TECHNICAL FIELD

The present invention relates to novel compounds, pharmaceutical compositions comprising the compounds, processes for their preparation, as well as to the use of the compounds in the inhibition of histories, in particular histone HI. The invention also relates to the compounds for use in medicine and particularly in the prevention and/or treatment of a disease, selected from: a neurodegenerative disease including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), neurodegeneration, dementia, vascular dementia, multiple sclerosis, a neuroinflammatory or inflammatory condition, or a disease state associated with aberrant histone levels.

The present invention also relates to use in the prevention and/or treatment of conditions that affect any organ or tissue that results in cellular damage and the release of histones due to pathology, infection, trauma, infarct/ischaemia, or sequelae arising from bacterial, fungal or viral infection or sepsis. As stated above, such conditions include neurological disorders and conditions.

BACKGROUND OF THE INVENTION

Histones are highly basic proteins that function in the nucleus to package chromatin and regulate transcription, It is usually assumed that they are restricted to the nucleus but a growing body of evidence indicates that certain histones and histone-derived peptides have cytoplasmic and extracellular locations, reviewed In [1]. When brain cells die they release histones into the extracellular environment where it is most probable that they exert toxic effects on neighbouring nerve cells.

Several lines of evidence support the idea that extracellular histones contribute to human disease mechanisms. Anti-histone antibodies are present in vascular dementia, pre-senile Alzheimer's disease (AD) and senile dementia of the Alzheimer's type [2]. These authors also demonstrated an inverse relationship between the levels of circulating antihistone autoantibodies in AD and cognitive ability. In systemic lupus erythematosus (SLE), many differeat nuclear antigens are released which provoke high titres of anti-histone autoantibodies and activate T and B cells [3]. Indeed, a significant proportion of patients with SLE have neurodegeneration. Both nucleosomes and free histones, H2A, H2B H3 and H4 are released from cultured human tonsil lymphoid tissue and exert significant immunoproliferative effects on both human and murine lymphocytes, resulting in increased synthesis of DNA and immunoglobulins [4], These authors also suggested that it was possible that apoptotic death in vivo may be augmented by the release of nucleosomes. Significantly, nucleosomes have been detected in the serum of patients who have suffered cerebral ischaemic strokes [5],

It has been known for some time that histones can be cytotoxic to a variety of microorganisms and mammalian cells [5-8]. In addition, it has been shown that histones can be toxic to malignant cells when applied exqgenously and it has been suggested that this extracellular action could have therapeutic implications [9].

However, chemotherapy using soluble native histones might be limited by their high toxicity when administered intravenously or intraperitoneal ly.

[10, 11]. The mechanisms of this toxicity are unclear but it has been shown that extracellularly applied histones are toxic to breast cancer cells because they cause large increases in intracellular calcium release and calcium influx [12].

Free histones have been detected in blood [13] and it is entirely reasonable to expect that their levels would increase (as do those of nucleosomes) following neuronal cell death from whatever cause.

Crucially however, histones are toxic to cortical neurons [ 14-16}. Under cold stress, cortical slices lose their ability to increase respiration in response to electrical stimulation. This is accompanied by migration of histones from the nucleus into the cytoplasm where they associate with microsomes and mitochondria (which swell). The loss of response is most likely to result from inhibition of oxidative metabolism of ATP and is completely reproduced by addition of soluble histones to the culture medium. Thus, under certain conditions histones can be liberated from the nucleus and taken up with both events leading to neuronal toxicity.

Almost nothing is known of the mechanisms or circumstances by which histones appear in the extracellular space. It is possible, that there are clearance mechanisms for histones following cell death/apoptosis but that under certain conditions these become overloaded. Whether or not histones are actively released from cells in AD is unknown. However, histone up-regulation is known to occur in AD, where ectopic non-nuclear H I is found at the neuronal cell-surface and in activated astrocytes [17].

Histones are also implicated in the formation of insoluble protein deposits. They bind to the Parkinson's disease-associated protein o-synuclein and increase its fibrillation rate [21-23] , Histones also bind Alzheimer β- amyloid precursor protein (APP) and to β-amyloid with high affinity [23, 24]. Since histones are found extraceilulariy within amyloid plaques in AO brain [25] an increase in their expression could be a catalyst for neuronal death and would be consistent with the findings of raised antibody titres. As such histones are likely to be important and overlooked therapeutic targets that can be used to delay the progression of AD.

In particular, the target(s) can be members of the highly conserved histone family of proteins (HI, H2A, H2B, H3, H4) and their isoforms. Histones are highly basic and are normally present in the nucleus where they form the major protein components of eukaryotic chromatin. Histones H2A, H2B, H3 and H4 are 'core' histones, have relatively similar structures and form an octameric arrangement termed the nucleosome, which packages genomic DNA. The linker histone Hi binds between nucleosomes and are much more variable in their sequences. Histones, which are released following apoptosis or necrosis of neurons and other cells are released into the extracellular space where they are neurotoxic and induce reactive gliosis, a hallmark of AD. Very little is known of the mechanisms by which extracellular histones mediate neurotoxicity/neuroinflammation but a requirement for glycosaminoglycan-containing ceil surface molecules has now been found for their endocytosis and toxicity.

