SELENO-COMPOUNDS AND THERAPEUTIC USES THEREOF

The present invention relates to compounds and compositions useful as antioxidants and in particular to selenium containing compounds of formula (I): wherein n is 1, 2, or 3; m is 2, 3, 4, or 5; and each R] is independently —(optionally substituted C1-C3 alkylene) p-OH, where p is 0 or 1, or a salt thereof. The invention also relates to the use of these seleno-compounds in the treatment of diseases or conditions associated with increased levels of oxidants produced by myeloperoxidase (MPO), such as for instance, atherosclerosis.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “oxidative stress” refers to an abnormal level of reactive oxygen species (ROS). Oxidative stress may be induced by, for example an increase in the levels of free radicals such as hydroxyl, nitric acid or superoxide or an increase in the levels of non-radicals such as hydrogen peroxide, lipid peroxide and hypohalous acid which may themselves be a source of free-radicals. Increased ROS levels may occur as a result of a number of activities or conditions including infections, inflammation, ageing, UV radiation, pollution, excessive alcohol consumption, and cigarette smoking. Oxidative stress may lead to oxidative damage of particular molecules such as proteins and lipids with consequential injury to cells, tissues or organs. Thus, oxidative stress is involved in a number of diseases including cancer, ischemia-reperfusion injury, infectious disease, inflammatory disease, autoimmune diseases, cardiovascular diseases. For a review on oxidative stress and related conditions/diseases the reader is referred to J. Ocul. Pharmacol Ther. 2000 April; 16(2):193-201 which is incorporated herein by reference.

For example, LDL (low density lipoprotein) may become oxidised during periods of oxidative stress and induce the formation of macrophage-derived foam cells. These foam cells are present in pre-atherosclerotic fatty-streak lesions and advanced atherosclerotic plaques. This link between oxidative stress and atherosclerosis is supported by findings that the antihyperlipidemic drug probucol exhibits an antioxidative activity and is effective for the treatment of arterial sclerosis.

In addition, the heme enzyme myeloperoxidase (MPO) is released at sites of inflammation by activated leukocytes. A key function of MPO is the production of hypohalous acids (HOX, X=Cl, Br), which are strong oxidants with potent antibacterial properties. However, HOX can also damage host tissue when produced at the wrong place, time or concentration; this has been implicated in several human diseases (e.g. atherosclerosis, some cancers). Thus, elevated blood and leukocyte levels of MPO are significant independent risk factors for atherosclerosis, while specific markers of HOX-mediated protein oxidation are often present at elevated levels in patients with inflammatory diseases. HOX react readily with amino acids, proteins, carbohydrates, lipids, nucleobases and antioxidants. Sulfur-containing amino acids (Cys, Met, cystine) and amines on amino acids, nucleobases, sugars and lipids are the major targets for HOX. Reaction with amines generates chloramines (RNHCl) and bromamines (RNHBr), which are more selective oxidants than HOX and are key intermediates in HOX biochemistry. These species are known to be formed in high yield on a range of protein targets, including proteins in human plasma, on exposure to HOCl. As such it is important to develop therapeutic compounds that can also scavenge these materials in a rapid and effective manner.

“Alkylene” refers to a divalent alkyl group. Examples of such alkylene groups include methylene (—CH2—), ethylene (—CH2CH2—), and the propylene isomers (e.g., —CH2CH2CH2—and —CH(CH3)CH2—).

“Optionally substituted” in the context of the present invention is taken to mean that a hydrogen atom on the alkylene chain may be replaced with a group selected from hydroxyl, amino, or thio. More preferably the substituent is hydroxyl.

In a preferred aspect the present invention provides stable, aqueous soluble 5, 6 and 7 membered selenocycles of formula (I) wherein the compound is not metabolisable or derivatisable (to any great extent) by the body. In this regard as there are no known mammalian enzymes that process L-sugars, in particular L-gulose and L-idose, in a further preferred aspect the seleno-cycles of formula (I) are seleno-derivatives of L-sugars.

In an embodiment n is 1.

In an embodiment n is 2.

In an embodiment n is 3.

In an embodiment n is 1 and m is 2, 3, or 4.

In an embodiment n is 2 and m is 2, 3, 4, or 5.

In an embodiment n is 3 and m is 2, 3, 4, and 5.

In an embodiment n is 1 or 2 and m is 2, 3, or 4.

In an embodiment n is 1 or 2, m is 2, 3, or 4 and at least one R1is (optionally substituted (C1-C3) alkylene)p-OH where p=1.

In an embodiment n is 1 or 2, m is 2, 3, or 4 and one R1is (optionally substituted C1-C3alkylene)p-OH where p=1.

In an embodiment n is 2, m is 4, and one R1is (optionally substituted C1-C3alkylene)p-OH where p=1.

In the above embodiments preferably the (optionally substituted C1-C3alkylene)p-OH group is optionally substituted C2-alkylene-OH or C1-alkylene-OH. More preferably the group is —CH2—OH.

In the above embodiments where the C1-C3alkylene group is substituted it is substituted with a hydroxyl group, for example —CH(OH)—CH2OH.

Examples of seleno-compounds of formula (I) include:

In an embodiment the seleno-compound of formula (I) is represented by

In a further embodiment the seleno-compound of formula (I) is represented by

The compounds of the invention may be in crystalline form either as the free compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

It will also be recognised that compounds of the invention may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or mixtures may be resolved by conventional methods, eg., chromatography, or use of a resolving agent.

