentration of glucose, fructose, or sorbitol found i n a mammalian subject of the same species not suffering from AD, wherein an elevated amount or concentration of encephalic glucose, encephalic sorbitol, or encephalic fructose i n the sample is indicative of the presence of AD in the mammalian subject.

[0062] In one embodiment, an elevated concentration of encephalic glucose in a sample from a human subject indicative of the presence of AD is above about 10 μιτιοΙ glucose/g wet weight.

[0063] In a further aspect, the invention relates to the use of

in the treatment of AD.

[0064] In another aspect, the invention relates to the use of

a) one or more copper binding tetramine compounds, one or more compounds of

Formula (I), and/or one or more compounds of Formula (II), and

in the preparation of a medicament for the treatment of AD.

[0065] In various embodiments, the invention concerns pharmaceutical compositions containing such agents, articles and kits and delivery devices containing such agents, and tablets and capsules and formulations comprising such agents or compositions. Particularly contemplated are articles, kits and delivery devices enabling the separate, sequential or simultaneous administration of (a) the one or more copper binding tetramine compounds, one or more compounds of Formula (I), and/or one or more compounds of Formula (II), and of (b) the one or more agents effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephal ic fructose in a mammalian subject.

[0066] Pharmaceutical compositions also comprise a pharmaceutically acceptable carrier or dil uent.

[0067] Useful copper chelating compounds include pharmaceutically acceptable polyami nes, including copper-binding polyamines. Polyamines may include, for example, spermidine, as well as spermine and other tetramines. Tetramines also include, for example, triethylenetetramine (for example, 2,2,2 tetramine or 2,3,2 tetrami ne), as well as salts, active metabolites, derivatives, and prodrugs thereof. Salts incl ude, for example, triethylenetetrami ne hydrochloride salts (e.g., triethylenetetrami ne dihydrochloride, triethylenetetramine tetrahydrochloride,) and succinate salts (e.g., triethylenetetramine disuccinate), as well as maleate salts (e.g., triethylenetetramine tetramaleate) and fumarate salts (e.g.,

triethylenetetramine tetrafumarate). Metabolites include, for example, acetylated metabolites, such as N-acetyl triethylenetetramine (e.g ., monoacetyl- triethylenetetramine). Derivatives include, for example, PEG-modified tetramines, including PEG-modified triethylenetetramines. Other useful compounds incl ude pharmaceutically acceptable compounds of Formula I and Formula II herein .

Suitable copper antagonists i ncl ude, for example, penicillamine, N-methylglycine, N- acetylpenicillamine, tetrathiomolybdate, l,8-diamino-3, 6, 10, 13, 16, 19-hexa- azabicyclo[6.6.6]icosane, Ν, Ν'-diethyldithiocarbamate, bathocuproinedisulfonic acid, and bathocuprinedisulfonate.

[0068] Other suitable copper chelating compounds include, for example, pharmaceutically acceptable linear or branched tetramines capable of binding copper.

[0069] Such compounds may be administered i n amounts, for example, that are effective to chelate encephalic Cu(II). Such compositions include, for example, tablets, capsules, solutions and suspensions for parenteral and oral delivery forms and formulations.

[0070] In various embodiments, the therapeutic methods described herein are carried out in conjunction with dietary or lifestyle modifications. The simplest of these regimens can be the provision to a subject with AD of motivation to i mplement such a lifestyle change, for example, dietary adjustments to reduce foods

contributing to elevated brain urea concentration observed in AD, such as high protein foods.

[0071] These and other aspects of the invention, which are not limited to or by the information in this Brief Summary, are provided below. [0072] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). [0073] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combi nations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0074] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is general ly for the purpose of providing a context for discussing the features of the present invention. Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

[0075] The term "comprisi ng" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

BRIEF DESCRIPTION OF TH E DRAWINGS

[0076] The invention will now be described by way of example only and with reference to the drawings in which :

[0077] Figure 1 shows regional A) brain copper, and B) mean glucose levels in the cerebel lum (CB), cingulate gyrus (CG), entorhinal cortex (ENT), hippocampus (HP), motor cortex (MCx), middle temporal gyrus (MTG), and sensory cortex (SCx) of patients with AD (n = 7-9/region; on right) and control patients (n = 7-9/region on left).

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention combines copper chelation with treatment to lower levels of glucose, fructose and/or sorbitol in the brain to treat or prevent AD.

[0079] As presented in the Examples, the applicant has demonstrated that 1) levels of glucose, sorbitol and fructose are elevated across many regions of the AD brain, and 2) the AD brain is copper-deficient. [0080] Without wishing to be bound by any theory, the applicant believes that elevated levels of gl ucose, fructose and sorbitol in the brain are l ikely to be toxic via the formation of advanced glycation endproducts (AGEs) such as N-epsilon-carboxy- methylysine (CML).

[0081] The formation of AGEs in the brain creates pathogenic copper (II) binding sites in the extracellular matrix (ECM) (Kamalov et al., 2015. Org. Biomol. Chem. 14: 13( 1) : 3058-3063). Copper bound to AGEs such as CML is catalytically active and therefore toxic.

[0082] The applicant believes that ECM-bound copper (II) causes deficient neuronal copper uptake via suppression of membrane-bound copper uptake mediated by copper transporter CTR1 and intracellular copper transport via copper chaperones (see Zhang et al ., 2014 Cardiovascular Diabetology 13: 100).

[0083] It has been shown that CML-bound copper itself catalyses glucose- mediated AGE formation in a feed-forward reaction (Brings et al ., 2015. Biochim. Biophys. Acta 1852(8) : 1610-1618).

[0084] Physiological copper cations are crucial to the function of enzymes involved in processes including tissue antioxidant defence, suppression of inflammation and effective utilisation of metabolic fuels. Enzymes that are targeted and repaired by copper chelation include superoxide dismutase I (SODl), superoxide dismutase III (SOD3), cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit II (COII) .

[0085] Reduction of copper avai lable to be incorporated into these enzymes as seen in the AD brain results in impaired function of the enzymes, which may result in mitochondrial dysfunction. For example, a reduction in cytochrome c oxidase activity leads to increased electron leaki ng from the mitochondrial and oxidative stress.

[0086] The combination of copper chelation and glucose-lowering treatment inhibits AGE formation and binding of copper to AGEs such as CML, thus reducing AGE-bound copper-induced suppression of copper transport in the brain. The method of the invention thus suppresses AGE formation, ameliorates copper deficiency and restores enzyme and mitochondrial function in the brain .

[0087] As used herein, a "copper antagonist" is a pharmaceutically acceptable compound that binds or chelates copper in vivo for removal . Copper chelators are presently preferred copper antagonists. Copper (II) chelators, and copper (II) specific chelators {i.e., those that preferentially bind copper (II) over other forms of copper such as copper (I)), are especially preferred.

[0088] As used herein, "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids the like. When a compound is basic, for example, salts may be prepared from pharmaceutically acceptable nontoxic acids, includi ng i norganic and organic acids. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are hydrochloric and succinic acid copper antagonist salts. Succinic acid copper antagonist salts are most preferred, particularly for those copper antagonist salts that are not anhydrous. [0089] As used herein, "preventing" means preventi ng in whole or in part, or ameliorating or controlling.

[0090] As used herein, a "therapeutically effective amount" in reference to the compounds or compositions of the instant invention refers to the amount sufficient to induce a desired biological, pharmaceutical, or therapeutic result. That result can be alleviation of the signs, symptoms, or causes of a disease or disorder or condition, or any other desired alteration of a biological system. In one aspect of the present inventions, the result wil l involve the prevention, decrease, or reversal of AD, in whole or in part, and prevention and/or treatment of related conditions, i ncluding those referenced herein. [0091] As used herein, the term "treati ng" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those prone to having the disorder, or those diagnosed with the disorder, or those in which the disorder is to be prevented .

[0092] As used herein, encephal ic glucose refers the glucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly

contemplates glucose present in one or more tissues or regions of the brai n. Methods to determine the amount or concentration of encephalic glucose are known i n the art, and exemplary methods are provided herein in the Examples. [0093] As used herein, encephal ic fructose refers the gl ucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly

contemplates fructose present i n one or more tissues or regions of the brain. Methods to determine the amount or concentration of encephal ic fructose are known in the art, and exemplary methods are provided herein in the Examples.

[0094] As used herein, encephal ic sorbitol refers the glucose present in the brain and/or encephalic cavity of a mammalian subject, and particularly

contemplates sorbitol present in one or more tissues or regions of the brain . Method to determine the amount or concentration of encephal ic sorbitol are known in the art, and exemplary methods are provided herein in the Examples.

[0095] It will be appreciated that agents that are effective to lower encephalic glucose, sorbitol, or fructose without a concomitant risk of hypoglycaemia are preferred . Representative examples of such agents are discussed below.

Amylin and amylin analogues [0096] Amylin is a small peptide hormone released into the bloodstream by the pancreatic β-cells after a meal, and reportedly modulates blood glucose by slowing gastric emptying, promoting satiety, and inhibiting inappropriate secretion of glucagon. Amyl in analogues, such as symlin (Pramlintide™, AstraZeneca), are used to augment endogenous amylin, or to replace amylin's function in diabetics who do not naturally produce amylin.

GLP-1 agonists

[0097] GLP-1 is a naturally-occurring peptide that is released within minutes of eating a meal . It has been reported to suppress glucagon secretion from pancreatic alpha cells and sti mulate insulin secretion by pancreatic beta cells. GLP-1 receptor agonists are generally used in the treatment of type 2 diabetes. Representative GLP-

1 agonists suitable for use in the methods and compositions described herein include exenatide (Byetta™/Bydureon™, AstraZeneca, approved in 2005/2012), l iraglutide (Victoza™, Saxenda™, Novo Nordisk, FDA approved 2010), lixisenatide (Lyxumia™, Zealand Pharma & Sanofi, approved i n EU 2013), albiglutide (Eperzan™, Tanzeum™, GSK, FDA approved in 2014), and dulaglutide (Trulicity™, Eli Lily, FDA approved in

2014). Selective dipeptidyl peptidase (DPP-IV) inhibitors

[0098] Dipeptidyl peptidase (DPP-IV) inhibitors have been reported to reduce glucagon and blood glucose levels via increasing insulin levels. Representative DPP- IV inhibitors suitable for use i n the method and compositions described herein include Sitaglipti n (FDA approved 2006, marketed by Merck & Co. as Januvia™),

Vildagliptin (EU approved 2007, marketed in the EU by Novartis as Galvus™), Saxaglipti n (FDA approved in 2009, marketed as Onglyza™), Linagliptin (FDA approved in 2011, marketed as Tradjenta™ by Eli Lilly Co and Boehri nger

Ingelheim), Anagl iptin (approved in Japan in 2012, marketed by Sanwa Kagaku Kenkyusho Co., Ltd . and Kowa Company, Ltd .), Teneligliptin (approved in Japan i n

2012), Alogliptin (FDA approved 2013, marketed by Takeda Pharmaceutical

Company), Trelagliptin (approved for use in Japan i n 2015), Gemigliptin (LG Life Sciences), Dutogliptin (Phenomix Corporation), and Omarigliptin (approved in Japan in 2015, developed by Merck & Co.). Copper binding compounds

[0099] Suitable copper-chelating compounds i nclude copper binding polyamine compound, polyamine compounds that bind Cu⁺², and preferably polyamine compounds that are specific for Cu⁺² over Cu^{+ 1}. Polyamine compounds may incl ude, for example, spermine, as well as spermidine and other tetramines. Preferred tetrami ne compounds i nclude triethylenetetramine (2,2,2 tetramine), 2,3,2 tetramine and 3,3,3 tetramine as well as salts, active metabolites, derivatives, and prodrugs thereof. Other pharmaceutically acceptable polyamines are also contemplated .