It has been widely reported that GAGs, such as low molecular weight heparin (L WH) derivatives are neuroprotective In vivo and in vitro [26- 30].

It has now been found that it is possible to block the cell-surface binding and endocytosis of f!uorescently-labeled histones using certain heparan sulphate proteoglycans (HSPG) and chondroitin sulphate proteoglycans (CSPG) and it has now been found that these effects correlate strictly with the blockade of neurotoxicity. Histones appear to bind to cell surface receptors that contain CSPG/HSPG modifications since ceils that are defective in these receptors do not take up histones.

In all neurodegenerative diseases neurons die and cells of the immune system called microglia become activated and also contribute to neuronal death. It has now been found that nanomolar concentrations of extracellular histones, especially HI which is released on cell death, kill neurons and activate microglia and that certain heparin and chondroitin carbohydrates can reduce these effects in culture.

However, despite indications that histone inhibitors may be useful in the treatment of a variety of neurodegenerative diseases, the development of inhibitors with good activity, selectivity and pharmacokinetic profiles is needed to fully exploit the clinical potential of this target..

Based upon a comprehensive structure and activity relation (SAR) study, novel carbohydrates have now been found and synthesised which can be used for neuro-protection. These compounds are advantageous because, in contrast to the known compounds, they are chemically homogenous as they are not derived from animals or plants. Thus, the present invention seeks to address the problems mentioned above and is focused to the identification of potent and selective histone inhibitors.

STATEMENT OF THE INVENTION

Remarkably, the present invention provides compounds which interact selectively with at. least one histone, in particular histone HI. Preferably, the histone is selected from mammalian histones,- more preferably human histones, including at least one of histone HI, H2A, H2B _y 3 OF 4 or a- combination thereof.

In a first aspect, the invention provides a compound selected from the group of compounds represented by the genera! formula :

or 

7 PCT/GB2012/000468



OMe

O e

4 ^m -Sulfo-Fucoseal-3 (4"-sulfo)-Fucoseal-3 (4'-5ulfo-Fucoseai-4- Glucuronic acid βΐ-θ- Methyl

or

4" ^, '-Sulfo-Fucoseal-3 (4"'-Sulfo)-FLicoseal-3 (4"-≤ulfo)-Fucoseal-3 (4'- Sulfo-Fucoseal-4-G!ucuronic acid β i-O- Methyl wherein X is sulfate {-S0 ₃ H) or phosphate (-P0 ₃ H);

Ri is H, alkyl, or cydoalkyi; alkyl or cydoalkyi optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, amino, an amino acid or a polypeptidyl residue, or by aryl or heteroary! optionally substituted with alkyi, hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, or an amino group; and when Ri is alkyl or cydoalkyi, one to three non-adjacent CH ₂ groups may be optionally replaced by NR', S or 0, where R" is H or alkyl, cydoalkyi or a sugar residue; or one to three non-adjacent CH ₂ groups may be optionally replaced by aryl or heteroaryi optionally substituted with aikyl, hydroxy, chloro, bromo, alkoxy, alkenyl, alkynyl, nitro, or an amino group;

Rz has the general formula:

-CO-R3 R ₃ is alkyi, or cycloalkyi; alkyi or cycloalkyi optionally substituted with one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, alkenyl, alkynyl, nitro, amino, an amino acid or a pclypeptidyl residue, or by aryl, heteroaryl optionally substituted with alkyi, hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, or an amino group; and when R ₃ is alky! or cycloalkyi, one to three non-adjacent CH ₂ groups may be optionally replaced by NfV, S or 0 where - is H or a!kyl or cycloalkyi; or when ^ is alkyi or cycloalkyi, one to three non-adjacent CH ₂ groups may be optionally replaced by aryl or heteroaryl substituted with alkyi hydroxy, halogen, alkoxy, alkenyl, aikynyl, nitro, or an amino group;

Su is sulfate;

Me is methyl;

Sulfation is preferred at at least one of the arrowed positions, more preferably at at least two of the arrowed positions, more preferably at at least three of the arrowed positions, more preferably at at least four of the arrowed positions, even more preferably at all five of the arrowed positions; or a pharmaceutically acceptable salt, hydrate, solvate, geometrical isomer, tautomer, optical isomer, or prodrug form thereof.

In a preferred embodiment X is sulfate (-S0 ₃ H) or phosphate (-P0 ₃ H);

Ri is H, alkyi, or cycloalkyi; alkyi or cycloalkyi optionally substituted with one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, nitro, amino, an amino acid or a poiypeptidyl residue, cr by phenyl substituted with lower alkyi hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when i is alkyi or cycioalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by IMR', S or 0, where R' is H or alkyi, cycioalkyl or a sugar residue; or one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl substituted with lower alkyi hydroxy, chloro, bromo, alkoxy, nitro _r or an amino group;

R ₂ has the general formula ;

-CO-R ₃

R ₃ is alkyi, or cycioalkyl; alkyi or cycioalkyl optionally substituted with one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, nitro, amino, an amino acid or a polypeptidyl residue, or by phenyl optionally substituted with alkyi, hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when R ₃ is alkyi or cycioalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by NR\ S or O where R' is H or alkyi or cycioalkyl; or when 3 is alkyi or cycioalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl substituted with lower alkyi hydroxy, chloro, bromo, alkoxy, nitro, or an amino group.