Alternatively, enantiomerically pure seleno-compounds of formula (I) may be prepared from carbohydrates. In this regard preferred compounds of the present invention may be representative seleno-derivatives of known monosaccharides where the selenium is in the ring position. Examples of suitable seleno-compounds of this sort may be derived from either D- or L-aldoses such as ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, and talose. Preferably the seleno-compounds are derivatives of L-aldoses. Representative examples include:

(shown as mixtures of α and β anomers)

In an embodiment the compound is selected from one of following:1,5-anhydro-5-seleno-L-gulitol1,5-anhydro-5-seleno-L-mannitol1,5-anhydro-5-seleno-L-iditol1,5-anhydro-5-seleno-L-glucitol1,5-anhydro-5-seleno-L-galitol1,5-anhydro-5-seleno-L-talitol1,5-anhydro-5-seleno-L-allitol1,5-anhydro-5-seleno-L-altritol

In an embodiment the compound is selected from one of the following:1,5-anhydro-5-seleno-D-gulitol1,5-anhydro-5-seleno-D-mannitol1,5-anhydro-5-seleno-D-iditol1,5-anhydro-5-seleno-D-glucitol1,5-anhydro-5-seleno-D-galitol1,5-anhydro-5-seleno-D-talitol1,5-anhydro-5-seleno-D-allitol1,5-anhydro-5-seleno-D-altritol

In an embodiment the compound is selected from one of the following:1,4-anhydro-4-seleno-L-gulitol1,4-anhydro-4-seleno-L-mannitol1,4-anhydro-4-seleno-L-iditol1,4-anhydro-4-seleno-L-glucitol1,4-anhydro-4-seleno-L-galitol1,4-anhydro-4-seleno-L-talitol1,4-anhydro-4-seleno-L-allitol1,4-anhydro-4-seleno-L-altritol

In another embodiment the compound is selected from one of the following:1,4-anhydro-4-seleno-D-gulitol1,4-anhydro-4-seleno-D-mannitol1,4-anhydro-4-seleno-D-iditol1,4-anhydro-4-seleno-D-glucitol1,4-anhydro-4-seleno-D-galitol1,4-anhydro-4-seleno-D-talitol1,4-anhydro-4-seleno-D-allitol1,4-anhydro-4-seleno-D-altritol

The seleno-compounds of the present invention can be prepared based on the modification of the synthetic procedures described in, for example, M. A. Lucas et al.,Tetrahedron, 2000, 56:3995-4000 and C. Storkey et al.,Chem. Comm.,2011, 47, 9693-9695.

In respect of compounds of formula (I) some examples of suitable synthetic approaches are depicted in the below schemes.

It will be appreciated from the above schemes, that various other seleno containing carbohydrates may be obtained by following the procedures using different starting carbohydrates.

During the reactions a number of the moieties may need to be protected. Suitable protecting groups are well known in industry and have been described in many references such as Protecting Groups in Organic Synthesis, Greene T W, Wiley-Interscience, New York, 1981.

In another aspect, the present invention provides pharmaceutical compositions for use as free-radical scavengers, more particularly as antioxidants, the composition comprising an effective amount of a seleno-compound of the present invention or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or diluent.

The term “composition” is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

The pharmaceutical compositions or formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

The seleno-compounds of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.

Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.

The seleno-compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention.

The compounds of the present invention may be administered to a subject as a pharmaceutically acceptable salt. It will be appreciated however that non-pharmaceutically acceptable salts also fall within the scope of the present invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.

In powders, the carrier is a finely divided solid that is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.

Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as an admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

Sterile liquid form compositions include sterile solutions, suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent or a mixture of both.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired.

Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.

Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.

In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 5 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation.

When desired, formulations adapted to give sustained release of the active ingredient may be employed.

The invention also includes the compounds in the absence of carrier where the compounds are in unit dosage form.

The amount of the seleno-compound which is to be administered may be in the range from about 10 mg to 2000 mg per day, depending on the activity of the compound and the disease to be treated.

Liquids or powders for intranasal administration, tablets or capsules for oral administration and liquids for intravenous administration are the preferred compositions.

The compositions may further contain one or more other antioxidants or be administered along with another active agent such as for instance an antihypertensive agent.

As discussed above the present inventors have found that seleno-compounds act as effective oxidant scavengers in plasma. Accordingly, the components of the present invention may be used in therapies where antioxidants have proven to be effective such as treating conditions associated with oxidative stress.

Thus, in another aspect the invention provides a method for scavenging oxidants in plasma comprising administering to a subject an effective amount of a compound of formula (I).

Humans consume approximately 250 grams of oxygen per day and a typical human cell metabolises about 1012molecules of oxygen per day. An inevitable consequence of our dependence on oxygen is that small amounts of highly reactive radical and non-radical derivatives of diatomic oxygen (ROS), such as O2.−, H2O2, .OH, RO2., ROOH, HOCl, HOBr, HOSCN and ONOO−, are generated in vivo.

The main source of ROS within the arterial wall is a form of the enzyme NAD(P)H oxidase. This enzyme generated superoxide radicals by catalysing the reduction of O2(see scheme 10). Superoxide radicals can subsequently be converted to more potent ROS. For example, dismutation provides hydrogen peroxide and reaction with nitric oxide affords peroxynitrite (see scheme 10).

Living organisms utilise ROS as inter- and intracellular mediators of signal transduction. However, ROS can oxidise all major classes of biomolecules and are harmful at high concentrations. Living organisms are protected against ROS by a group of antioxidant compounds and enzymes. Notable antioxidant enzymes are the enzymes glutathione peroxidase (GPx) and thioredoxin reductase which both contain selenium.