[00100] Nitrogen-containing copper antagonists, for example, such as, for example, triethylenetetramine, that can be delivered as a salt(s) (such as acid addition salts, e.g., triethylenetetramine disuccinate or triethylenetetramine dihydrochloride) act as copper-chelating agents or antagonists, which aids the elimination of copper from the body by formi ng a stable soluble complex that is readily excreted by the kidney. Thus inorganic acids can be used, e.g., sulfuric acid, nitric acid, hydrohal ic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid . This is not an exhaustive list.

Other organic acids can be used to prepare suitable salt forms, in particular aliphatic, alicyclic, arali phatic, aromatic or heterocyclic mono-or polybasic carboxyl ic, sulfonic or sulfuric acids, (e.g., formic acid, acetic acid, propionic acid, pival ic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenemono-and-disulfonic acids, and laurylsulfuric acid) . Hydrochloric acid and succinic acid salts are preferred, and succinic acid salts are most preferred . Those i n the art will be able to prepare other suitable salt forms.

[00101] Nitrogen-containing copper antagonists, for example, such as, for example, triethylenetetramine, can also be in the form of quarternary ammonium salts in which the nitrogen atom carries a suitable organic group such as an al kyl, alkenyl, alkynyl or aralkyl moiety. In one embodiment such nitrogen-containing copper antagonists are in the form of a compound or buffered in solution and/or suspension to a near neutral pH much lower than the pH 14 of a solution of triethylenetetramine itself.

[00102] Other copper antagonists include derivatives, for example,

triethylenetetramine in combination with picolinic acid (2-pyridi necarboxylic acid). These derivatives include, for example, triethylenetetramine picolinate and salts of triethylenetetramine picoli nate, for example, triethylenetetramine picolinate HCI . They also include, for example, triethylenetetramine di-picolinate and salts of triethylenetetramine di-picoli nate, for example, triethylenetetramine di-picolinate HCI . Picolinic acid moieties may be attached to triethylenetetramine, for example one or more of the CH2 moieties, using chemical techniques known in the art. Those in the art wil l be able to prepare other suitable derivatives, for example, triethylenetetramine-PEG derivatives, which may be useful for particular dosage forms including oral dosage forms having increased bioavailability.

[00103] Other compounds include cyclic and acycl ic compounds according to the following formulae, for example:

FORMULA I

[00104] Tetra-heteroatom acyclic compounds within Formula I are provided where Xi, X2, X3, and X4 are independently chosen from the atoms N, S or 0, such that, (a) for a four-nitrogen series, i.e., when Xi, X2, X3, and X4 are N then : Ri, R2, R3, R4, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched al kyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and, R7, Rs, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 al kyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or several of Ri, R2, R3, R4, R5, or Rs may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- C10 alkyl-NH-protein, C1-C10 alkyl-N H-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S- protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization i nclude but are not limited to Cl- C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

(b) for a first three-nitrogen series, i.e. , when Xi, X2, X3, are N and X4 is S or 0 then : Rs does not exist; Ri, R2, R3, R4 and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and, R7, Rs, R9, Rio, Rn, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or several of Ri, R2, R3, R4, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein .

(c) for a second three-nitrogen series, i. e., when Xi, X2, and X4 are N and X3 is 0 or S then : R4 does not exist and Ri, R2, R3, R5, and Rs are independently chosen from H, CH3, C2- CIO straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and, R7, Rs, R9, Rio, Rn, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or several of Ri, R2, R3, Rs, or Rs may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein .

(d) for a first two-nitrogen series, i.e., when X2 and X3 are N and Xi and X4 are O or S then : Ri and Rs do not exist; R2, R3, R4, and Rs are independently chosen from H, CH3, C2- C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, Rn, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyl heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of R2, R3, F , or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to Cl-ClO alkyl-CO-peptide, Cl-ClO alkyl-CO-protein, Cl-ClO alkyl-CO- PEG, Cl-ClO alkyl-NH-peptide, Cl-ClO alkyl-N H-protein, Cl-ClO alkyl-NH-CO-PEG, Cl-ClO alkyl-S-peptide, Cl-ClO alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to Cl-ClO alkyl-CO-peptide, Cl-ClO alkyl-CO- protein, Cl-ClO alkyl-CO-PEG, Cl-ClO alkyl-N H-peptide, Cl-ClO alkyl-NH-protein, Cl-ClO alkyl-NH-CO-PEG, Cl-ClO alkyl-S-peptide, and Cl-ClO alkyl-S-protein .

(e) for a second two-nitrogen series, i.e. , when Xi and X3 are N and X2 and X4 are 0 or

S then : R3 and Rs do not exist; Ri, R2, R4, and R5 are independently chosen from H, CH3, C2- C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, Cl- C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 al kyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, Rn, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyl heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of Ri, R2, R4, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities i n order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to Cl-ClO alkyl-CO-peptide, Cl-ClO alkyl-CO-protein, Cl-ClO alkyl-CO- PEG, Cl-ClO alkyl-NH-peptide, Cl-ClO alkyl-N H-protein, Cl-ClO alkyl-NH-CO-PEG, Cl-ClO alkyl-S-peptide, and Cl-ClO alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to Cl-ClO alkyl-CO-peptide, Cl-ClO alkyl-CO- protein, Cl-ClO alkyl-CO-PEG, Cl-ClO alkyl-N H-peptide, Cl-ClO alkyl-NH-protein, Cl-ClO alkyl-NH-CO-PEG, Cl-ClO alkyl-S-peptide, and Cl-ClO alkyl-S-protein .

(f) for a third two-nitrogen series, i.e., when Xi, and X2 are N and X3 and X4 are 0 or S then : R4 and Rs do not exist; Ri, R2, R3, and R5 are independently chosen from H, CH3, C2- CIO straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, R11, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyi heteroaryl, C1-C6 alkyi fused aryl. In addition, one or several of Ri, R2, R3, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities i n order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, R11, or R12 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein .

(g) for a fourth two-nitrogen series, i.e., when Xi and X4 are N and X2 and X3 are 0 or S then : R3 and R4 do not exist; Ri, R2, R5 and Rs are independently chosen from H, CH3, C2- C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, n2, and n3 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, R11, and R12 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, Cl- C5 alkyi heteroaryl, C1-C6 alkyi fused aryl. In addition, one or several of Ri, R2, R5, or Rs may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities i n order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, R11, or R12 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein .

[00105] Second, for a tetra-heteroatom series of cyclic analogues, one of Ri and R2 and one of Rs and Rs are joined together to form the bridging group (CRi3Ri4)n4, and Xi, X2, X3, and X4 are independently chosen from the atoms N, S or 0 such that,

(a) for a four-nitrogen series, i.e., when Xi, X2, X3, and X4 are N then : R2, R3, R4, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloal kyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH) ; n l, n2, n3, and n4 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl . In addition, one or several of R2, R3, R4, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization i nclude but are not limited to Cl- C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, Cl- CIO alkyl-S-protein. Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

(b) for a three-nitrogen series, i.e., when Xi, X2, X3, are N and X4 is S or 0 then : R5 does not exist; R2, R3, and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ; nl, n2, n3, and n4 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, Rn, R12, R13 and R14 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloal kyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 al kyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or several of R2, R3 or F may be functional ized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half-lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, Cl- C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S- peptide, and C1-C10 al kyl-S-protein. Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or Ri4 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein .

(c) for a first two-nitrogen series, i.e. , when X2 and X3 are N and Xi and X4 are 0 or S then : R2 and R5 do not exist; R3 and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ; nl, n2, n3, and n4 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, Rn, R12, R13 and R14 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloal kyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or both of R3, or R4 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half-lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, CI- CIO alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl- S-protein .

(d) for a second two-nitrogen series, i.e., when Xi and X3 are N and X2 and X4 are 0 or S then : R3 and R5 do not exist; R2 and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyi, C1-C6 alkyl C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, Cl- C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 al kyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ; nl, n2, n3, and n4 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloal kyi, C1-C6 alkyl C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl . In addition, one or both of R2, or R4 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half-lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO- protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- CIO alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl- S-protein .

(e) for a one-nitrogen series, i.e., when Xi is N and X2, X3 and X4 are 0 or S then : R3, R4 and R5 do not exist; R2 is i ndependently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyi, C1-C6 alkyl C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ; nl, n2, n3, and n4 are independently chosen to be 2 or 3; and R7, Rs, R9, Rio, Rn, R12, R13 and R14 are independently chosen from H, CH₃, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyi, C1-C6 alkyl C3-C10 cycloal kyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 al kyl heteroaryl, C1-C6 alkyl fused aryl. In addition, R2 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl- S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functional ized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, Cl- C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S- peptide, and C1-C10 alkyl-S-protein.

FORMULA II

[00106] Tri-heteroatom compounds within Formula II are provided where Xi, X2, and X3 are independently chosen from the atoms N, S or 0 such that,

(a) for a three-nitrogen series, when Xi, X2, and X3 are N then : Ri, R2, R3, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloal kyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl, and n2 are independently chosen to be 2 or 3; and R₇, Rs, R9, and Rio are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl . In addition, one or several of Ri, R2, R3, Rs or Rs may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl- CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, Cl- CIO alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half-l ives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl- CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, Cl- C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

(b) for a first two-nitrogen series, when Xi and X2 are N and X3 is S or 0 then : R3 does not exist; Ri, R2, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH); n l, and n2 are independently chosen to be 2 or 3; and R7, Rs, R9, and Rio are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 al kyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of Ri, R2, R5 or Rs may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl- S-protein . Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half- lives of the constructs. Examples of such functionalization i nclude but are not limited to Cl- C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

(c) for a second, two-nitrogen series, when Xi and X2 are N and X3 is 0 or S then : R5 does not exist; Ri, R2, R3, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 al kyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH); nl and n2 are independently chosen to be 2 or 3; and R7, Rs, R9, and Rio are i ndependently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, Cl- C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 al kyl fused aryl . In addition, one or several of Ri, R2, R5, or Rs may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization i nclude but are not limited to Cl- C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half-lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, Cl- C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S- peptide, and C1-C10 alkyl-S-protein.

[00107] A series of tri-heteroatom cyclic analogues according to the above Formula II are provided in which Ri and Rs are joined together to form the bridging group (CRnRi2)n3, and Xi, X2 and X3 are independently chosen from the atoms N, S or 0 such that:

(a) for a three-nitrogen series, when Xi, X2, and X3 are N then : R2, R3, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloal kyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH2PO(OH)2, CH2P(CH3)0(OH) ; nl, n2, and n3 are independently chosen to be 2 or 3; and R7, Re, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 al kyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or several of R2, R3, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl- NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein . Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization i nclude but are not limited to Cl- CIO alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-N H- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. (b) for a two-nitrogen series, when Xi and X2 are N and X3 is S or 0 then : R5 does not exist; R2, and R3 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH) ; nl, n2, and n3 are independently chosen to be 2 or 3; and R7, R8, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 al kyi heteroaryl, C1-C6 alkyi fused aryl . In addition, one or both of R2 or R3 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverabil ity and/or half-l ives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl- CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, Cl- C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl- CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl- S-protein .

(c) for a one-nitrogen series, when Xi is N and X2 and X3 are 0 or S then :

R3 and R5 do not exist; R2 is independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ; nl, n2, and n3 are independently chosen to be 2 or 3; and R₇,

Rs, R9, Rio, Rn, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl . In addition, R2 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl- NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n. Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, Cl- C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

[00108] The compounds of the i nvention, includi ng triethylenetetramine active agents, may be made using any of a variety of chemical synthesis, isolation, and purification methods known in the art.