In an even more preferred embodiment, the invention provides a compound selected from the group consisting of compounds represented by the general forumula:

wherein the compound is sulfated at at least one of the positions indicated with an arrow.

The compounds of the invention, including but not limited to those specified in the examples, possess the ability to inhibit the activity of at least one histone. The histone is preferably selected from a mammalian histone, more preferably a human histone, even more preferably HI, H2A, H2B, H3 or H4 or a combination thereof. Such compounds may be useful in the treatment of a wide variety of conditions including a neurodegenerative disease including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), neurodegeneratlon, dementia, vascular dementia, multiple sclerosis, a neuroinflammatory or inflammatory condition, or a disease state associated with aberrant histone levels.

The present invention also relates to use in the prevention and/or treatment of conditions that affect any organ or tissue that results in cellular damage and the release of histories due to pathology, infection, trauma, infarct/ischaemia, or sequelae arising from bacterial, fungal or viral infection or sepsis. As stated above, such conditions include neurological disorders and conditions. In addition compounds of the invention may be useful in the prevention of medical conditions described above either when used alone or in combination with other chemotherapeutic agents.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the appearance of histones in AD, The right and far left panels show the abnormal appearance of histones in human AD. The middle panel shows histone HI in glia and in amyloid plaques in a transgenic AD mouse.

Phosphory!ated histone H3 is a nuclear marker for the M-phase of normal cell division but becomes activated and re-localised to the cytoplasm of neurons in AD [18] see far left panel in Fig. 2. There is compelling evidence that aberrant re-entry into the cell cycle may be a significant cause of neuronal cell death in AD [19]. Moreover, following UV-induced damage, the histone isoform, HI.2, translocates from the nucleus to the cytoplasm and triggers apoptosis [20]. Thus, (consistent with 14-16) one source of extracellular histones might be those re-localised in the cytoplasm while another could be that released from the nuclei of disintegrating cells. Potentially, extracellular histones could act both at the cell surface and within the cytoplasm following uptake.

Figure 2 has three panels which show cultures of mixed cartica! neurons and glia which have been exposed to 7.5 nM of fluorescently labelled histone protein HI for 24 hrs.

The left hand panel shows trafficking of histone Hi to ER surrounding the nucleus in neuron (this route is taken by numerous toxins and viruses). The chromatin {DAPI blue) is condensing, the neuron is under ER stress and is dying.

In contrast (middle panel) there, is endocytosis and cytoplasmic uptake in glial cells with no ER invoivement. The right hand panel shows endocytosed histone Hi and differentiation to reactive GFAP-positive astrocyte

It has now been shown that fluorescently labelled histone HI is taken up b/ neurons, astrocytes, HeLa and CHO cells but that only neurons die. Hence an accumulation of extracellular histones could exacerbate neurotoxicity and as cell death increases ou!d produce an auto-catalytic cascade of neuronal cell death.

Figure -3 has three panels. On the left is a confocal z-projection of a section of temporal cortex stained for histone HI that came from a 68 year old neurologically normal woman who died from a perforated ulcer. There is no extranuclear expression of histone.

The middle panel shows temporal cortex of a 70 year old woman who died from AD and demonstrates extensive extracellular deposits of histone Hi.

The right hand panel shows that histone HI is also distributed extracellu!arly in a transgenic AD model (Swedish and London point mutations to human Amyloid Precursor Protein) expressed under the Thy 1 promoter.

Figure 4 has two panels. Both panels show mixed neuronal and glial cultutes of rat neocortex. The left hand panel shows the effects of 30nM fluorescently labelled histone H I (red) application for 24hrs. Histone has killed the majority of the neurons whose disintegrating nuclei are labelled with fluorescent HI. Glial cells are seen in blue.

The right hand panel shows that histone toxicity is prevented by a GAG, chondroitin sulphate C.

This indicates that GAGs bind to soluble histones preventing their uptake and is consistent with observations that histones are GAG binding factors [31-35]. Although the interaction sites have not been mapped precisely, a number of consensus heparin binding sites can be predicted which strongly suggests that the GAG-histone interaction is direct.

Figure 5 shows possible points of attack in an histone amplification cascade using drugs and humanised antibodies. Histones are present abnormally in AD and contribute to disease progression through the amplification cascade. Histones bind to Alzheimer β-amyloid precursor protein (APP) and to β-amyioid with high affinity and are found within amyloid plaques in AD brain. Thus, an increase in their expression could be a catalyst for neuronal death and gliosis.

Figure 6 shows a schematic representation of likely cell surface glycoprotein histohe receptors.

Histones bind to CSPG/HSPG glycan decorations (wavy lines) on cell- suface proteoglycan receptors. It has now been shown that co-application of histones and soluble sulphated GAGs prevent histone binding and uptake, whereas pre-treatment with a GAG does not (indicating that the . GAG acts as decoy receptor and binds direct!y to the histone) . Furthermore, histones do not bind to a CSPG/HSPG-defident cell-line or cells that have had their GAG decorations stripped away using heparinase or chondroitinase.