Antioxidants prevent the formation of ROS or intercept ROS and exclude them from further activity. In healthy aerobic organisms, ROS production is counterbalanced by antioxidant defence networks and ROS levels are tightly regulated. However, sometimes the endogenous antioxidant defence network becomes overwhelmed by excess ROS. This imbalance between ROS and antioxidants in favour of ROS is referred to as oxidative stress and it has been implicated in the pathology of a vast array of diseases including, hyperlipidemia, diabetes mellitus, ischemic heart disease, atherosclerosis and chronic heart failure. There is a growing body of evidence which suggests that oxidative stress is also involved in the pathogenesis of hypertension. This is because one of the many effects of angiotensin II is to stimulate NAD(P)H oxidase and thereby increase the amount of NAD(P)H oxidase derived ROS present in the vasculature. The numerous mechanisms via which these ROS proceed to bring about hypertension are yet to be fully elucidated. It is thought that hydrogen peroxide may increase the concentration of calcium cations in vascular cells and calcium cations are known to induce vasoconstriction. Alternatively, ROS may activate genes and transcription factors mediated oxidation of arachidonic acid to F2-isoprostanes, which are prostaglandin-like compounds that are potent vasoconstrictors.

Furthermore, mtDNA diseases such as cardiomyopathy, heart failure, heart block, arrhythmia, diabetes, pancreatitis, retinopathy, optic neuropathy, renal failure, Kearns Sayre Syndrome, Sudden Infant Death Syndrome, dementia and epilepsy, stroke may also be effectively treated using the compounds of the present invention.

Other conditions such-as inflammation, ischaemic-reperfusion tissue injury in strokes, heart attacks, organ transplantation and surgery, edema, atherosclerosis, may also be beneficially treated with the compounds of the present invention.

For certain of the above mentioned conditions/diseases it is clear that the compounds may be used prophylactically as well as for the alleviation of acute symptoms. References herein to “treatment” or the like are to be understood to include such prophylactic treatment, as well as treatment of acute conditions.

From the above discussion it would be evident that one of the other main advantages of the seleno-compounds of the present invention will be their ability to provide cardioprotective qualities. Accordingly, the present seleno-compounds are seen to be beneficial in the context of increasing the bodies natural ability to prevent (or enhance the prevention of) tissue damage in the cardiovascular system.

The invention will now be described in the following Examples. The Examples are not to be construed as limiting the invention in any way.

EXAMPLES

General Experimental Techniques

1H NMR spectra were recorded on Varian Inova 400 (400 MHz) or Varian Inova 500 (500 MHz) instruments at room temperature, using CDCl3(or other indicated solvents) as internal reference deuterium lock, CDCl3at δ 7.26 ppm, CH3OD at δ 3.31 ppm. The chemical shift data for each signal are given as δ in units of parts per million (ppm). The multiplicity of each signal is indicated by: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets) and m (multiplet). The number of protons(n) for a given resonance is indicated by n H. Coupling constants (J) are quoted in Hz and are recorded to the nearest 0.1 Hz.

13C NMR spectra were recorded on Varian Inova 400 (400 MHz) or Varian Inova 500 (500 MHz) instruments using the central resonance of the triplet of CDCl3at δ 77.23 ppm as an internal reference. The chemical shift data for each signal are given as δ in units of parts per million (ppm).

77Se NMR spectra were recorded on a Varian Inova 500 (500 MHz) instrument with proton decoupling. The chemical shift data for each signal are given as δ in units of ppm relative to (SePh)2.

Infrared spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer in the region 4000-650 cm−1. The samples were analysed as thin films from dichloromethane or as solutions in the indicated solvents.

Mass spectra were recorded at the Bio21 Institute, The University of Melbourne. Low resolution spectra were recorded on a Waters Micromass Quattro II instrument (EI and CI). All high resolution mass spectrometry experiments were conducted using a commercially available hybrid linear ion trap and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Finnigan LTQ-FT San Jose, Calif.), which is equipped with ESI. The ions of interest were mass selected in the LTQ using standard procedures and were then analyzed in the FT-ICR MS to generate the high resolution tandem mass spectrum.

Optical specific rotations were measured using a Jasco DIP-1000 digital polarimeter, in a cell of 1 dm path length. The concentration (c) is expressed in g/100 cm3(equivalent to g/0.1 dm3). Specific rotations are denoted [α]DTand given in implied units of ° dm2g−1(T=temperature in ° C.).

Analytical thin layer chromatography (TLC) was carried out on pre-coated 0.25 mm thick Merck 60 F254silica gel plates. Visualisation was by absorption of UV light, or thermal development after dipping in an ethanolic solution of phosphomolybdic acid (PMA) or sulfuric acid (H2SO4). Flash chromatography was carried out, on silica gel [Merck Kieselgel 60 (230-400 mesh)] under a pressure of nitrogen.

Hydrogenation was carried out in a Büchi GlasUster “miniclave drive” stainless steel vessel, 100 ml, with a maximum operation pressure of 60 bar. Teflon inserts were used and reactions were stirred using magnetic stirrer bars.

Dry DMF was distilled from sodium hydride. Anhydrous THF, diethyl ether, and dichloromethane were dried by passage through a packed column of activated neutral alumina under a nitrogen atmosphere, and toluene being passed through a coloumn with additional R3-11 copper-based catalyst (BASF Australia). Petroleum ether refers to the fraction of boiling point range 40-60° C. Procedures using moisture or air sensitive reagents were undertaken in a nitrogen-filled dual manifold employing standard Schlenk line techniques.