[00109] In sti ll further embodiments, methods are provided for treating AD by administering one or more copper binding tetramine compounds, compounds of

Formula (I), or compounds of Formula (II), in the form of a pharmaceutical composition. Thus, pharmaceutical compositions are also provided comprising one or more copper binding tetramine compounds, compounds of Formula (I), or compounds of Formula (II), in combination with a pharmaceutically acceptable carrier or dil uent.

[00110] Copper antagonists useful in the invention also include copper chelators that have been pre-complexed with a non-copper metal ion prior to administration for therapy. Metal ions used for pre-complexing have a lower association constant for the copper antagonist than that of copper. For example, a metal ion for pre- complexing a copper antagonist that chelates Cu²⁺ is one that has a lower bi nding affinity for the copper antagonist than Cu²⁺. Preferred metal ions for precomplexing include calcium (e.g. , Ca²⁺), magnesium (e.g., Mg²⁺), chromium (e.g., Cr²⁺ and Cr³⁺), manganese (e.g. , Mn²⁺), zinc (e.g. , Zn²⁺), selenium (e.g., Se⁴⁺), and iron (e.g. , Fe²⁺ and Fe³⁺). Most preferred metal ions for precomplexing are calci um, zinc, and iron. Other metals include, for example, cobalt (e.g. , Co²⁺), nickel (e.g. , Ni²⁺), si lver (e.g. , Ag¹⁺), and bismuth (e.g., Bi³⁺). Metals are chosen with regard, for example, to their relative binding to the copper antagonist, and relative to toxicity and the dose of the copper antagonist to be administered.

[00111] Also encompassed are metal complexes comprising copper antagonists and non-copper metals (that have lower binding affinities than copper for the copper antagonist) and one or more additional ligands than typically found in complexes of that metal . These additional ligands may serve to block sites of entry i nto the complex for water, oxygen, hydroxide, or other species that may undesirably complex with the metal ion and can cause degradation of the copper antagonist. For example, copper complexes of triethylenetetramine have been found to form pentacoordinate complexes with a tetracoordinated triethylenetetrami ne and a chloride ligand when crystallized from a salt solution rather than a tetracoordi nate Cu²⁺ triethylenetetramine complex. In this regard, 219 mg of triethylenetetramine ^■ 2 HCI were dissolved in 50 ml, and 170 mg of CuC ^■ 2H20 were dissolved in 25 ml ethanol (95%) . After addition of the CuC solution to the triethylenetetrami ne solution, the color changed from light to dark blue and white crystals precipitated.

The crystals were dissolved by addition of a solution of 80 mg NaOH i n 15 ml H20. After the solvent was evaporated, the residue was dissolved in ethanol, and two equivalents of ammonium-hexafluorophosphate were added . Blue crystals could be obtained after reduction of the solvent. Crystals were found that were suitable for x- ray structure determination . X-ray crystallography revealed a

[Cu(triethylenetetramine)CI] complex. Other coordinated complexes may be formed from or between copper antagonists, for example, copper chelators (such as Cu2+ chelators, spermidine, spermine, tetracyclam, etc.), particularly those subject to degradative pathways such as those noted above, by providing additional complexing agents (such as anions in solution, for example, I^", Br, F\ (SO4)^2", (CO3)^2", BF⁴\ NO^3"

, ethylene, pyridine, etc.) in solutions of such complexes. This may be particularly desirable for complexes with more accessi ble metal ions, such as planar complexes or complexes having four or fewer coordinating agents, where one or more additional complexing agents could provide additional shielding to the metal from undesirable ligands that might otherwise access the metal and displace a desired complexing agent.

[00112] General synthetic chemistry protocols are somewhat different for these classes of molecules due to their propensity to chelate with metallic cations, includi ng copper. Glassware should be cleaned and silanized prior to use. Plasticware should be chosen specifically to have mi nimal presence of metal ions. Metal implements such as spatulas should be excluded from any chemistry protocol involving chelators. Water used should be purified by sequential carbon filteri ng, ion exchange and reverse osmosis to the highest level of purity possible, not by distillation. All organic solvents used should be rigorously purified to exclude any possible traces of metal ion contamination.

[00113] Care must also be taken with purification of such derivatives due to their propensity to chelate with a variety of cations, includi ng copper, which may be present in trace amounts in water, on the surface of glass or plastic vessels. Once again, glassware should be cleaned and silanized prior to use. Plasticware should be chosen specifically to have minimal presence of metal ions. Metal implements such as spatulas should be avoided, and water used should be purified by sequential carbon filtering, ion exchange and reverse osmosis to the highest level of purity possible, and not by distillation. All organic solvents used should be rigorously purified to exclude any possi ble traces of metal ion contami nation . Ion exchange chromatography followed by lyophilization is typically the best way to obtain pure solid materials of these classes of molecules. Ion exchange resins should be washed clean of any possible metal contamination.

[00114] Many of the synthetic routes allow for control of the particular R groups introduced . For synthetic methods incorporating amino acids, synthetic amino acids can be used to incorporate a variety of substituent R groups. The dichloroethane synthetic schemes also allow for the incorporation of a wide variety of R groups by using dichlorinated ethane derivatives. It will be appreciated that many of these synthetic schemes can lead to isomeric forms of the compounds; such isomers can be separated using techniques known in the art.

[00115] Documents descri bing aspects of these synthetic schemes i ncl ude the following : ( 1) A W von Hoffman, Berichte 23, 3711 (1890); (2) The Polymerization Of Ethylenimine, Giffin D. Jones, Arne Langsjoen, Sister Mary Marguerite Christine

Neumann, Jack Zomlefer, J. Org . Chem., 1944; 9(2) ; 125-147; (3) The peptide way to macrocyclic bifunctional chelati ng agents: synthesis of 2-(p-nitrobenzyl)-l, 4,7, 10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid and study of its yttrium(III) complex, Min K. Moi et al. , J. Am. Chem. Soc , 1988; 110(18); 6266-6267; (4) Synthesis of a kinetically stable ⁹⁰Y labelled macrocycle-antibody conjugate, Jonathan

P L Cox, et al., J. Chem. Soc. Chem. Comm. , 797 (1989); (5) Specific and stable labeling of antibodies with technetium-99m with a diamide dithiolate chelating agent, Fritzberg AR, Abrams PG, Beaumier PL, Kasina S, Morgan AC, Rao TN, Reno JM, Sanderson JA, Srinivasan A, Wil bur DS, et a/., Proc. Natl. Acad. Sc.i U. S. A.

85(l l) :4025-4029 ( 1988 Jun) ; (6) Towards tumour imaging with ⁱⁿIn labelled macrocycle-antibody conj ugates, Andrew S Craig et al ., J . Chem. Soc. Chem. Comm., 794 (1989); (7) Synthesis of C- and N-functionalised derivatives of NOTA, DOTA, and DTPA: bifunctional complexing agents for the derivitisation of antibodies, Jonathan P L Cox et al .,, J . Chem. Soc. Perkin . I, 2567 ( 1990) ; (8) Macrocyclic chelators as anticancer agents in radioimmunotherapy, N R A Beeley and P R J Ansell, Current

Opinions in Therapeutic Patents, 2: 1539-1553 (1992); and (9) Synthesis of new macrocyclic amino-phosphi nic acid complexing agents and their C- and P- functionalised derivatives for protein linkage, Christopher J Broan et al ., Synthesis, 63 (1992).

[00116] Acyclic and cyclic compounds of the invention and exemplary synthetic methods and existing syntheses from the art include the followi ng :

For tetra-heteroatom acyclic examples of Formula I:

Xi, X2, X3, and X4 are independently chosen from the atoms N, S or 0 such that: 4N SERIES: when Xi, X2, X3, and X4 are N then :

i, R2, R3, R4, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 al kyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ;

nl, n2, and n3 are independently chosen to be 2 or 3, and each repeat of any of nl, n2, and n3 may be the same as or different than any other repeat; and

R7, Rs, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00117] In addition, one or several of Rl, R2, R3, R4, R5, or R6 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

[00118] Furthermore one or several of R7, R8, R9, RIO, Rl l, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to Cl-ClO alkyl-CO-peptide, Cl-ClO alkyl-CO-protein, Cl-ClO alkyl-CO-PEG, Cl-ClO alkyl-NH-peptide, Cl-ClO alkyl-NH- protein, Cl-ClO alkyl-NH-CO-PEG, Cl-ClO alkyl-S-peptide, and Cl-ClO alkyl-S- protein.

[00119] Also provided are embodiments wherein one, two, three or four of Rl through R12 are other than hydrogen.

[00120] In some embodiments, the compounds of Formula I or II are selective for a particular oxidation state of copper. For example, the compounds may be selected so that they preferentially bind oxidized copper, or copper (II). Copper selectivity can be assayed using methods known in the art. Competition assays can be done using isotopes of copper (I) and copper (II) to determine the ability of the compounds to selectively bind one form of copper.

[00121] In some embodiments, the compounds of Formula I or II may be chosen to avoid excessive lipophilicity, for example by avoiding large or numerous alkyl substituents. Excessive lipophilicity can cause the compounds to bind to and/or pass through cellular membranes, thereby decreasing the amount of compound available for chelating copper, particularly for extracellular copper, which may be

predominantly in the oxidized form of copper (II).

Synthesis of examples of the open chain 4N series of Formula I

[00122] Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diamine with 1,2-dichloro ethane to give triethylenetetramine directly (1). Modification of this procedure by using starting materials with appropriate Ra and Rb groups (where Ra, Rb = R7, R8 or Rll, R12) would lead to symmetrically substituted open chain 4N examples as shown below:

[00123] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifically towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the tetra-aza series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (3) should be used . Standard peptide synthesis using the Ri nk resin along with FMOC protected natural and un-natural amino acids which can be conveniently cleaved at the penultimate step of the synthesis generates a tri- peptide C-terminal amide. This is reduced using Diborane in THF to give the open chain tetra-aza compounds as shown below:

Rl 1.^R12

O R₉ Rio ^H O BH₃ in THF R₉ Ri₀ ^H

[00124] The incorporation of Rl, R2, R5 and R6 can be accomplished with this chemistry by standard procedures.

[00125] The reverse Rink approach, shown above, also leads to this class of tetra-aza derivatives and may be useful in cases where peptide coupling of a sterical ly hindered amino acid requires multiple coupli ng attempts in order to achieve success in the initial Rink approach.

[00126] The oxalamide approach, shown above, also can lead to successful syntheses of this class of compounds, although the central substituents are always going to be hydrogen or its isotopes with this kind of chemistry. This particular variant makes use of the trichloroethyl ester group to protect one of the carbolxylic acid functions of oxalic acid but other protecti ng groups are also envisaged . Reaction of an amino acid amide derived from a natural or unnatural amino acid with a differentially protected oxalyl mono chloride gives the mono-oxalamide shown which can be reacted under standard peptide coupling condition to give the un-symmetrical bis-oxalamide which can then be reduced with di borane to give the desired tetra-aza derivative.

1. 3NX series 1:

when Xi, X2, X3, are N and X4 is S or 0 then :

Rs does not exist

Ri, R2, R3, R4 and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 al kyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ;

[00127] In addition, one or several of Rl, R2, R3, R4, or R5 may be

functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco- kinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00128] Furthermore one or several of R7, R8, R9, RIO, Rl l, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the open chain 3NX series 1 of Formula I:

[00129] Variations of the syntheses used for the 4N series provide examples of the 3N series 1 class of compounds. The chemistry described by Meares et al (3) can be modified to give examples of the 3NX series of compounds.

[00130] Standard peptide synthesis according to the so-called reverse Rink approach as shown above using FMOC protected natural and un-natural amino acids which can be conveniently cleaved at the penultimate step of the synthesis generates a modified tri-peptide C-terminal amide. The cases where X4 is 0 are incorporated by the use of an alpha-substituted carboxylic acid in the last coupling step. This is reduced using Di borane in THF to give the open chain tetra-aza compounds.