The major candidate receptors are members of the glypican (GPI-linked, orange circle) or syndecan (transmembrane, blue rectangle) families. Another strong candidate is APP, which contains a single HSPG modification and is a known histone-binding protein. Rece tor- mediated uptake in neurons results in cell death but elicits proliferation and differentiation of glial cells (astrocytes), which contribute to neuropathogenesis. A therapeutic compound according to the invention targets histones in the extracellular space and prevents receptor binding.

DETAILED DISCLOSURE OF THE INVENTION In- a first aspect the invention provides a compound selected from the group of compounds represented by the general formula :

or

or 20

21

4 ^,,, -Sulfo-Fucoseal~3 (4"-sulfo)-Fucoseal-3 (4'-Sulfo-FucoseQi-4- Glucuronic acid βΐ-θ- Methyl

or

4""-Sulfo-Fucoseal-3 (4 ^* "-Sulfo)-Fucoseal-3 (4"-sulfo)-Fucoseal-3 4'- Sulfo-Fucoseal-4~Glucuronic acid β 1-0- Methyl wherein X is sulfate (-S0 ₃ H) or phosphate (-P0 ₃ H);

Ri is H, alkyl, or cycloaikyi; alkyl or cycloaikyi optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, amine, an amino acid or a polypeptidyl residue, or by aryl ar heteroaryl optionally substituted with alkyl, hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, or an amino group; and when Ri is alkyl or cycloaikyi, one to three non-adjacent CH ₂ groups may be optionally replaced by NR', S or 0, where R' is H or alkyl, cycloaikyi or a sugar residue; or one to three non-adjacent CH ₂ groups may be optionally replaced by aryl or heteroaryl optionally substituted with alkyl, hydroxy, chloro, bromo, alkoxy, alkenyl, alkynyl, nitro, or an amino group;

R ₂ has the general formula :

-CO-R ₃ R ₃ is alky!, or cycloalkyl; alkyl or cydoalkyl optionaliy substituted with one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, alkenyl, alkynyl, nitro, amino, an amino acid or a po!ypeptidyl residue, or by aryl, heteroaryl optionally substituted with alkyl, hydroxy, halogen, atkoxy, alkenyl, alkynyl, nitro, or an amino group; and when j is alkyl or cycloalkyl, one to three non-adjacent CHj groups may be optionally replaced by NR', S or 0 where R' is H cr alkyl or cycloalkyl; or when R ₃ is alkyl or cycloalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by aryl or heteroaryl substituted with alkyl hydroxy, halogen, alkoxy, alkenyl, alkynyl, nitro, or an amino group;

Su is sulfate;

Me is methyl;

Sulfation is most likely at the arrowed positions or a pharmaceutically acceptable derivative, analogue, salt, hydrate, solvate, geometrical isomer, tautomer, optical isomer, or prodrug form thereof.

In a preferred embodiment X is sulfate (-50 ₃ Η) or phosphate ( -P0 ₃ H);

Rj is H, alkyl, or cycloalkyl; alkyl or cycloalkyl optionally substituted with one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, nitro, amino, an amino acid or a polypeptidyl residue, or by phenyl substituted with lower alkyl hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when i is alkyl or cycloalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by NR', S or O, where R' is H or alkyl, cycloalkyl or a sugar residue; or one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl substituted with lower alkyl hydroxy, chloro, bromo, alkoxy, nitro, or an amino group;

R ₂ has the general formula:

-CO-R3

R ₃ is alkyl, or cycloalkyl; alkyl or cycloalkyl optionaliy substituted with one or more substituents selected from the group consisting of hydroxy, chloro, rom , alkoxy,, nitro, amino, an amino acid or a polypeptidyl residue, or by phenyl optionally substituted with alkyl, hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when R ₃ is alkyl or cycloalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by NR' ₍ S or O where R' is H or alkyl or cycloalkyl; or when R3 is alkyl or cycloalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl, substituted with lower alkyl hydroxy, chloro, bromo, alkoxy, nitro, or an amino group.

Preferred compounds comprise less than about 10 sugars, more

preferably less than about 6 sugars, more preferably they comprise four or five sugars. Preferably, the sugars are selected from L-Fticose alpha linked, D-Glucosamine beta linked, D-Glucose alpha or beta linked, D- Mannose alpha linked, D-Galactose alpha or beta linked to other sugars.

Preferred compounds are given in the Examples and listed in Tables 1, 2 and 3.

Any known compound having a structural formula identical to any one of the compounds covered by Formula (1) is hereby explicitly disclaimed per se.

In a second aspect the invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable diluent or carrier. In a third aspect the invention provides a process for the preparation of a compound of the invention as described below, which comprises at least one of the steps of:

a) reacting starting materials to form a first intermediate compound and with one or more reagents to arrive at a second intermediate compound;

b) reacting the second intermediate compound with one or more reagents;

c) obtaining a compound according to the invention.

In a preferred embodiment, the process comprises all of steps a) to c).

In a fourth aspect the invention provides a method for the prophylaxis or treatment of a disorder associated with aberrant histone level which comprises administering to a subject in need of such treatment an effective amount of a compound or a pharmaceutical formulation of the invention.