Melting points were determined with an Electrothermal Engineering IA9100 or a Büchi 510 melting point apparatus and are uncorrected.

Synthesis of Selenium Containing Carbohydrates

Synthesis of 1,5-anhydro-5-seleno-L-gulitol

To a suspension ofD-mannose (10 g, 55.5, mmol) and p-toluenesulfonic acid monohydrate (1.06 g, 5.55 mmol) over 4 Å molecular sieves in dry DMF (100 mL) at 0° C. was added 2-methoxypropene (10.6 mL, 8.0 g, 222 mmol) dropwise over 30 minutes. The suspension was maintained at 0° C. for 8 hours and allowed to warm to room temperature. The resulting pale yellow solution was quenched by the addition of NaCO3(2 g). Filtration and removal of the solvent in vacuo gave a yellow oil. The residue was partitioned between ethyl acetate (200 mL) and water (200 mL) and the organic layer was separated. The aqueous phase was extracted with ethyl acetate (3×100 mL) and the combined organic extracts washed with brine (2×80 mL) and dried over MgSO4. Evaporation afforded the crude di-isopropylidene as the major of three products, two of which were virtually inseperable by column chromatography Rf0.18 (hexane:ethyl acetate) (3:1). The crude mixture was then dissolved in anhydrous methanol (100 mL) under nitrogen at 0° C. before the portionwise addition of sodiumborohydride (2.9 g, 77 mmol). Vigorous effervescence occurred and the solution was stirred at 0° C. for 30 min and then at room temperature for 4 hours. Two new products were observed by TLC, the major of which being the desired diol 1 (Rf0.36), the minor product (Rf0.52) (ethyl acetate:hexanes) (2:1) was now able to be separated by column chromatography The solvent was removed in vacuo and the residue was partitioned between ethyl acetate (150 mL) and water (150 mL) and the organic layer was separated. The aqueous phase was extracted with ethyl acetate (5×50 mL) and the combined organic extracts washed with brine (2×50 mL) and dried over MgSO4. Evaporation and chromatography (25%-67% ethyl acetate in petroleum ether) afforded the diol (1) as a colourless oil (9.36 g, 36 mmol, 67% over 2 steps). Rf0.36 (Hex:EtOAc 1:2); [α]D22=−12.8° (c 1.0 in DCM);1H NMR (500 MHz, CDCl3) δ 4.47 (dd, J=2.3, 6.7 Hz, 1H), 4.31 (dt, J=4.8, 6.7 Hz, 1H), 3.97-3.89 (m, 2H), 3.81 (m, 2H), 3.70 (dd, J=2.3, 8.8 Hz, 1H), 3.64 (td, J=2.6, 10.3 Hz, 1H), 1.53 (s, 3H), 1.49 (s, 3H), 1.41 (s, 3H), 1.38 (s, 3H);13C NMR (125 MHz, CDCl3) δ 109.10, 99.43, 77.88, 74.90, 72.58, 64.94, 63.98, 61.66, 28.29, 26.90, 25.80, 19.65; IR (neat)/cm−1: 3433, 2986, 1217, 1066; MS (ESI+) m/z (rel intensity) 263.09 [100, (M+Na)+]; HRMS (ESI+) m/z 263.1489 (263.1489 calcd for C12H22O6Na). These data agree with the published literature values (H. Liu and B. M. Pinto,Can. J. Chem.,2006, 84, 4, 497-505).

Synthesis of 1,5-anhydro-5-seleno-D-mannitol

Synthesis of 1,5-anhydro-5-seleno-L-iditol

To a suspension of D-Glucose (10 g, 83 mmol) in acetic anhydride (33 mL) under nitrogen at room temperature was added hydrobromic acid (8 ml, 33% HBr in acetic acid) dropwise. The suspension was stirred for 1 hour during which time the glucose dissolved into the solution. After this time a further amount of hydrobromic acid was added (42 mL, 33% HBr in acetic acid) dropwise and the reaction was stirred at room temperature overnight. Sodium acetate (10 g) was then added and the solution was stirred for a further 30 minutes before the addition of CH2Cl2(200 mL). The organic layer was washed with saturated NaHCO3(5×100 mL), brine (50 mL), dried over MgSO4, and concentrated in vacuo to afforded desired product (11) (21.7 g, 79 mmol, 95%), which was used without further purification.

Synthesis of 1,5-anhydro-5-seleno-D-glucitol

Alternative synthesis of 1,5-anhydro-5-seleno-L-iditol

Synthesis of 1,5-anhydro-5-seleno-D-glucitol

Synthesis of 1,4-anhydro-4-seleno-l-talitol

1.2 Biological Data

1.2.1 Seleno Sugars as Potent Scavengers of Hypochlorous and Hypobromous Acid

The kinetics of the reactions of HOBr (10 μM) with the seleno-sugar derivatives (0.75 mM-0.02 mM) were investigated in competition with N-acetyl tyrosine (1 mM) at 22° C. by the methods described by Davies and co-workers (M. J. Davies et al.,Antioxid. Redox Signaling,2008, 10, 7, 1199-1234). The assay examines the conversion of N-acetyl tyrosine to the corresponding N-acetyl-3-bromotyrosine, in the absence and presence of an oxidation scavenger (e.g. Se-sugar). The yields of N-acetyl-3-bromotyrosine at increasing carbohydrate derivative concentration (yieldquench) were determined by HPLC and compared to the maximal yield in the absence of added quencher (yieldmax). Using competition kinetics, the yields of the products of reaction with HOX are related by equation (1), and rearrangement of this equation results in the linear form (y=mx+c), given in equation (2).