[00131] The incorporation of Rl, R2, R5 and R6 can be accomplished with this chemistry by standard procedures.

[00132] For the cases where X4 = S a si milar approach using standard peptide synthesis according to the so-called reverse Rink approach as shown above can be used . Coupling with FMOC protected natural and un-natural amino acids, which can be conveniently cleaved at the penultimate step of the synthesis, generates a modified tri-peptide C-terminal amide. The incorporation of X4 = S is achieved by the use of an alpha-substituted carboxylic acid in the last coupling step. This is reduced using Diborane in THF to give the open chain tetra-aza compounds. [00133] The incorporation of Rl, R2, R5 and R6 can be accomplished with this chemistry by standard procedures.

[00134] The oxalamide approach, shown above, can also lead to successful syntheses of this class of compounds, although the central substituents are always going to be hydrogen or its isotopes with this kind of chemistry. This particular variant makes use of the trichloroethyl ester group to protect one of the carbolxylic acid functions of oxalic acid but other protecti ng groups are also envisaged . Reaction of an amino acid amide derived from a natural or unnatural amino acid with a differentially protected oxalyl mono chloride gives the mono-oxalamide shown which can be reacted under standard peptide coupling conditions with an ethanolamine or ethanethiolamine derivative to give the un-symmetrical bis-oxalamide which can then be reduced with diborane as shown to give the desired tri-aza derivative. 3NX series 2: when Xi, X2, and X4 are N and X3 is 0 or S then :

F does not exist, and

i, R2, R3, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 al kyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

[00135] In addition, one or several of Rl, R2, R3, R5, or R6 may be

functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00136] Furthermore one or several of R7, R8, R9, RIO, Rl l, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of examples of the open chain 3NX series 2 of Formula I:

[00137] A different approach can be used for the synthesis of the 3N series 2 class of compounds. The key component is the incorporation in the synthesis of an appropriately substituted and protected ethanolamine or ethanethiolamine derivative, which is readily available from both natural and un-natural amino acids, as shown below.

[00138] The BOC protected ethanolamine or ethanethiolamine is reacted with an appropriate benzyl protected alpha chloroacid . After hydrogenation to deprotect the ester function, standard peptide coupli ng with a natural or unnatural ami no acid amide fol lowed by deprotection and reduction with diborane in THF gives the open chain tri-aza compounds. If hydrogenation is not compatible with other functionality in the molecule then alternative combinations of protecting groups can be used such as trichloroethyloxy carbonyl and t-butyl .

[00139] The incorporation of Rl, R2, R5 and R6 can be accomplished with this chemistry by standard procedures.

2N2X series 1:

when X2 and X3 are N and Xi and X4 are 0 or S then :

Ri and Rs do not exist;

R2, R3, R4, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

R7, R8, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, CI- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00140] In addition, one or several of R2, R3, R4, or R5 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n. [00141] Furthermore one or several of R7, R8, R9, RIO, Rl l, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the open chain 2N2X series 1 of Formula I:

[00142] The oxalamide approach, shown above, can lead to successful syntheses of this class of compounds. This particular variant makes use of the trichloroethyl ester group to protect one of the carbolxylic acid functions of oxalic acid but other protecting groups are also envisaged . Reaction of an aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid with a

differentially protected oxalyl mono chloride gives the mono-oxalamide shown which can be reacted under standard peptide coupling condition to give the un-symmetrical bis-oxalamide which can then be reduced with di borane to give the desired tetra-aza derivative.

X-, = O or S

X₄ = O or S R-11 R-I2

[00143] A variant of the dichloroethanee approach, shown above, can also lead to successful syntheses of this class of compounds. Reaction of an ami noalcohol or aminothiol derivative readily available from a natural or unnatural amino acid with an O-protected 1-chloro, 2-hydroxy ethane derivative followed by deprotection and substitution with chloride gives the mono-chloro compound shown which can be further reacted with an appropriate aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid to give the un-symmetrical desired product.

2N2X series 2:

when Xi and X3 are N and X2 and X4 are 0 or S then :

R3 and Rs do not exist;

Ri, R2, R4, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

R7, Rs, R9, Rio, R11, and R12 are independently chosen from H, C H3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00144] In addition, one or several of Ri, R2, R4, or R5 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00145] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of the open chain 2N2X series 2 of Formula I:

[00146] A variant of the dichloroethane approach, shown above, can lead to successful syntheses of this class of compounds. Reaction of an aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid with an O-protected 1-chloro, 2-hydroxy ethane derivative followed by deprotection and substitution with chloride gives the mono-chloro compound shown which can be further reacted with an appropriately protected aminoalcohol or aminothiol derivative, readily available from a natural or unnatural amino acid, to give the un- symmetrical desired product after de-protection.

2N2X series 3:

when Xi and X2 are N and X3 and X4 are 0 or S then :

R4 and Rs do not exist;

Ri, R2, R3, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH) ; nl, n2, and n3 are independently chosen to be 2 or 3, and each repeat of any of nl, n2, and n3 may be the same as or different than any other repeat; and

R7, Re, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, Cl- C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 al kyl fused aryl .

[00147] In addition, one or several of Ri, R2, R3, or R5 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl- CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO- PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

[00148] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of the open chain 2N2X series 3:

[00149] A variant of the dichloroethanee approach, shown above, can lead to successful syntheses of this class of compounds. Reaction of a monoprotected ethylene diamine derivative, readily available from a natural or unnatural ami no acid with an O-protected 1-chloro, 2-hydroxy ethane derivative followed by deprotection and substitution with chloride gives the mono-chloro compound shown which can be further reacted with an appropriately protected bis-alcohol or bis thiol derivative, readily avai lable from a natural or unnatural amino acid, to give the un-symmetrical desired product after de-protection.

2N2X series 4:

when Xi and X4 are N and X2 and X3 are 0 or S then :

R3 and F do not exist;

Ri, R2, R5 and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

[00150] In addition, one or several of Ri, R2, R5, or Rs may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n. [00151] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of the open chain 2N2X series 4 of Formula I:

[00152] A variant of the dichloroethanee approach, shown above, can lead to successful syntheses of this class of compounds. Reaction of a an appropriately protected bis-alcohol or bis thiol derivative, readily available from a natural or unnatural amino acid, with an O-protected 1-chloro, 2-hydroxy ethane derivative followed by deprotection and substitution with chloride gives the mono-chloro compound shown which can be further reacted with an appropriately protected bis- alcohol or bis thiol derivative, readily available from a natural or unnatural amino acid, to give the un-symmetrical desired product after de-protection.

For the Tetra-heteroatom cyclic series:

One of Ri and R2 (if Ri does not exist) and one of R5 (if Rs does not exist) and Rs are joined together to form the bridging group (CRi3Ri4)n4;

Xi, X2, X3, and X4 are independently chosen from the atoms N, S or 0 such that: 4N macrocyclic series:

when Xi, X2, X3, and X4 are N then :

R2, R3, R4, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) ₂, CH₂P(CH₃)0(OH);

nl, n2, n3, and n4 are independently chosen to be 2 or 3, and each repeat of any of n l, n2, n3 and n4 may be the same as or different than any other repeat; and

R7, Rs, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2- CIO straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00153] In addition, one or several of R2, R3, R4, or R5 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

[00154] Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the macrocyclic 4N series of Formula I:

[00155] Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diami ne with 1,2-dichloro ethane to give triethylenetetramine directly ( 1). Possible side products from this synthesis include the 12N4 macrocycle shown below, which could also be synthesized directly from Triethylenetetramine by reaction with a further equivalent of 1,2-dichloro ethane under appropriately dilute concentrations to provide the 12N4 macrocycle shown . Modification of this procedure by using starting materials with appropriate Ra and Rb (where Ra, Rb correspond to R7, Rs or Rn, R12) groups would lead to symmetrically substituted 12N4 macrocycle examples as shown below:

[00156] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the tetra-aza series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (3) should be used . Standard peptide synthesis usi ng the Merrifield approach or the SASRIN resin along with FMOC protected natural and unnatural amino acids which can be conveniently cleaved at a later step of the synthesis generates a fully protected tetra-peptide C-terminal SASRIN derivative. Cleavage of the N terminal FMOC protecting group followed by direct cyclization upon concomitant cleavage from the resin gives the macrocyclic tetrapeptide. This is reduced using Di borane in THF to give the 12N4 series of compounds as shown below:

[00157] The incorporation of Ri, R2, R5 and Rs can be accomplished with this chemistry by standard procedures.

[00158] The reverse Merrifield/SASRIN approach, shown above, also leads to this class of tetra-aza derivatives and may be useful in cases where peptide coupling of a sterically hindered amino acid requires multiple coupling attempts in order to achieve success in the initial Merrifield approach.

[00159] The oxalamide approach, shown above, also can lead to successful syntheses of this class of compounds. This particular variant makes use of the trichloroethyl ester group to protect one of the carbolxylic acid functions of oxalic acid but other protecting groups are also envisaged . Reaction of an amino acid amide derived from a natural or unnatural amino acid with a differentially protected oxalyl mono chloride gives the mono-oxalamide shown which can be reacted under standard peptide coupling condition to give the un-symmetrical bis-oxalamide which can then be reduced with diborane to give the desired tetra-aza derivative. Further reaction with oxalic acid gives the cyclic derivative, which can then be reduced once again with diborane to give the 12N4 series of compounds.

3NX series:

when Xi, X2, X3, are N and X4 is S or 0 then :

R5 does not exist;

R2, R3, and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

R7, Re, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2- C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, CI- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00160] In addition, one or several of R2, R3 or F may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

[00161] Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the macrocyclic 3NX series of Formula I:

[00162] Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diami ne with 1,2-dichloro ethane to give triethylenetetramine directly ( 1). Possible side products from this synthesis include the 12N4 macrocycle shown below, which could also be synthesized directly from Triethylenetetramine by reaction with a further equivalent of 1,2-dichloro ethane under appropriately dilute concentrations to provide the 12N4 macrocycle shown . Modification of this procedure by using starting materials with appropriate R groups leads to symmetrically substituted 12N4 macrocycle examples as shown below:

X₄ = O or S

[00163] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the tri-aza X series. In order to obtain alternative un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (3) could be used . Standard peptide synthesis using the Merrifield approach or the SASRIN resin along with FMOC protected natural and un-natural amino acids which can be conveniently cleaved at a later step of the synthesis generates a tri-peptide C-terminal SASRIN derivative which can be further elaborated with an appropriate BOCO or BOCS compound the give the resin bound 3NX compound shown. Reduction with diborane followed by Tosylation would give the 3NX OTosyl linear compound, which, upon deprotection and cyclization would give the desired 3NX macrocycle as shown below:

[00164] The incorporation of Ri, R2, R5 and Rs can be accomplished with this chemistry by standard procedures.

[00165] The reverse Merrifield/SASRIN approach, shown above, also leads to this class of tetra-aza derivatives and may be useful in cases where peptide coupling of a sterical ly hindered amino acid requires multiple coupli ng attempts in order to achieve success in the initial Merrifield approach.

2N2X series 1:

when X2 and X3 are N and Xi and X4 are 0 or S then :

R2 and R5 do not exist

R3 and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ;

R7, Re, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2- C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl

[00166] In addition, one or both of R3, or F may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00167] Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the macrocyclic 2N2X series 1 of Formula I:

[00168] The oxalamide approach, shown above, again can lead to successful syntheses of this class of compounds, although the central substituents are always going to be hydrogen or its isotopes with this kind of chemistry. This particular variant makes use of the trichloroethyl ester group to protect one of the carboxylic acid functions of oxalic acid but other protecti ng groups are also envisaged . Reaction of an aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid with a differentially protected oxalyl mono chloride gives the mono-oxalamide shown which can be reacted under standard peptide coupling condition to give the un-symmetrical bis-oxalamide which can then be reduced with diborane to give the desired di-aza derivative. Deprotection followed by cyclization would give the 12N2X2 analogs.