In a preferred embodiment the condition is selected from a neurodegenerative disease including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), ^; neurodegeneration, dementia, vascular dementia, multiple sclerosis, a neuroinflammatory or inflammatory condition, or a disease state associated with aberrant histone levels.

The present invention also relates to use in the prevention and/or ^' treatment , of conditions that affect any organ or tissue that results in cellufer damage and the release of histones due to pathology, infection, trauma, infarct/ischaemia, or sequelae arising from bacterial, fungal or viral infection or sepsis. As stated above, such conditions include neurological disorders and conditions.

In . a preferred embodiment of the method, the compound, or the pharmaceutical formulation of the invention, is administered in combination with other chemotherapeuttc agents. In a fifth aspect the invention provides a method for modulating histone activity which comprises administering to a subject in need of such treatment an effective amount of a compound or a pharmaceutical formulation of the Invention.

In a sixth aspect the invention provides a compound of the invention for use in therapy, especially for use in the prophylaxis or treatment of a disorder associated with aberrant histone levels.

A further aspect of the invention is the use of a compound as mentioned above for the manufacture of a medicament for use in the prophylaxis or treatment of a disorder associated with aberrant histone !evel.

The compound may be administered in combination, with other chemotherapeutic agents.

Examples of disorders associated with aberrant histone level include a disease or condition selected from a neurodegenerative disease including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), neurodegeneration, dementia, vascular dementia, multiple sclerosis, a neuroinflarnmatory or inflammatory condition, or a disease state associated with aberrant histone levels.

The present invention also relates to use in the prevention and/or treatment of conditions that affect any organ or tissue that results in cellular damage and the release of histones due to pathology, infection, trauma, infarct/tschaemia, or sequelae arising from bacterial, fungal or viral infection or sepsis. As stated above, such conditions include neurological disorders and conditions,

Definitions

The following definitions shall apply throughout the specification and the appended claims. Within the context of the present application, the term "comprises" is taker to mean "includes among other things", and is not taken to mean "consists of only".

Embodiments have been described herein in a concise way. It should be appreciated that features of these embodiments may be variously separated or combined within the invention.

Unless otherwise stated or indicated, the term "alkyl" denotes a straight or branched alky! group. Preferably said alkyl group is a "lower alkyl" having from 1 to 5 carbon atoms ("G-s-alkyl"). Examples of said lower alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl and straight- and branched-chain pentyl and hexyl. For parts of the range all subgroups thereof are contemplated such as Ci.5-alkyl, C^-alky), C _1- alkyi, C _J-2 -alkyl, C ₂ -6-alkyl, C ₂ .s-alkyi, C _z . ^-alkyl, C _2- j-alkyl, C ₃ . ₆ -alkyl, C4.5-alk.yl, etc, "Halo-C^-alkyl" means a C _1-6 - alkyl group substituted with one or more halogen atoms. Likewise, "aryl- Cx. _f i-alkyi" means a C _1-6 -alkyl group substituted with one or more aryl groups.

Unless otherwise stated or indicated, the term "cycloalkyl" denotes a cyclic alkyl group preferably having a ring size from 3 to 8 carbon atoms (' ^, C ₃ .g-cyc!oa!ky!' ^r }. Examples of said cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexy!, cycloheptyl, and cyclooctyl. For parts of the range "C ₃ . _$ -cycloalkyl" all subgroups thereof are contemplated such as C ₃ .7-cycioalkyl, C ₃ . _s -cycloalkyl, C _3-5 -cycioalkyl, C _3- 4-cycloalky(, G,.$-cycloalkyl, Q-7-cycloalkyl, C^e-cycloalkyl, C4-5- cycloalkyl, C ₅ - ₇ -cycloalkyl, C ₆ . ₇ -cycloalkyl, etc.

Unless otherwise stated or indicated, the term "alkoxy" denotes a straight or branched alkoxy group. Preferably said alkoxy group is a "lower alkoxy" group having from 1 to 6 carbon atoms ("G. ₆ alkoxy"). Examples of said lower alkoxy include methoxy, ethoxy, n-propoxy, iso-propoxy, n- butoxy, iso-butoxy, sec-butoxy, t-butoxy and straight- and branched- chain pentoxy and- hexoxy. For parts of the range ^¾ Ci _-$ -alkoxy" all subgroups thereof are contemplated such as C _1-5 -alkoxy, C^-a!koxy, Ci-3- alkoxy, G^-alkoxy, C ₂ .6-alkoxy, G-s-alkoxy, C ₂ ^-aikoxy, Cz-3-alkoxy, C ₃ -6- alkoxy, Gt _-5 -alkoxy _; etc.

Unless otherwise stated or indicated, the term "alkenyl" means a straight chain or branched a!kenyl radical preferably of 2 to 6 carbon atoms and containing one or more carbon-carbon double bonds and includes but is net limited to ethylene, n-propyi-l-ene, n-propyl-2-ene, isopropylene, etc.