From a plot of yieldmax[N-acetyl tyrosine]/yieldquenchagainst increasing concentration of quencher ([quencher]) the gradient of the corresponding line can determine the value of kquencherusing the known value of kTyrwith a set y-intercept equal to [N-acetyl tyrosine]. The results are depicted inFIG. 3AandFIG. 3C.

Experimental Procedures

Competitive Kinetic Studies for Seleno-Sugars Against HOBr Using N-Acetyl-Tyrosine

HOBr was prepared by mixing HOCl (40 mM in water, pH 13) with NaBr (45 mM in water) in equal volumes. The reaction was left for 1 minute before dilution with 0.1 M phosphate buffer (pH 7.4) to the required concentration of HOBr (typically 0.2-2.0 mM). As HOBr disproportionates slowly to form Br−and BrO2−, fresh solutions were prepared for each kinetic run and used within 30 minutes. To investigate whether Br2formed in the presence of excess Br−contributed to the observed reaction kinetics, HOBr solutions were also prepared with increasing concentrations of NaBr (45-250 mM). At neutral pH, hypobromous acid exists primarily as HOBr with low concentrations of−OBr also present (pKa8.7).

1.1.1.1 HPLC Instrumentation and Methods

Analysis and quantification of N-acetyl-tyrosine and its reaction products with HOBr were carried out on a Shimadzu LC-10A HPLC system (Shimadzu, South Rydalmere, NSW, Australia). The reaction mixtures were separated on a Zorbax reverse-phase HPLC column (25 cm×4.6 mm, 5 μM particle size; Rockland Technologies, Newport, Del.) packed with octadecyl silanized silica, equipped with a Pelliguard guard column (2 cm; Supelco). The column was maintained at 30° C. using a column oven (Waters Corp., Milford, Mass.). The mobile phase was comprised of a gradient of solvent A (10 mM phosphoric acid with 100 mM sodium perchlorate at pH 2.0) and solvent B [80% (v/v) MeOH in nanopure water] eluting at 1 mL min−1. The gradient was programmed as follows: 20% solvent B and 80% solvent A at 0 min increasing to 80% solvent Bover 10 mins; over the next 5 minutes the proportion of solvent B was held at 80%, before the proportion of solvent B was reduced to 20%, and the column was allowed to re-equilibrate for 6 minutes prior to injection of the next sample. The eluent was monitored in series by a UV detector (280 nm) and an electrochemical detector (Antec Leyden Intro). The channel of the electrochemical detector was set to an oxidation potential of +1200 mV to quantify the halogenated N-acetyl-tyrosine products. Peak areas were quantified using Class VP 7.4 Sp1 software (Shimadzu) and compared to authentic standards when required. Using these conditions, N-acetyl-tyrosine was detected in the +1200 mV electrochemical channel at a retention time of 8.3 min, N-acetyl-3-bromotyrosine at 11.4 min, and N-acetyl-3,5-dibromotyrosine at 13.4 min. A small impurity peak present in the parent compound was also detected with a retention time of 5.6 min, but this was not characterized further.

1.1.1.2 Sample Preparation for HOBr Analysis

Varying concentrations of Se-sugars (0.75 mM-0.02 mM) were added to solutions of known N-acetyl-tyrosine (1 mM).

200 μL of each sample (Se1-Se10) was subsequently added to a solution of HOBr (20 μL of 0.1 mM HOBr). Samples, with a final volume of 200 μL, were then mixed, filtered (0.2 μM cut-off filters) and placed in a glass HPLC vials for HPLC analysis.

Competitive Kinetic Studies for Seleno-Sugars Against HOCl Using FMoc-Methionine

The kinetics of the reactions of HOCl (1 μM) with the seleno-sugar derivatives (1.2 μM-20 μM) were investigated in competition with FMoc-methionine (5 μM) at 22° C. by adapting the methods described by Davies and co-workers (M. J. Davies et al.,Antioxid. Redox Signaling,2008, 10, 7, 1199-1234). The assay examines the conversion of FMoc-methionine to the corresponding FMoc-methionine sulfoxide, in the absence and presence of an oxidation scavenger (e.g. Se-sugar). The yields of FMoc-methionine sulfoxide at increasing carbohydrate derivative concentration (yieldquench) were determined by HPLC and compared to the maximal yield in the absence of added quencher (yieldmax). Using competition kinetics, the yields of the products of reaction with HOX are related by equation (1), and rearrangement of this equation results in the linear form (y=mx+c), given in equation (2).

From a plot of yieldmax[FMoc-methionine]/yieldquenchagainst increasing concentration of quencher ([quencher]) the gradient of the corresponding line can determine the value of kquencherusing the known value of kMetwith a set y-intercept equal to [FMoc-methionine]. The results are depicted inFIG. 3BandFIG. 3C.

Experimental Procedures

All chemicals were obtained from Sigma/Aldrich/Fluka and were used as received, with the exception of sodium hypochlorite (in 0.1 M NaOH, low in bromine; BDH Chemicals). The HOCl was standardized by measuring the absorbance at 292 nm at pH 12 [ε-292 (−OCl) 350 M−1cm−1]. All studies were performed in 10 mM phosphate buffer (pH 7.4). All phosphate buffers were prepared using Milli Q water and treated with Chelex resin (Bio-Rad) to remove contaminating transition metal ions. The pH values of solutions were adjusted, where necessary, to pH 7.4 using 100 mM H2SO4or 100 mM NaOH.