[00169] A variant of the dichloroethane approach, shown above, can also lead to successful syntheses of this class of compounds. Reaction of an aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid with an O-protected 1-chloro, 2-hydroxy ethane derivative followed by deprotection and substitution with chloride gives the mono-chloro compound shown which can be further reacted with an appropriate aminoalcohol or aminothiol derivative readily available from a natural or unnatural amino acid to give the un-symmetrical product shown. Deprotection followed by cyclization with a dichloroethane derivative would give a mixture of the two position isomers shown.

2N2X series 2:

when Xi and X3 are N and X2 and X4 are 0 or S then :

R3 and R5 do not exist

R2 and R4 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂2PO(OH)₂, CH₂P(CH₃0(OH);

R7, R8, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2-

C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00170] In addition, one or both of R2, or R4 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG,

C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00171] Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco- kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of examples of the macrocyclic 2N2X series 2 of Formula I:

[00172] Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diami ne with 1,2-dichloro ethane to give triethylenetetramine directly ( 1). Possi ble side products from this synthesis include the 12N4 macrocycle shown below, which could also be synthesized directly from Triethylenetetramine by reaction with a further equivalent of 1,2-dichloro ethane under appropriately dilute concentrations to provide the 12N4 macrocycle shown . Modification of this procedure by using starting materials with appropriate R groups would lead to symmetrically substituted 12N4 macrocycle examples as shown below:

X₂ = O or S

X₄ = O or S

[00173] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group and an appropriate 0 or S protecting group allows the chemistry to be directed specifically towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the di-aza 2X series. A variant of this approach using substituted dichloroethanee derivatives could be used to access more complex substitution patterns. This would lead to mixtures of position isomers, which can be separated by

HPLC.

4 position isomers

1N3X series:

when Xi is N and X2, X3 and X4 are 0 or S then :

R3, R4 and R5 do not exist;

R2 is independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH); nl, n2, n3, and n4 are independently chosen to be 2 or 3, and each repeat of any of n l, n2, n3 and n4 may be the same as or different than any other repeat; and

7, Re, R9, Rio, R11, R12, R13 and R14 are independently chosen from H, CH3, C2- C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, Cl- C6 alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 al kyl fused aryl .

[00174] In addition, R2 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n. [00175] Furthermore one or several of R7, Rs, R9, Rio, Rn, R12, R13 or R14 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of examples of the macrocyclic 1N3X series of Formula I:

[00176] Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diami ne with 1,2-dichloro ethane to give triethylenetetramine directly ( 1). Possi ble side products from this synthesis include the 12N4 macrocycle shown below, which could also be synthesized directly from Triethylenetetramine by reaction with a further equivalent of 1,2-dichloro ethane under appropriately dilute concentrations to provide the 12N4 macrocycle shown . Modification of this procedure by using starting materials with appropriate R groups would lead to substituted

12NX3 macrocycle examples as shown below:

X₂, X₃, X₄ = O or S

[00177] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group and an appropriate 0 or S protecting group allows the chemistry to be directed specifically towards the substitution pattern shown.

Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the mono-aza 3X series. A variant of this approach using substituted dichloroethane derivatives could be used to access more complex substitution patterns. This would lead to mixtures of position isomers, which can be separated by HPLC.

2 position isomers

For the tri-heteroatom acyclic examples of Formula II:

Xi, X2, and X3 are independently chosen from the atoms N, S or 0 such that: 3N series:

when Xi, X2, and X3 are N then :

Ri, R2, R3, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 al kyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ;

nl and n2 are independently chosen to be 2 or 3, and each repeat of any of nl and n2 may be the same as or different than any other repeat; and R7, Re, R9, and Rio are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl .

[00178] In addition, one or several of Ri, R2, R3, R5 or Rs may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

[00179] Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

Synthesis of the open chain 3N series of Formula II:

[00180] As mentioned above Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diamine with 1,2-dichloro ethane to give Triethylenetetramine directly (1). A variant of this procedure by using starting materials with appropriate R groups and l-ami no,2-chloro ethane would lead to some open chai n 3N examples as shown below:

[00181] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the tri-aza series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (2) could be used . Standard peptide synthesis using the Ri nk resin along with FMOC protected natural and un-natural amino acids which can be conveniently cleaved at the penultimate step of the synthesis generates a di- peptide C-terminal amide. This can be reduced using Diborane in THF to give the open chain tri-aza compounds as shown below:

[00182] The reverse Rink approach may also be useful where peptide coupli ng is slowed for a particular substitution pattern as shown below. Agai n the incorporation of Ri, R2, R5 and Rs can be accomplished with this chemistry by standard procedures:

2NX series 1:

when Xi and X3 are N and X2 is S or 0 then :

R3 does not exist

Ri, R2, R5, and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH) ;

nl and n2 are independently chosen to be 2 or 3, and each repeat of any of nl and n2 may be the same as or different than any other repeat; and R7, Re, R9, and Rio are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyl, C1-C6 alkyi C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl

[00183] In addition, one or several of Ri, R2, R5 or Rs may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

[00184] Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overal l pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl- CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO- PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

Synthesis of the open chain 2NX series 1 of Formula II:

[00185] The synthesis of the 2NX series 1 compounds can be readily achieved as shown above. The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifically towards the substitution pattern shown above. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the tri-aza X series. 2NX series 2

when Xi and X2 are N and X3 is 0 or S then :

Rs does not exist;

Ri, R2, R3 and Rs are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)2, CH₂P(CH₃)0(OH) ;

nl and n2 are independently chosen to be 2 or 3, and each repeat of any of nl and n2 may be the same as or different than any other repeat; and

R7, Rs, R9, and Rio are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl .

[00186] In addition, one or several of Ri, R2, R5, or Rs may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl- CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO- PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

[00187] Furthermore one or several of R7, Rs, R9, or Rio may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overal l pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl- CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO- PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protein.

Synthesis of the open chain 2NX series 2 of Formula II:

[00188] For the cases where X3 = 0 or S a similar approach usi ng standard peptide synthesis according to the Rink approach as shown above can be used .

Coupling of a suitably protected alpha thiolo or hydroxy carboxylic acid with a Rink resin amino acid derivative followed by cleavage gives the desired linear di-amide, which can be reduced with Diborane in THF to give the open chain 2NX compounds.

[00189] The incorporation of Rl, R2, R5 and R6 can be accomplished with this chemistry by standard procedures.

[00190] The reverse Rink version is also feasible and again the incorporation of Rl, R2, R5 and R6 can be accompl ished with this chemistry by standard procedures

Tri-heteroatom cyclic series of Formula II:

Ri and Rs form a bridging group (CRnRi2)n3; and

when Xi, X2 and X3 are N then :

R2, R3, and R5 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH) ;

nl, n2, and n3 are independently chosen to be 2 or 3, and each repeat of any of nl, n2 and n3 may be the same as or different than any other repeat; and

R7, R8, R9, Rio, R11, and R12 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, Cl- C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 al kyi fused aryl .

[00191] In addition, one or several of R2, R3, or R5 may be functional ized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

[00192] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.

Synthesis of examples of the macrocyclic 3N series of Formula II:

[00193] As mentioned above Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diamine with 1,2-dichloro ethane to give Triethylenetetramine directly (1). A variant of this procedure by using starting materials with appropriate R groups and l-ami no,2-chloro ethane would lead to open chain 3N examples which could then be cyclized by reaction with an appropriate 1,2 dichloroethane derivative as shown below:

[00194] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the macrocyclic tri-aza series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (3) could be used. Standard peptide synthesis using the Merrifield approach/SASRIN resin along with FMOC protected natural and un-natural amino acids which can be conveniently cleaved at the penultimate step of the synthesis generates a tri-peptide attached to resin via it's C-terminus. This can be cyclized during concomitant cleavage from the resin followed by reduction using Diborane in THF to give the cyclic tri-aza compounds as shown below:

[00195] The incorporation of Ri, R2, and R5 can be accomplished with this chemistry by standard procedures.

[00196] The reverse Rink approach may also be useful where peptide coupling is slowed for a particular substitution pattern as shown below. Again the incorporation of Ri, R2, R5 and Rs can be accomplished with this chemistry by standard procedures:

2NX series:

when Xi and X2 are N and X3 is S or 0 then :

Rs does not exist;

R2 and R3 are independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH) 2, CH₂P(CH₃)0(OH);

[00197] In addition, one or both of R2 or R3 may be functionalized for

attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO- PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

[00198] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, protei ns, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, delive rability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of examples of the macrocyclic 2NX series of Formula II:

[00199] As mentioned above Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diamine with 1,2-dichloro ethane to give Triethylenetetramine directly (1). A variant of this procedure by using starting materials with appropriate R groups and l-ami no,2-chloro ethane would lead to open chain 2NX examples which could then be cyclized by reaction with an appropriate 1,2 dichloroethanee derivative as shown below:

[00200] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the macrocyclic di-aza X series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry described by Meares et al (3) could be used . Standard peptide synthesis using the Merrifield approach/SASRIN resin along with FMOC protected natural and un-natural amino acids which can be conveniently cleaved at the penultimate step of the synthesis generates a tri-peptide attached to resin via it's C-termi nus. This can be cyclized during concomitant cleavage from the resin followed by reduction using Diborane in THF to give the cyclic tri-aza compounds as shown below:

[00201] The incorporation of Ri, and R2 can be accomplished with this chemistry by standard procedures.

[00202] The reverse Rink approach may also be useful where peptide coupli ng is slowed for a particular substitution pattern as shown below. Again the incorporation of Ri, and R2 can be accomplished with this chemistry by standard procedures:

1N2X series:

when Xi is N and X2 and X3 are 0 or S then :

R3 and R5 do not exist;

R2 is independently chosen from H, CH3, C2-C10 straight chain or branched alkyi, C3-C10 cycloalkyi, C1-C6 alkyi C3-C10 cycloalkyi, aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6 alkyi aryl, C1-C6 alkyi mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyi heteroaryl, C1-C6 alkyi fused aryl, CH2COOH, CH2SO3H, CH₂PO(OH)₂, CH₂P(CH₃)0(OH);

nl, n2, and n3 are independently chosen to be 2 or 3, and each repeat of any of nl, n2 and n3 may be the same as or different than any other repeat;

[00203] In addition, R2 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmaco-kinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not li mited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH- peptide, C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S-protei n.

[00204] Furthermore one or several of R7, Rs, R9, Rio, Rn, or R12 may be functionalized for attachment, for example, to peptides, proteins, polyethylene glycols and other such chemical entities in order to modify the overall pharmacokinetics, deliverability and/or half lives of the constructs. Examples of such functionalization include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protei n, C1-C10 al kyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-N H- protein, C1-C10 alkyl-NH-CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10 alkyl-S- protein.

Synthesis of examples of the macrocyclic 1N2X series of Formula II:

[00205] As mentioned above Triethylenetetramine itself has been synthesized by reaction of 2 equivalents of ethylene diamine with 1,2-dichloro ethane to give Triethylenetetramine directly (1). A variant of this procedure by using starting materials with appropriate R groups and l-amino,2-chloro ethane would lead to open chain 1N2X examples which could then be cyclized by reaction with an appropriate 1,2 dichloroethanee derivative as shown below:

X₂ X₃ = S or O

[00206] The judicious use of protecting group chemistry such as the widely used BOC (t-butyloxycarbonyl) group allows the chemistry to be directed specifical ly towards the substitution pattern shown. Other approaches such as via the chemistry of ethyleneimine (2) may also lead to a subset of the macrocyclic aza di-X series. In order to obtain the un-symmetrically substituted derivatives a variant of some chemistry above could be used :

[00207] The incorporation of Ri and R2 can be accomplished with this chemistry by standard procedures. [00208] Methods of preparing triethylenetetramines particularly suited for use as described herei n are presented i n U .S. Patent No. 7,582,796 (Jonas et al ., issued 1 September 2009), herein incorporated by reference in its entirety.