Unless otherwise stated or indicated, the term "alkynyl" means a straight chain or branched alkynyl radical preferably of 2 to 6 carbon atoms and containing one or more carbo -carbon triple bonds and includes but is not limited to ethynyl, 2-methylethynyl etc. -

Unless otherwise stated or indicated, the term "aryl" refers to a hydrocarbon ring system, which is preferably a 3-10, membered ring system, having at least one aromatic ring or being fused to one or more saturated or unsaturated rings including, but not limited to phenyl, pentaienyl, indenyl, indanyl, isoindolinyl, chromanyl, na ^' phthyl, fluorenyl, anthryl, phenanthryl and pyrenyl, The aryl rings may optionally be substituted with G. ₆ -alkyl. Examples of substituted aryl groups are benzyl and 2-methylphenyl. Likewise, aryloxy refers to an aryl group banded to an oxygen atom .

Unless otherwise stated or indicated, the term "heteroaryl" refers to a hydrocarbon ring system having at least one aromatic ring which contains at least one heteroatom such as 0, N, or S, Preferably the hydrocarbon ring system is a 3-10 membered ring system. Examples of heteroaryl groups include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, thia2olyl, isothiazolyl, pyridinyl, pyrimidinyl, quinazolinyi, indoiyl, pyrazolyl, pyridazinyl, quinolinyl, benzofuranyl, dihydrobenzofuranyl, benzodioxolyl, benzodioxinyi, benzothiazclyl, , benzothiadiazolyl, and benzotriazolyl groups. ^" "Heterocyclyl" means a preferably 3-10 membered ring system containing one or more heteroatoms selected from N, 0 or S and includes heteroaryl. The heterocyclyl system can contain one ring or may be fused to one or more saturated or unsaturated rings; the heterocyclyl can be fully saturated, partially saturated or unsaturated and includes but is not limited to heteroaryl and heterocarbocyclyl. Examples of carbocyclyl or heterocyclyl groups include but are not limited to cyclohexyl, phenyl, acridlne, benzimidazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, carbazole, cinnoiine, dioxln, dioxane, dioxolane, dithiane, dithia2ine, dithiazoie, dithiolane, furan, imidazole, imidazoline, imidazolidine, indole, indoline, indolizine, indazole, isoindole, isoquinoline, isoxazole, isothiazole, morpholine, napthyridine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, piperidine, pteridine, purine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, p rroline, quino!ine, quinoxaiine, quinazoline, quinolizine, tetrahydrofuran, tetrazine, tetrazole, thiophene, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thianaphtbafene, thiopyran, triazine, triazole, and trithiane.

Unless otherwise stated or indicated, the term "halogen" ^' shall mean fluorine, chlorine, bromine or iodine.

The term "leaving group" refers to a group to be displaced from a molecule during a nudeophiiic displacement reaction. Examples of leaving groups are bromide, chloride and methanesulfonate, especially bromide and methanesulfonate

"interact" means affecting the binding or activity of a molecule. This includes competitive binding, agonism and antagonism .

"Pharmaceutically acceptable" means being useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes being useful for veterinary use as well as human pharmaceutical use, "Treatment" as used herein includes prophylaxis of the named disorder or condition, or amelioration or elimination of the disorder once it has been established,

"An effective amount" refers to an amount of a compound that confers a therapeutic effect on the treated subject The therapeutic effect may be objective (i.e. , measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).

The term "prodrug forms" means a pharmacologically acceptable derivative, such as an ester or an amide, which derivative is biotransformed in the body to form the active drug. Reference is made to Goodman and Gilman ^' s, The Pharmacological basis of Therapeutics, 8 ^th ed., Mc- ^' Graw-Hill, Int. Ed. 1992, "Biotransformation of Drugs" p. 13- 15.

The following abbreviations have been used:

Ac means acetyl

ACN means acetonitrile,

DEA means diethylamide,

, DEPT means distortion enhancement polarisation transfer,

D SO means dimethyl sulfoxide,

ELS means electron light scattering,

H means hydrogen

HPLC means high performance liquid chromatography,

Me means methyl

t means retention time,

TFA means trifluoroacetic acid,

THF means tetrahydrofuran,

TLC means thin layer chromatography.

All diastereomeric forms possible (pure enantiomers, tautomers, racemic mixtures and unequal mixtures of two or more enantiomers) are within the scope of the invention. Such compounds can also occur as cis- or trans-, - or Z- double bond isomer forms. All isomeric forms and mixtures thereof are contemplated,

Neurodegenerative diseases are chronic degenerative diseases of the Central Nervous System (CNS) that often cause dementia, Although for the most part the causes and mechanisms of this collection of brain diseases are not well known, they are increasing in incidence in the developed as well as the underdeveloped world and are often found in the aging population. These diseases are characterized by molecular changes in nerve cells that result in nerve cell degeneration and ultimately nerve dysfunction and cell death, resulting in neurological signs and symptoms and in extreme cases dementia. There appears to be a genetic link to neurodegenerative diseases, but the genetic changes that occur and the changes in gene expression that are found in these diseases are complex. One of the types of change found in essentially all neurological degenerative diseases is the over-expression of oxidative free radical compounds (oxidative stress) that cause lipid, protein and genetic structural changes. In addition to genetics and changes in gene expression, it is thought that nutritional deficiencies, head trauma, environmental toxins, chronic bacterial and viral infections, autoimmune immunological responses, vascular diseases, accumulation of fluid in the brain, changes in neurotransmitter concentrations and other causes are involved in various neurodegenerative diseases.