1.1.1.3 HPLC Instrumentation and Methods

Analysis and quantification of FMoc-methionine and its reaction products with HOCl were carried out on a Shimadzu Nexera UPLC system (Shimadzu, South Rydalmere, NSW, Australia). The reaction mixtures were separated on a Shim-pack XR-ODS (Shimadzu, 100×4.6 mm, 2.2 μM) column. The column was maintained at 40° C. with a flow rate of 1.2 mL.min−1. The mobile phase was comprised of a gradient of solvent A[(MeOH (20%), THF (2.5%), NaOAc (5%) and H2O (72.5%)] and solvent B [MeOH (80%), THF (2.5%) and NaOAc (5%), H2O (12.5%)]. The gradient was programmed as follows: 75% solvent B and 25% solvent A at 0 min, increasing to 87.5% solvent B over 5 min, followed by a further increase to 100% solvent B over the next 0.5 min and a wash with 100% solvent B for 2.5 min, before returning to 75% solvent B over the next 0.5 min with 3.5 min of re-equilibrating preceding the next injection. The eluent was monitored by fluorescence detection (RF-20Axs; λex, 265 nm; λem, 310 nm), with peak areas determined using Lab solutions 5.32 SP1 software (Shimadzu) and compared to authentic standards when required. Using these conditions, FMoc-methionine sulfoxide was detected in the fluorescence channel (λex, 265 nm; λem, 310 nm) at a retention time of 1.7 min, and FMoc-methionine at 2.8 min.

1.1.1.4 Sample Preparation for HOCl Analysis

Varying concentrations of Se-sugars (1.2 μM-20 μM) were added to solutions of known FMoc-methionine (5 μM).

2. Sample compositions for HOCl (1 μM) rate determination for Se-sugars

250 μL of 2 μM HOCl was added to each sample (Se0-Se8) except the Blank. Samples, with a final volume of 500 μL, were then mixed, filtered (0.2 μM cut-off filters) and placed in a glass HPLC vials for HPLC analysis.

HPLC Amino Acid Analysis of HOCl Oxidized BSA and Plasma

1.1.1.5 Sample Preparation for Protein Hydrolysis

Varying concentrations of Se-sugars (1.0 mM-0.05 mM) were added to solutions containing 0.1 mg.mL−1of protein (BSA or Plasma).

3.Example of sample compositions for protein protection against HOCl (0.76 mM) by Se-sugars

150 μL of each sample (Se1BSA1—Control) were added to 50 μL of 3 mM HOCl. Samples, with a final volume of 200 μL, were placed in a glass vial (8×40 mm, 1 mL, No. 98212, Alltech) labeled by etching with a diamond tipped pen or engraver. Proteins (0.1 mg in 200 L) were delipidated and precipitated by the addition of 25 μL 0.3% (w/v) deoxycholic acid and 50 μL of 50% (w/v) TCA, with incubation on ice for 5 min. The glass vials containing samples were placed in 1.5 mL centrifuge tubes (with caps removed) for 2 minutes at 9000 rpm at 5° C. (Eppendorf 5415R centrifuge) to pellet protein. Protein pellets were washed once with 5% (w/v) TCA, and twice with ice cold acetone (stored in −20° C. freezer) with 2 min, 9000 rpm, spins between washes in each case to settle pellets. Samples were then re-suspended in 150 μL of 4 M methanesulfonic acid (MSA) containing 0.2% w/v tryptamine, before the addition of 5 μL of homo-Arg (10 mM) as an internal standard. The samples were then transferred to PicoTag hydrolysis vessels and placed under vacuum in the oven at 110° C. for 16.-18 hours. The PicoTag vessels were removed from oven and allowed to cool before releasing vacuum. Samples were neutralized by the addition of 150 μL freshly prepared 4 M NaOH and filtered (centrifuge at 10,000 rpm for 2 minutes through a PVDF 0.22 μm membrane, 0.5 mL volume, No. UFC30GVNB, Millipore) to remove any insoluble precipitate. The samples were diluted into water (10-fold), before transferring 40 μL to HPLC vials.

1.1.1.6 Preparation of OPA and Amino Acid Standards

OPA reagent (Sigma-Aldrich, P7914) was activated immediately before use by addition of 5 μl of 2-mercaptoethanol to 1 mL of OPA reagent in a HPLC vial. The derivatization method involved 20 μL injections of activated OPA reagent per sample. A solution of 5 μM standards was prepared by addition of 10 μL Sigma-Aldrich amino acid standards (A9781, 500 μM stock), 5 μL MetSO (1 mM stock), and 5 μL homo-Arg (1 mM stock) to 980 μL water. These stock solutions were diluted to give 1, 2, 3, 4, and 5 μM standards. 40 μL of each standard was transferred to HPLC vials containing 0.2 mL inserts and placed in the auto injector.

1.1.1.7 Preparation of HPLC Mobile Phase

A 1.0 M stock solution of sodium acetate trihydrate was prepared by the addition of 136.08 g of this compound to 900 mL of water, before pH adjustment to 5.0 with glacial acetic acid (˜29 mL) before addition of water to a final volume of 1 L. Buffer A contained 400 mL MeOH, 50 mL tetrahydrofuran; 1450 mL water, and 100 mL of 1.0 M sodium acetate, pH 5.0 (to give 50 mM final). Buffer B contains 1600 mL MeOH, 50 mL tetrahydrofuran, 250 mL water, and 100 mL of 1 M sodium acetate, pH 5.0 (to give 50 mM final). Both buffers were filtered through 0.2 μm membrane filters (e.g., VacuCap 90 filter unit with 0.2 μm Supor membrane, No. 4622, Pall Corporation), and degassed prior to running HPLC analysis.