EXAMPLES EXAMPLE 1

[00209] This example investigates metabolic perturbations in various brain regions of AD sufferers.

2. Methods

Acquisition of human brains [00210] Whole brai ns from patients and matched controls were obtained from the

New Zealand Neurological Foundation Human Brai n Bank, in the Centre for Brain Research, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand . All procedures in this study were approved by the University of Auckland Human Partici pants Ethics Committee with informed consent from all families. The quality of AD brain tissue acquired by the Human Brain Bank was uniformly high and only those with very short post-mortem delays (~4-13 h) were used for the current study.

Sampling of human-brain tissue

[00211] After receipt into the Human Brain Bank, brains were dissected under the supervision of neuroanatomists, to ensure accurate identification of each of the seven brain regions targeted i n this study. The brain regions included three regions known to undergo severe neuronal damage in AD (the hippocampus (HP), entorhinal cortex (ENT) and middle-temporal gyrus (MTG)) three regions known to be less severely affected (the sensory cortex (SCx), motor cortex (MCx) and cingulate gyrus (CG)), and one region known to be relatively spared (the cerebellum (CB)) . Tissue samples of 50 ± 5 mg were dissected from each region and stored at -80 °C until analysis.

Diagnosis and severity of AD

[00212] Al l AD patients had clinical dementia, whereas controls did not. Control brains were selected from the Human Brain Bank by matching for age, sex and post- mortem delay as shown in Table 1. A consultant neuropathologist diagnosed or excluded AD by applying the Consortium to Establish a Registry for AD (CERAD) criteria .

Table 1: Group characteristics of brains used in study

Variable Control AD

Number 9 9

Age (± SD) 70.1 (±6.7) 70.3 (±7.1)

Male sex, n (%) 5 (55.6) 5 (55.6)

Post-mortem delay (h, range) 9 (5.5-13.0) 7 (4.0-12.0)

Brain weight (g, range) 1260 (1094-1461) 1062* (831-1355)

*P=0.005 compared with Control ; al l other differences were non-significant.

Tissue extraction

[00213] Brain tissues placed in "Safe-Lok" microfuge tubes (Eppendorf AG;

Hamburg, Germany) were held at -80 °C until extraction. They then underwent a Folch-style extraction using a TissueLyser batch bead homogeniser (Qiagen;

Manchester, UK). Briefly, each sample containing 50 ± 5 mg of brain tissue was extracted in 0.8 ml 50: 50 (v/v) methanok chloroform, to which a solution of the labelled internal standards in methanol had been added to achieve a final concentration of 0.016 mg/ml of each internal standard in the extraction solvent (kept at -20°C until used) . A set of seven isotopically-labelled standards (Citric acid- 04, ¹³Cs-D-fructose, Ltryptophan-cfe, L-alanine-c 7, stearic acid-c/35, benzoic acid-c 5, and leucine-c jo) purchased from Cambridge Isotopes Inc (Tewksbury, MA) were used in this study. Extraction was performed for 10 min at 25 Hz with a single 3-mm tungsten carbide bead per tube. Samples corresponding to the same brain region were handled as single separate batches for this and all subsequent procedures. Separation of phases was achieved by addition of 0.4-ml water followed by vortex- mixing ( 10-15 s) and centrifugation (2,400 g, 15 min) . After separation, tissue debris lay at the interface between the lower (non-polar, chloroform) phase and the upper (polar, methanok water) phase containing the target molecules for the current study. For each batch, extraction blanks were prepared by processing tubes containing solvent and bead, but no tissue sample. This procedure produced clean polar extracts with low levels of lipid and protein content, which are known to otherwise cause response-instabil ity in the GC-MS method . Sample preparation

[00214] Chloroform in extraction tubes was removed usi ng a 500-μΙ HPLC syringe (Sigma Aldrich, MO, USA) . Tubes were then centrifuged (16,000 g, 15 min) to encourage tissue debris to form a coherent pellet. From the methanol : water supernatant, 200-μΙ aliquots were transferred to pre-labelled tubes containing 600 μΙ of methanol, to precipitate residual protein . A quality-control (QC) pool was made by combining 200-μΙ aliquots from each extraction. The pooled samples were gently mixed and 200-μΙ portions dispensed into tubes containing 600-μΙ methanol. Both sample and QC tubes were centrifuged (16,000 g, 15 min) and 750-μΙ al iquots were transferred to a final set of pre-labelled tubes which were processed to dryness in a Speedvac centrifugal concentrator (~30 °C, 16-18 h (Savant; SPD331 DDA, Thermo Scientific)) . Dried residues were held in sealed tubes at 4 °C for up to one week (shown to be stable for eight weeks for serum previously stored) until derivatization for GC-MS analysis.

GC-MS Analysis

[00215] Methyloxime/trimethylsilyl derivatives were prepared by a two-step procedure. GC-MS analysis was performed using an MPS2 autosampler (Gerstel ; Mulheim an der Ruhr, Germany), a 7890A Gas Chromatograph with Split/Splitless inlet (Agilent; Santa Clara, CA, USA), and a Pegasus HT time-of-f l ight mass spectrometer (LECO; Stockport, UK).

[00216] Gas chromatography was performed using an Agilent/J&W DB-17MS column (30 m x 0.25 mm x 0.25 μιτι ; Agilent: # 122-4732) with a 3-m deactivated Fused Silica retention gap (0.25 mm; Agilent: #No 160-2256-10), and helium carrier gas ( 1.4 ml/min, constant flow mode). l-μΙ sample injections were made in Pulse Spl itless mode at an inlet temperature of 270 °C, using an "empty, hot-needle" technique. The initial column temperature (50 °C) was held for 6 min and then increased to 300 °C at 10 °C /min and held for a further 4 min. This resulted in a total cycle time of 42 min between injections. After an initial 450-s solvent delay (to allow solvent and reagents to elute without damaging the detector), mass spectral data were acquired at 10 spectra/s for the range 45-800 Da, detecti ng a range of amino acids, sugars, sugar alcohols and organic acids as their TMS derivatives. Standard 70-eV electron energy was employed, at a source temperature of 220 °C.

[00217] The study was performed i n a series of single-batch experiments, where each specific brai n region constituted a batch. Within each batch, individual cases and controls were randomized, and run in a sequence interleaved with injections of the pooled QC samples (one per four study samples) and extraction blanks (two per batch) . A lead-in sequence of six QC injections at the start of each batch was used to condition the chromatographic system. Extraction blanks were inspected visually to confirm absence of carryover, but not included in subsequent data analysis. Data reduction

[00218] Data were prepared using the 'Reference Compare' method withi n ChromaTOF 4.5 (LECO) .

[00219] Briefly, the software was used to perform a global peak deconvolution of parameters to compi le a list of nominated 'metabolites', and search mass-spectral libraries to generate putative identities. Databases we employed were: the NIST

Mass Spectral Reference Library (NIST08/2008; National Institute of Standards and Technology/Environmental Protection Agency/National Institutes of Health Spectral Library; NIST, Gaithersburg, MD, USA); the Golm Metabolome Database (Max Planck Institute of Molecular Plant Physiology, Potsdam- Golm, Germany); and an in-house library developed at the University of Manchester. Chromatographic retention-time data were available from reference standard compounds for a subset of the identities. Withi n this subset, matching of both mass spectra and expected retention time(s) was interpreted to constitute a defi nitive (D) molecular identification. Matching of mass spectra and retention time with reported data was interpreted as confident (C) identification. Matching of mass spectra only was interpreted as a putative (P) identification. From the list of nominations, we compiled reference tables comprising expected mass spectra and retention-time windows. These were then applied as target l ists of features to be searched across al l the study samples. As the pooled QC samples should contain al l metabolite features encountered in the experiment, these were suitable candidates for compilation of reference tables. To provide a robust reference table, the initial list of nominations was edited to remove ambiguous and low quality spectra prior to application. Global deconvolution was performed on several (3-4) pooled QC injections across the entire experi ment to improve identifications. By displaying these overlaid while editing the list, reproducible spectra were more readily distinguished from lower quality candidates. The same target list was used for all brain regions.

Metabolite abundance reporting

[00220] In order to use the edited reference table as a reporting tool, appropriate parameters such as mass spectral match thresholds and tolerable retention-time deviations (6 sec) were specified, and the table initial ized using a pooled QC sample to provide reference m/z peak areas. Improved reproducibility was achieved by the use of internal standard ratios rather than raw peak areas.

[00221] The most suitable standard was assigned to each metabolite by determini ng which internal standard yielded the lowest variance for a given metabolite across all the QC injections.

[00222] The resulting data for each experiment were compiled into a matrix of metabolite-intensity data, which was merged with experimental metadata for visualization and statistical analysis. Although the automated procedure was highly reliable (estimated return of correct peak areas for >95% of features measured), data sets were also curated manually to remove possible integration errors which were mostly associated with metabolites showing non-ideal peak shape.

Statistics

[00223] The merged metadata were used for data analysis. A pri nci pal- components analysis (PCA) was performed for visualization to confirm overall data integrity, using SIMCA-P software (UMetrics AB, Umea, Sweden). Calculation of relative fold-change and statistical analysis were performed in log space using multiple t-tests (GraphPad Prism 6). Data were considered for multiple comparison analysis applyi ng an FDR (10%) correction . The fold-changes were converted to linear space for presentation and metabolites identified in≥ 5 samples in each group were reported.

Results

[00224] There was about 16% median brai n weight decline in AD; median (range) brain weight was 1,062 g (831-1,355) in AD and 1,260 g ( 1,094-1,461 ; P<0.005) in controls (see Table 1).

[00225] PCA of GC-MS data revealed : 1) excellent class separation in all brain regions between AD and control samples; 2) greater biological than technical variation; and 3) absence of run-order effects in this study (data not shown). One control sample clustered more closely to the AD samples. The brain from which this sample originated had the lowest brain-weight ( 1,094 g) among the controls and was assessed as Braak stage II by neuropathological examination. On this basis, this control sample was reclassified as a case of preclinical AD case and has been excluded from the subsequent analysis for reasons of clarity. [00226] 69 metabolite features were categorised per brain region. The metabolites for which an altered abundance in one or more regions of the AD was observed are shown in Table 2. N umbers indicate fold-change (AD/controls). Changes with P<0.05 (10% FDR) were considered significant and were shown in bold italic font. 55 features were shown to change in at least one brain region (FDR corrected multiple t-test (p < 0.05)), with individual regions showing between 16 and 33 metabolites identified as significantly changed .

[00227] Metabolites for which no altered abundance was observed for any brain region are shown in Table 3. Table 2: Relative fold change in metabolites with altered abundance in the AD brain.