The compounds of the invention may be used as such or, where appropriate, as pharmacologically acceptable salts (acid or base addition salts) thereof. The pharmacologically acceptable addition salts mentioned above are meant to comprise the therapeutically active non-toxic acid and base addition salt forms that the compounds are able to form. Compounds that have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating the base form with an appropriate acid. Exemplary acids include inorganic acids, such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric, acid, phosphoric acid; and organic acids such as formic acid, acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, glycolic acid, maleic acid, malonic acid, oxalic acid, benzenesulfonic acid, to!uenesulfonlc acid, methanesulfonic acid, trifluoroacetic acid, fumaric acid, succinic acid, malic acid, tartaric acid, citric acid, salicylic acid, p-aminosalicylic acid, pamoic acid, benzoic acid, ascorbic acid and the tike. Exemplary base addition salt forms are the sodium, potassium, calcium salts, and salts with pharmaceutically acceptable amines such as, for example, ammonia, alkylamines, benzathine, and amino acids, such as, e.g. arginine and lysine. The term addition salt as used herein also comprises solvates which the compounds and salts thereof are able to form, such as, for example, hydrates, alcohoiates and the like.

For clinical use, the compounds of the invention are formulated into pharmaceutical formulations for oral, rectal, parenteral or other mode of administration. Pharmaceutical formulations are usually prepared by mixing the active substance, or a pharmaceutically acceptable salt thereof, with a conventional pharmaceutical excipient (a pharmaceutically acceptable diluent or carrier). Examples of excipients are water, gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch giycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide, and the like. Such formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like. The use of such excipients for pharmaceutical active substances is well known in the art. Except in so far as conventional excipient is incompatible with the active compound, use thereof in the pharmaceutical formulation of the invention is contemplated.

Such formulations may also contain other pharmacologically active agents, and conventional additives, such as stabilizers, wetting agents, emulsifiers, flavouring agents, buffers, and the like,

The formulations can be further prepared by known methods such as granulation, compression, microencapsulation, spray coating, etc. The formulations may be prepared by conventional methods in the dosage form of tablets, capsules, granules, powders, syrups, suspensions, suppositories or injections. Liquid formulations may be prepared by dissolving or suspending the active substance in water or other suitable vehicles. Tablets and granules may be coated in a conventional manner.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as ben2yl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, poiyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like}, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, 'chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcoho|s such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum mono stearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a compound according to an embodiment of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients . from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, palyanhydrides, polyglycolic acid, collagen, polyorthoesters, and po!ylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated ; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the iimitations inherent in the art of compounding such an active compound for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected ceils and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animai models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information cart be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography,

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

In a further aspect the invention relates to methods of making compounds of any of the formulae herein comprising reacting any one or more of the compounds of the formulae delineated herein, including any processes delineated herein. The compounds of the invention may be prepared by, or in analogy with, conventional methods.

The processes described above may be carried out to give a compound of the invention in the form of a free base or as an acid addition salt. A pharmaceutically acceptable acid addition salt may be obtained by dissolving the free base in a suitable organic solvent and treating the solution with an acid, in accordance with conventional procedures for preparing acid addition slats from base compounds. Examples of addition salt forming acids are mentioned above.

The compounds of the invention may possess one or more chiral carbon atoms, and they ma therefore be obtained in the form of optical isomers, e.g. as a pure enantiomer, or as a mixture of enantiomers (racemate) or as a mixture containing diastereomers. The separation of mixtures of optical isomers to obtain pure enantiomers is well known in the art and may, for example, be achieved by fractional crystallization of salts with optically active (chira!) acids or by chromatographic separation on chiral columns.

The necessary starting materials for preparing the compounds of the invention are either known or may be prepared in analogy with the preparation of known compounds. The dose level and frequency of dosage of the specific compound will vary depending on a variety of factors including the potency of the specific compound employed, the metabolic stability and length of action of that compound, the patient's age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the condition to be treated, and the patient undergoing therapy. The daily dosage may, for example, range from about 0.001 mg to about 100 mg per kilo of body weight, administered singly or multiply in doses, e.g. from about 0.01 mg to about 25 mg each. Normally, such a dosage is given orally but parenteral administration may also be chosen.

In accordance with methods of treatment and pharmaceutical compositions of the invention, the compounds can be administered alone or in combination with other anticancer agents

Within this specification embodiments have been described in a way that enables a clear and concise specification to be written, but it will be appreciated that embodiments may be variously combined or separated without parting from the invention.

The invention will now be further illustrated by the following non-limiting examples,

EXAMPLES

Experimental Methods

All reagents were commercia! grade and were used as received without further purification, unless otherwise specified. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases, unless otherwise specified.

Preferred compounds of the present invention are shown in Table 1, Table 2 and Table 3.