1.1.1.8 HPLC Conditions, Method and Results

The auto injector was programmed to add 20 μL activated OPA reagent to the specified sample (40 μL), followed by 3 mixing cycles, and a 1 minute incubation period. After the incubation step, 15 μL of the final reaction mixture was injected. A flow rate of 1 mL min−1was used, with the column oven set at 30° C. and fluorescence detector set with λEX340 nm, λEM440 nm. The concentration of each amino acid in the samples was determined from linear plots of the HPLC peak area versus concentration from the standards. Any variation in derivatization efficiency was taken into account by expressing the results as a ratio with the internal standard homo-Arg. Any variation in the efficiency of hydrolysis or sample recovery after the precipitation and washing steps was taken into account by expressing the concentration of the amino acids of interest as a ratio with an amino acid that is not modified by the particular oxidant treatment. The results showing protection of individual amino acid residues present on BSA are depicted inFIGS. 4A-E, and analogous data for the protection of amino acid residues present on proteins in human plasma are shown inFIGS. 4F-J.

Analysis of 3-Chlorotyrosine Using LCMS

1.1.1.9 Sample Preparation for Protein Hydrolysis

Varying concentrations of Se-sugars (1.0 mM-0.05 mM) were added to solutions containing 0.1 mg.mL−1of protein (BSA or Plasma).

4.Example of sample compositions for Cl-Ty prevention against HOCl (0.76 mM) by Se-sugars

150 μL of each sample (Se1HSA1—Control) were added to 50 μL of 3 mM HOCl. Samples, with a final volume of 200 μL, were placed in a glass vial (8×40 mm, 1 mL, No. 98212, Alltech) labeled by etching with a diamond tipped pen or engraver. Proteins (0.1 mg in 200 μL) were delipidated and precipitated by the addition of 25 μL 0.3% (w/v) deoxycholic acid and 50 μL of 50% (w/v) TCA, with incubation on ice for 5 min. The glass vials containing samples were placed in 1.5 mL centrifuge tubes for 2 minutes at 9000 rpm at 5° C. (Eppendorf 5415R centrifuge) to pellet protein. Protein pellets were washed once with 5% (w/v) TCA, and twice with ice cold acetone (stored in −20° C. freezer) with 2 min, 9000 rpm, spins between washes in each case to settle pellets. The samples were then transferred to PicoTag hydrolysis vessels before the addition of 150 μL of 6 M HCl and 50 μL of thioglycolic acid into the PicoTag vessel and placed under vacuum in the oven at 110° C. for 16-18 hours. The PicoTag vessels were removed from oven and allowed to cool before releasing vacuum. The sample vials were then placed in 1.5 mL centrifuge tubes and dried under vacuum, using centrifuge speedy vacuum system (3 hours at maximum vacuum). Each sample was then re-suspended in 50 μL of water and filtered (centrifuge at 10,000 rpm for 2 minutes through a PVDF 0.22 μm membrane, 0.5 mL volume, No. UFC30GVNB, Millipore) to remove any insoluble precipitate. The samples were then transferred to HPLC vials for LCMS analysis.

1.1.1.1.10 Preparation of Standards

A standard solution of 100 μM tyrosine and 2.5 mM 3-chlorotyrosine was prepared in buffer. Each stock was diluted to give 1:1 mixtures of tyrosine:chlorotyrosine with concentrations of 100-500 pmol in 20 μM. 40 μL of each standard was transferred to HPLC vials for LCMS analysis

1.1.1.1.11 Sample Analysis

L-Tyrosine, 3-chlorotyrosine and di-tyrosine were analysed by LC-MS in the positive ion mode with a Finigan LCQ Deca XP ion-trap instrument coupled to a Finnigan surveyor HPLC system. Tyrosine residues were separated on a Thermo hypercarb ODS column (100 mm×2.1 mm; 5 μm particle size) at 30° C. with a flow rate of 0.2 mL.min−1. Solvent A contained 0.1% TFA in water and solvent B contained 0.1% TFA in acetonitrile. The tyrosine residues were eluted using the following gradient: 5% to 50% B over 20 minutes, then 50-80% B over 2 minutes, followed by isocratic elution of 80% B for 5 minutes before decreasing to 5% B for 3 minutes and re-equilibrating to 5% B for 20 minutes. The electrospray needle was held at 4500 V. Helium was used as the collision gas and nitrogen was used as the sheath and sweep gas set to 50 and 32 units respectively. The temperature of the heated capillary was 325° C. The results are shown inFIGS. 5A and 5B.

Scavenging of HOCl and Chloramines Using TMB Assay

1.1.1.12 Basis of TMB Assay

The developing reagent was prepared by dissolving 4.8 mg of TMB in 1 mL of dimethylformamide, followed by the addition of 9 mL of 0.44 M pH 5.4 sodium acetate buffer and 50 uL of 2 mM sodium iodide solution. The developing reagent was prepared immediately prior to addition to the standards and samples to avoid any unwanted oxidation of TMB. Standard curves were produced by adding varying amounts, between 0 and 100 uL, 200 uM HOCl, to 100 uL of 10 mM taurine solution in a 96-well plate. The volume in each well was made up to 200 uL with 0.1 M pH 7.4 phosphate buffer. The standards were incubated for 0.5 minutes before the addition of developing reagent. The solution was incubated for another 5 minutes before the absorbance at 645 nm was determined using BioRad Benchmark Plus microplate spectrophotometer. Standards were produced substituting 10 mM taurine solution with 10 mM solutions of glycine and N-acetyl-lysine, 200 uM solution of N-acetyl-histidine and 0.5 mg/mL solution of bovine serum albumin or human plasma.