Metabolite H P ENT MTG sex MCX CG CB

Glucose and related metabolites & pentose phosphate pathway components

Glucose (D) 12.3 9.3 16.9 10.8 8.0 9.9 6.8

Glucose-6-phosphate (D) 5.9 4.9 8.5 4.8 6.0 4.8 3.8

Sorbitol (D) 3.4 3.8 3.9 5.0 5.3 5.0 4.6

Fructose (D) 4.6 4.8 7.0 6.8 7.0 7.0 7.1

Fructose-6-phosphate (D) 1.2 0.6 5.3 1.6 2.5 7.7 2.3

Pentonic acid A (P) 1.1 1.3 1.3 1.2 1.4 1.4 1.3

Pentonic acid B (P) 2.1 2.0 1.9 1.7 1.7 1.9 1.7

Arabinose (P) 3.5 N M 3.6 3.5 6.8 3.9 NM

Ribose-5-phosphate (D) 0.8 0.6 0.8 1.1 1.1 1.0 0.9

Erythronic acid (P) 1.6 1.8 1.1 1.2 1.2 1.4 1.5

Alternative fuel source

Butanediol (D) 4.2 4.3 1.6 4.2 9.3 4.1 4.1 β-Hydroxybutyric acid (D) 1.6 2.5 1.5 3.4 1.8 2.5 1.6

Lactic acid (D) 2.6 0.5 1.3 3.4 7.3 1.7 0.6

2-hydroxy-3-methylbutyric acid (P) 7.7 2.3 2.8 4.3 2.5 4.7 3.4

Threitol (D) 2.0 2.3 1.8 2.4 2.3 2.4 3.2

Xyl itol (D) 1.7 1.3 1.2 1.2 1.1 1.4 1.7

Disaccharide (D) 4.8 2.3 3.2 3.6 1.8 1.8 0.9

N-acetylglucosamine (C) 1.2 1.1 1.7 1.4 1.4 1.6 2.8 myo-Inositol (D) 1.1 1.9 1.1 1.2 0.8 0.7 0.9 myo-Inositol-l-phosphate (P) 2.3 2.0 4.1 1.7 2.2 3.9 3.5

Glycerol (D) 0.6 0.7 0.8 0.8 0.7 0.9 0.9 Glycerol-2-phosphate (P) 1.9 1.6 1.5 1.7 1.8 1.6 1.6

Glycerol-3-phosphate (D) 2.3 2.7 1.4 2.5 2.6 1.8 1.6

Glyceric acid (P) 1.3 1.1 2.8 1.5 1.1 2.6 1.8

TCA cycle & urea cycle

Citric acid (D) 1.7 2.1 1.7 1.9 1.1 1.1 1.1

Malic acid (C) 1.6 1.9 2.4 1 0.8 1.7 0.9

Fumaric acid (C) 1.8 1.3 1.7 1.2 0.8 1.4 1.3

Ornithine (D) 0.6 0.6 1.0 0.7 0.7 0.9 0.3

Urea (D) 6.5 5.6 4.7 4.9 5.0 5.3 4.9

N-acetylglutamic acid (D) 0.4 0.8 0.9 0.8 0.7 0.8 1.0

Creatinine (D) 1.1 1.0 1.5 1.0 1.0 1.2 1.2

Amino acids

Prol ine (D) 0.5 0.4 0.5 0.7 0.8 0.8 0.5

Lysine (D) 0.5 0.7 1.1 0.8 1.1 1.1 0.3

Glycine (D) 0.9 0.7 0.8 0.8 0.7 0.9 0.9

Seri ne (D) 0.7 0.6 1.0 0.7 0.8 1.0 0.7

Threonine (D) 0.8 1.3 1.5 1.1 2.2 1.4 1.7

Cysteine (D) 2.0 1.4 0.7 1.0 1.0 1.4 0.9 beta-Alanine (D) 1.2 1.0 1.3 1.1 1.1 1.3 0.9

Aspartic acid (D) 0.6 0.6 0.8 1.0 0.9 0.9 0.8

N-acetylaspartic acid (D) 0.7 0.8 0.7 1.0 0.9 0.9 1.0

Glutamic acid (P) 1.3 1.0 1.0 1.3 1.3 1.3 2.6

GABA (D) 1.3 0.6 0.5 0.7 0.7 0.9 0.8

4-hydroxybutyric acid (C) 0.6 0.6 1.1 0.8 0.8 0.7 0.7

Phenylalanine (D) 1.3 1.2 2.1 1.6 1.8 2.0 1.3

Tryptophan (D) 2.5 2.2 4.7 1.8 3.2 4.0 1.1

Nucleosides

Adenine (D) 1.0 1.0 0.9 1.0 0.9 0.9 1.7

Uracil (C) 0.6 0.5 0.6 0.6 0.5 0.6 0.7

Adenosine-5-monophosphate (P) 1.6 2.1 1.9 1.4 NM 2.4 1.3

Guanosine (D) 0.7 0.8 2.8 0.9 NM 2.5 1.6

Hypoxanthine (D) 0.7 0.7 0.7 0.7 0.7 0.8 0.7

Miscellaneous

Ethanolamine (D) 0.6 0.4 0.5 0.5 0.5 0.6 0.6

Methyl-phosphate (C) 0.7 0.4 0.4 0.7 0.6 0.5 0.8

Phosphoric acid (D) 1.2 1.1 0.5 0.8 0.7 0.6 0.9

2-Hydroxygl utaric acid (D) 1.9 2.2 1.9 1.6 1.6 1.8 1.4 Ascorbic acid (P) 2.0 1.7 1.1 1.8 2.2 1.6 1.5

Abbreviations: D, definitive; C, confident; P, putative. Changed with P<0.05 were considered significant and are shown in bold italic font.

Table 3: Relative fold change in metabolites with no statistically significant change in abundance in the AD brain.

Metabolite HP ENT MTG sex MCX CG CB

Hydroxylamine (P) 1.0 1.1 0.8 1.0 1.2 0.9 1.2

Alanine (D) 0.8 0.7 1.3 0.6 3.4 1.0 1.2

Pyruvic acid (D) 1.3 1.6 1.5 0.8 0.5 1.0 0.8

Valine (D) 1.6 0.9 2.8 1.0 1.0 1.5 0.5

Leucine (D) 0.9 0.9 1.4 1.0 1.0 1.4 0.8

Isoleucine (D) 0.8 0.8 1.3 0.8 1.0 0.7 1.0

Succinic acid (C) 1.3 0.6 1.1 1.2 1.3 1.2 1.2

Methionine (D) 1.0 0.9 1.3 0.9 1.0 1.4 0.7

Ribotol (P) 0.3 1.0 2.0 0.4 1.2 1.1 1.0

Pyroglutamic acid (D) 1.2 1.2 1.1 1.3 1.5 1.2 1.1

Mannitol (D) 1.1 1.2 1.3 1.3 1.2 1.2 1.8

Scyllo-inositol (D) 0.9 0.9 1.1 0.7 0.7 0.8 0.7

Tyrosine (D) 0.7 0.7 1.2 1.0 1.0 1.2 0.6

Adenosine (D) 2.0 1.3 0.9 1.0 1.0 0.8 1.0

Abbreviations: D, definitive; C, confident; P, putative.

[00228] This example demonstrates alterations in metabolites from several pathways involved in gl ucose clearance and utilisation.

EXAMPLE 2

[00229] This example investigates copper metal levels in various brain regions of AD sufferers.

1. Methods

[00230] Human brains were acquired and sampled as for Example 1. Study group characteristics are shown in Table 4.

[00231] The diagnosis and severity of AD was assessed as for Example 1. Table 4: Group characteristics of brains used in study

Variable Control AD

Number 13 9

Age (± SD) 70 (±6.3) 70.3 (±7.1)

Male sex, n (%) 7 (53.8) 5 (55.6)

Post-mortem delay (h, range) 12 (5.5-15.0) 7* (4.0-12.0)

Brain weight (g, range) 1260 (1094-1461) 1062* (831-1355)

Wet-wt/dry-wt 5.7 (5.6-5.9) 5.5 (5.4-5.6)

Plaques, n (%) 1 (7.7) 9 (100)**

Tangles, n (%) 1 (7.7) 9 (100)**

*P=0.005, **P<0.0001 compared with Control ; all other differences were non-significant.

Preparation of brain extracts [00232] Cu concentrations were determined on a dry-weight basis. Brain samples of 50 ± 5 mg wet-weight were first dried to constant weight in a centrifugal concentrator (Savant Speedvac; Thermo-Fisher, Waltham, MA). The precise weights of the dried tissue samples were measured to enable normalization of tissue-metal content by dry-weight (Table 1) . Samples were digested in 2-mL microcentrifuge tubes (eppendorf) as described below.

Digestion

[00233] Tissue was digested using concentrated nitric acid (A509 Trace Metal Grade; Fisher, Loughborough, UK) to which was added 5% (v/v) Agilent Internal Standard mixture (5183-4681 ; Agilent Technologies, Cheadle, UK). This internally- standardized acid was also used at appropriate dilutions to provide rinse and calibration solutions, at 2% (v/v) final nitric acid concentration.

[00234] Calibration solutions were produced by appropriate dilutions of

Environmental Cal ibration Standard (Agilent 5183-4688). Acid digestion was carried out using an Open-vessel' method. Tissue al iquots were briefly centrifuged to ensure that the tissue sat at the bottom of the tube. The tube l ids were punctured to prevent pressure build-up, and 0.2 mL standard-containing nitric acid added . Tubes were then inserted into a "Dri-block" heater which was i nitially at room temperature. Tubes with standard-containi ng acid but no sample were processed in each batch to provide "digestion" blanks. Temperature was then set to 60oC and the block switched on. After 30 min, the set temperature was increased to lOOoC, and digestion continued for a further 210 mi n. After digestion, the tubes were al lowed to cool overnight. [00235] Al iquots of 100 μί were taken from each digestion and added to 15-mL Falcon tubes (Greiner) containing 5 mL LC-MS grade water, to produce solutions for analysis at a final nitric acid concentration of 2% (v/v).

ICP-MS

[00236] Cu-metal concentrations were measured using an Agilent 7700x ICP-MS spectrometer equipped with a MicroMist nebulizer (Glass Expansion, Melbourne, Australia) and a Scott double-pass spray chamber. Nickel sample and skimmer cones were used . Sample introduction was performed using an Agilent Integrated autosampler (I-AS). Helium was used as the collision gas. A multi-element method including all elements present in the calibration solution was applied . Calibration solutions were produced by appropriate dilutions of Environmental Calibration Standard (Agilent 5183-4688). Scandium was used as the internal standard. Two collision cell gas modes were applied, Cu metal concentration was analysed in helium mode (5.0 mL. min-1 helium). Mode selection followed Agilent recommendations to minimize interference for measured elements by e.g . isobaric cluster ions.

Integration time was 0.3 s. For each analytical batch, multi-element calibration was performed using serial dilutions of the calibration standard . An intermediate concentration from this calibration series was used as a periodic quality-control (QC) sample throughout each analytical batch. Instrument and digestion blanks were also interspersed through each set of randomized samples. The detection limit for Cu was determined by comparison of cal ibration samples and blanks and any samples below this level were eliminated prior to reporti ng.

[00237] ICP-MS measures the amount of metals such as copper in thei r elemental state. In contrast, most metals are present in the human body as cations; in the case of copper, the physiological cations copper(I) and copper(II) .

Data analysis

[00238] Datasets were exported to Microsoft Excel worksheets and individual values of each sample were normalized by the corresponding sample weight (dry weight). Weight-adj usted datasets were then log-transformed for statistical analysis. Means (± 95% CI) of the log-transformed data were calculated and the significance of between-group differences was examined by unpaired t-test with Welch's correction to allow for unequal variances and sample sizes. Means (± 95% CI) were back-transformed to reflect the actual elemental concentrations of elements.

Statistical calculations were performed using GraphPad v6.04 (Prism; La Jolla, CA). P-values of <0.05 have been considered significant, and those of 0.05≤ P < 0.10 have also been tabulated.

2. Results

[00239] The concentration of the essential metal Cu was measured in seven regions of human post-mortem brains from nine AD and 13 control subjects matched for age and sex.

[00240] One control patient also had neuropathological findings consistent with AD (Braak Stage II) and was therefore diagnosed with premanifest disease: this finding is consistent with the known frequency of asymptomatic AD in similarly-aged groups in the study population. Wet-wt/dry-wt ratios did not differ significantly between cases or controls.