Chloramines were formed by adding 50 uL of 200 uM HOCl solution to 10 mM taurine solution and incubated for 5 minutes. Varying volumes, between 0 and 50 uL, of 400 uM potential antioxidant solution were then added to the wells, and the volume made up to 200 uL in each well with 0.1 M pH 7.4 phosphate buffer. The samples were incubated for 5 minutes before the addition of developing reagent. The solution was incubated for another 5 minutes before the absorbance at 645 nm was determined using BioRad Benchmark Plus microplate spectrophotometer. The method was repeated substituting the 10 mM taurine solution with 10 mM solutions of glycine and N-acetyl-lysine, 200 uM N-acetyl-histidine and 0.5 mg/mL bovine serum albumin or human plasma.

The results are shown inFIGS. 6A to 6E. The IC50 values for scavenging of the various chloramines by the compounds tested are given inFIG. 6F.

Recycling of Oxidized Seleno Compounds by Thiols

The purpose of these experiments was to determine whether thiols could reduce the selenoxides formed on oxidation of the seleno compounds. The ThioGlo assay was used to monitor the loss of thiol groups upon addition of selenoxides as this agent produces a fluorescent product in the presence of reduced thiols.

ThioGlo Assay Method

The ThioGlo reagent was prepared by diluting 30 uL of a stock solution (5 mg in 5.070 mL acetonitrile) in 2970 uL of 0.1 M pH 7.4 phosphate buffer. Preparation of the developing reagent was performed immediately prior to addition to standards or samples. Standard curves were prepared by addition of varying volumes, between 0 and 50 uL, of 10 uM GSH solution to wells in a 96-well plate. The volume in each well was made up to 50 uL with 0.1 M pH 7.4 phosphate buffer. 50 uL of ThioGlo reagent was added to each standard, and incubated in the dark for 5 minutes. The fluorescence was measured using a PerSeptive Biosystems CytoFluor II fluorescence multi-well plate reader with λex=360 nm λem=530 nm. Standards using 10 uM Cys and 2 mg/mL BSA were produced in the same method.

Solutions of 8 uM SeMetO were produced by mixing 20 uM SeMet and 16 uM HOCl together, and incubating for 30 minutes. Samples were prepared by adding 25 uL of 16 uM to wells of a 96-well plate. Varying volumes of 8 uM SeMetO, between 0 and 25 uL, were added to the samples, and the volume of each made up to 50 uL using 0.1 M pH 7.4 phosphate buffer. 50 uL of ThioGlo reagent was added to each sample, and incubated in the dark for 5 minutes. The fluorescence was measured using the PerSeptive Biosystems CytoFluor II fluorescence multi-well plate reader with λex=360 nm λem=530 nm. Samples using 16 uM cysteine and 3 mg/mL bovine serum albumin in place of 16 uM glutathione, were produced in the same method. Samples using 8 uM SeTalO, in place of 8 uM SeMetO, were produced in the same method. Standard curves for GSH, BSA and Cys had R2values>0.99.

The results are reported as a percentage of thiol remaining after selenoxide addition. GSH and Cys samples showed a dose dependent decreases in the amount of thiols after addition of the selenoxides from selenomethionine (SeMetO) and 1,4-anhydro-4-seleno-D-talitol (SeTalO) consistent with a dose dependent reduction of the pre-formed selenoxide back to the parent selenide. This reduction was less marked with the thiol group present on bovine serum albumin (FIGS. 7Aand B).

Cytotoxic Effects of Seleno-Compounds

C57Bl/6 mouse isolated glial cells and Chinese Hamster Ovary (CHO) were kindly donated by Dr Peter Crack (University of Melbourne) and Prof. Walter Thomas (University of Queensland, Australia), respectively. Cells were cultured in a tissue-culture flask containing Modified Eagles Medium (MEM) and 50% Foetal Bovine Serum (FBS). The cells were grown in a 5% CO2incubator (Forma Scientific, Marietta, Ohio, USA) at 37° C. until they were confluent. Once confluent, cells were plated onto a 96 well plate at a density of 30,000 cells per well.

Wells were incubated with phosphate buffered saline (PBS), SeTal (compound 38) (1 mm), SeGul (compound 4) (1 mM) or staurosporine (0.01, 0.1 or 1 μM) in quadruplicates for 48 h in a 5% CO2incubator. Drugs were made up fresh daily in PBS. After 48 h cells were incubated for 2 h with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 2 mg/ml). After 2 h the media was decanted and cells were then solubilised with 25% dimethyl sulfoxide (DMSO). The contents of each well was then transferred to a clean 96 well plate and the absorbance of the wells determined using spectrophotometry (Thermo Electron Corporation, Vantaa, Finland) at 595 nm λ (FIG. 8).

MTT is a yellow tetrazole which is converted by the mitochondrial reductase of living cells into a purple formazan. DMSO is added to each well to dissolve the insoluble purple formazan product into a coloured solution. Absorbance of the wells was averaged for each treatment group and expressed as a percentage of control wells (% control) which were incubated with PBS only. Differences in cell survival were compared using a one-sample test compared to control (100%; GraphPad, La Jolla, Calif., USA). The results are depicted inFIG. 9.