[00241] Mean Cu concentrations in control tissue were highest in CB (710 μιτιοΙ/kg dry-wt) and lowest in ENT (310 μιτιοΙ/kg dry-wt) as shown in Table 5. Cu levels were significantly decreased compared with corresponding regional-control values in all seven regions of AD-brain, consistent with the presence of pan-cerebral copper deficiency.

Table 5: Copper metal concentration and wet/wt/dry-wt ratios in seven brain regions of AD and control brains.

Cu (μιηοΙ/kg dry-wt, mean ± 95% CI)

Brain region Control AD p-value

HP 330 (260-419) 183 (128-261) 0.0066

ENT 309 (281-340) 202 (146-281) 0.018

356 (300-422) 239 (195-293) 0.0035

MTG

sex 382 (323-451) 268 (218-330) 0.0079

MCX 390 (319-478) 252 (184-347) 0.019

346 (264-454)

CG 198 (141-280) 0.010

CB 708 (617-811) 374 (323-434) < 0.0001

Reference isotope = ^S3Cu. Data are means (± 95% CI); P-values for significance of between-group differences were calculated by Welch's modified t-tests based on measurements from control (n = 13) and AD (n=9) brains.

[00242] This example demonstrates pan-cerebral copper-deficiency in seven regions of the AD brain. EXAMPLE 3

[00243] This example investigates levels of metabolites and trace metals in brain regions known to undergo varying degrees of damage i n AD.

1. Methods

[00244] Human brains were acquired and sampled as for Example 1. The study group was the same as that for Example 1. No patients had history consistent with type II diabetes.

[00245] The diagnosis and severity of AD was assessed as for Example 1.

Analytical methods

[00246] Metabolite levels were compared between cases and controls by GC-MS- based metabolomics in wet-tissue. Copper levels were measured i n dry-tissue by inductively-coupled-plasma mass spectrometry (ICP-MS).

Observational case-control study

[00247] Patients and controls without cognitive impairment were respectively recruited through memory clinics in Leeds and Dewsbury (England), and the Leeds Family Health Services Authority day hospitals and elderly medicine outpatient clinics in the Leeds area . All were of European Caucasian background and gave written informed consent (consent from relatives of the AD-patients was provided, where appropriate) . Diagnosis of probable AD was made in accordance with international diagnostic criteria (National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group: NINCDS-ADRDA). All participants underwent a standardised clinical evaluation : medical history, fasting plasma glucose (FPG) and HbAlc, and cognitive function assessment by Mini-Mental State Examination (MMSE).

[00248] AD samples were selected by excluding patients with diagnosed T1D or T2D including those on synthetic insulin . Patients with a previous medical history of other medical conditions were also excluded . Samples were then selected from the whole-study population for whom required measurements (FPG, HbAlc) were available. The resulting 42 AD patients were then age- and gender-matched to 43 controls. 2. Results

Glucose, sorbitol and fructose in AD brain regions

[00249] The relative fold-changes in glucose, sorbitol and fructose in seven brain regions is shown in Table 6.

[00250] Levels of glucose were elevated in all brain regions of AD patients. Levels of glucose tended to be higher in regions of the brain more severely affected by AD such as the middle temporal gyrus.

[00251] Sorbitol, formed from glucose, is the fi rst metabol ite in the polyol pathway, which usually accounts for a few percent at most of glucose utilization under normal conditions. Sorbitol was elevated in al l AD brain regions.

[00252] Fructose is the second metabolite in the polyol pathway. Brai n fructose levels were elevated in all AD-brain regions.

Table 6: Relative fold change in glucose, sorbitol and fructose in brain regions.

Lower Upper P- BH FDR

Metabolite Brain region Estimate

Bound Bound value Q-value

Glucose Cerebell um 5.2 1.4 18.6 0.015 0.015

Entorhinal cortex 7.4 2.4 23.2 0.0028 0.0041

Cingulate gyrus 8.0 2.4 26.1 0.0023 0.0037

Hippocampus 8.9 2.9 27.4 0.0016 0.0030

Sensory cortex 9.2 3.0 28.8 0.0014 0.0029

Motor cortex 11.8 3.7 37.7 0.0006 0.0019

Middle temporal gyrus 16.4 5.2 51.2 0.0002 0.0019

Sorbitol Cerebellum 3.7 1.6 8.6 0.0036 0.0044

Entorhinal cortex 4.3 2.0 9.1 0.0006 0.0019

Cingulate gyrus 3.1 1.5 6.5 0.0048 0.0053

Hippocampus 4.1 1.9 8.5 0.0009 0.0021

Sensory cortex 4.1 1.9 8.6 0.0009 0.0021

Motor cortex 3.0 1.4 6.6 0.0090 0.0094

Middle temporal gyrus 3.3 1.5 6.9 0.0039 0.0046

Fructose Cerebellum 5.3 2.0 14.4 0.0018 0.0031

Entorhinal cortex 5.7 2.4 13.5 0.0004 0.0019

Cingulate gyrus 3.9 1.7 9.2 0.0030 0.0041

Hippocampus 5.5 2.3 12.9 0.0005 0.0019 Sensory cortex 5.4 2.4 12.4 0.0004 0.0019

Motor cortex 4.2 1.7 10.2 0.0032 0.0041

Middle temporal gyrus 5.7 2.6 12.9 0.0003 0.0019

Estimates and their lower and upper bounds were derived by Bayesian modelling . Abbreviation : 'BH FDR Q-value' is the Benjamini-Hochberg False-Discovery Rate- adjusted P-value.

[00253] Elevated brain levels of glucose, sorbitol and fructose were present in one control patient, a 76 year-old female, who had no ante-mortem clinical evidence for brain disease or dementia, but had premanifest AD characterised by low brain weight ( 1,094 g) and positive post-mortem histology.

Copper levels in AD brain regions

[00254] Mean (±95% CI) brai n-copper levels summed across all seven regions were 256 (232-279) μιτιοΙ/dry kg in AD and 406 (363-449) μιτιοΙ/dry kg in controls (P= 1.6xl0^"s) : there was thus an overall decrease of ~40% in brain-copper levels in AD. Mean brain-copper levels tended to be lower in AD than controls in all brain regions exami ned as shown in Figure I B. In addition, there was moderate evidence for a trend in copper levels to be inversely proportional to tissue-glucose values in AD-patients (P=0.021 ; restricted iterative generalized least squares, RIGLS 23) but not in controls as shown in Figure 1 (A and B) . Plasma glucose and plasma copper levels in AD patients

[00255] In the case control study group, measured cognitive function (MMSE score) in the AD group was significantly lower than that of controls whereas the ApoE4 al lele was more prevalent (data not shown). Fasting plasma glucose (FPG) and serum HbAlc levels were equivalent between groups so there was no evidence for elevated rates of undiagnosed T2D or impaired glucose tolerance (IGT)/impaired fasting glucose (IFG) in this group of British patients with sporadic AD (data not shown). FPG and fasting plasma-copper levels did not differ significantly between the AD group and the controls (data not shown) .

[00256] This example demonstrates elevated glucose, fructose, sorbitol and copper levels i n the AD brain.

Claims

1. A method of treating Alzheimer's disease (AD) by administration to a mammalian subject in need thereof an effective amount of a copper binding tetramine compound and an effective amount of an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject.

2. The method of clai m 1 wherein the copper binding tetramine compound binds Cu(II).

3. The method of claim 2 wherein the copper binding tetramine compound is a tetramine compound that is specific for Cu(II) over Cu(I).

4. The method of claim 3 wherei n the tetramine compound is selected from the group comprising triethylenetetramine (2,2,2 tetrami ne), 2,3,2 tetramine, 3,3,3 tetramine, and salts, active metabolites, derivatives, and prodrugs thereof.

5. The method of claim 4 wherei n the tetramine compound is triethylenetetramine

disuccinate.

6. The method of any one of clai ms 1 to 5 wherein the agent effective to reduce the

amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

7. The method of claim 6 wherei n the amylin analogue is Symlin .

8. The method of claim 6 wherein the GLP-1 agonist is exenatide, liraglutide, lixisenatide, albiglutide, or dulaglutide, or any combination of two or more thereof.

9. The method of clai m 6 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagl iptin, Vildagliptin, Saxagl iptin, Linagliptin, Anagliptin, Teneligl iptin, Alogliptin, Trelagliptin, Gemigl iptin, Dutoglipti n, and

Omarigliptin.

10. The method of clai m 6 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagli ptin, Nesina, Tradjenta, Onglyza, and Januvia .

11. The method of any one of clai ms 1 to 10 wherein the administration of the copper bi nding tetramine compound and of the agent effective to reduce the amount or concentration of one or more of encephalic gl ucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is simultaneous, sequential, or separate.

12. The method of claim 1 wherein the administration is of triethylenetetramine

disuccinate and amylin or Symlin, and may be simultaneous, sequential, or separate administration.

13. The method of claim 1 wherein the administration is of triethylenetetramine

disuccinate and one or more agents selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligli ptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin, and may be si multaneous, sequential, or separate administration.

14. The method of claim 1 wherein the administration is of triethylenetetramine

disuccinate and one or more agents selected from the group comprising alogliptin, linagl iptin, saxaglipti n, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia, and may be simultaneous, sequential, or separate administration.

15. The method of any one of clai ms 1 to 14 wherein the mammalian subject is a non- diabetic subject or a subject who is not undergoing treatment for diabetes.

16. The method of any one of clai ms 1 to 15 wherein the mammalian subject does not suffer from Wilson's disease.

17. The method of any one of clai ms 1 to 16 wherein the mammalian subject is a human subject.

18. A pharmaceutical composition comprising one or more copper binding tetramine

compounds, additional ly comprising or formulated to be administered in conj unction in with an agent effective to reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in a mammalian subject, wherein the one or more pharmaceutical compositions comprises a pharmaceutically acceptable carrier or dil uent.

19. The pharmaceutical composition of claim 18 wherein the tetrami ne compound is

triethylenetetramine disuccinate.

20. The pharmaceutical composition of claim 18 or 19 wherein the agent effective to

reduce the amount or concentration of one or more of encephalic glucose, encephalic sorbitol, or encephalic fructose in the mammalian subject is selected from the group comprising amylin, an amylin analogue, a GLP-1 agonist, and a selective dipeptidyl peptidase (DPP-IV) inhibitor.

21. The pharmaceutical composition of claim 20 wherein the amylin analogue is Syml in .

22. The pharmaceutical composition of claim 20 wherein the GLP-1 agonist is exenatide, liragl utide, lixisenatide, albiglutide, or dulagl utide, or any combination of two or more thereof.

23. The pharmaceutical composition of claim 20 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagl iptin, Anagliptin, Tenel igliptin, Alogliptin, Trelaglipti n, Gemigliptin, Dutogliptin, and Omarigliptin.

24. The pharmaceutical composition of claim 20 wherein the selective dipeptidyl peptidase (DPP-IV) inhibitor is selected from the group comprising alogliptin, linagl iptin, saxagli ptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia .

25. The pharmaceutical composition of claim 18 comprising triethylenetetramine

disuccinate and amylin or Symlin.

26. The pharmaceutical composition of claim 18 comprisi ng triethylenetetramine

disuccinate and one or more agents selected from the group comprising Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Anagliptin, Teneligli ptin, Alogliptin, Trelagliptin, Gemigliptin, Dutogliptin, and Omarigliptin .

27. The pharmaceutical composition of claim 18 comprising triethylenetetramine

disuccinate and one or more agents selected from the group comprising alogliptin, linagliptin, saxagliptin, sitagliptin, Nesina, Tradjenta, Onglyza, and Januvia .