Table 1

Compound Core

Sequence

In table 1 X is sulfate (-S0 ₃ H) or phosphate (-P0 ₃ H);

R ₃ is H, alkyl, or cycioalkyl; alkyl or cycioalkyl optionally substituted with , one or more substituents selected from the group consisting of hydroxy, chloro, bromo, alkoxy, nitro, amino, an amino acid or a polypeptidyl residue, or by phenyl substituted with lower alkyl hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when Ri is alkyl or cycioalkyl, one to three non-adjacent CH _∑ groups may be optionally replaced by NR', S or O, where R' is H or alky), cycioalkyl or a sugar residue; or one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl substituted with lower alkyl hydroxy, chioro, bromo, aikoxy, nitro, or an amino group;

R ₂ has the genera! formula:

R3 Is alkyl, or cycioalkyl; alkyl or cycioalkyl optionally substituted with one or more substituents selected from the group consisting af hydroxy, chloro, bromo, alkoxy, nitro, amino, an amino acid or a poiypeptidyi residue, or by phenyl optionally substituted with alkyl, hydroxy, chloro, bromo, alkoxy, nitro, or an amino group; and when R ₃ is alkyl or cycioalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by NR', S or 0 where R' is H or alkyl or cycioalkyl; or when R ₃ is alkyl or cycioalkyl, one to three non-adjacent CH ₂ groups may be optionally replaced by phenyl or phenyl substituted with lower ^■ alkyl hydroxy, chloro, bromo, alkoxy, nitro, or an amino group.

Table 2

Fucaidan mimetic

Fucoidan mimetic

Fucoidan mimetic

0 ^OH

Fucoidan mimetic

OH ^Bu

Fucoidan mimetic

Fucoidan mimetic

Fucoidan mimetic

Fucoidan mimetic

4'"-Suifo-Fucoseai-3 <4"-sulfo)-FucoseaJ.-3 (4'-Sulfo-Fucoseal-4- Fucoidan Glucuronic acid βΐ-θ- Methyl mimetic

4"*'-Sulfo-Fucoseal-3 (4"'-Sulfo}-Fucoseal-3 (4"-sulfo)-Fucoseal-3 Fucoidan

(4'-Sulfo-Fucoseal-4-Glucuronic acid β 1-0- Methyl mimetic In Table 2, Su indicates a sulphated group; and Me indicates a methyl group.

Table 3

Remarkably, the present invention provides compounds which interact selectively with histones and show selectivity over other molecules, more specifically the key selectivity target histone H I.

STRUCTURE ACTIVITY RELATIONSHIP DATA

Novel neurotoxic factors have no been found, and available compounds have been systematically explored to reduce their toxicity in 96-well plate assays. Various compound libraries have been screened including high molecular weight giycosaminoglycan (GAG) species to the identification of lower molecular weight GAG 'hits'. To explore structure activity relationships (SAR) the research focus has been directed at heparin/heparan sulphate proteoglycans (HSPGs) due to the availability of modifications to the backbone e.g. sulphation and acety!ation states. From this information, smaller HSPG oligomeric species have been identified ( w <15O0Da).

The following description of 'hits' and Yion-hits' identified includes structural information.

Synopsis of compounds analysed

Glycogaminoglycans

Structurally complex polysaccharides with a multitude of possible permutations to a repeating backbone. GAGs commonly have a disaccharide repeat and are classified based on chemical similarities which determine their overall properties:

« Monosaccharide units

• Disaccharide repeat (some alternating with 3 types of monosaccharide)

• Type of glycosidic link e.g. a-(l,3), a-(i,4) links

• Branching

• Specific modifications to the backbone such as /V-acetylation, N- suiphation, O-suiphation and epimerization (stereoisomers of the same monosaccharide).

C on toitin sulphate

Disaccharide repeat IM-acetyl gaiactosamine (GlcNAc) and uronic acid (GlcUA) or the epimer iduronic acid (IdoUA) as in CSB

Glycosidic link c-{l,3)

Suiphation on C4 or C6 of GlcNAc

Chondroiti Sulphates are never N- sulphated CSA/chondroitin 4-sulphate (GlcUA-GalNAc-4S) Su!phated on C4 of the GlcNAc.

Uremic acid N-acetyl galactosamine

CSC/chondroitin 6-sulphate (GlcUA-GalNAc-6S) Sulphated on C6 of the GlcNAc.

Uronic acid N-acetyl galactosamine

Heparin/heparan sulphate

Disaccharide repeat: N-acetyl or IM sulpho-glucosamine and uronic acid (GlcUA) or the epimer iduronic acid (IdoUA)

Glycosidic link: α-{1,4)

Sulphation occurs: N-sulphation

O-sulphation: 2-0, 6-0 or the less common 3-0 De-N or De-0 has the selective removal of N- linked or Q-sulphate esters

Heparin disaccharide repeat and potential sites of sulphation e

Hyaluronic acid

Disaccharide repeat: N-acetyl glucosamine and uronic acid (GlcUA) Glycosidic link: Alternating β-(1,4) and β-(1,3) glycosidic bonds Sulphation: Un-su!phated making it one of the least complicated GAGs

Dextran

A complex, branched polysaccharide composed of glucose monosaccharides joined by a-(l,S) glycosidic linkages with branching occurring from o-(i _; 3) linkages.

SAR - hits

Fucoidan Hyaluronic acid K5002/EK008

(heparin polysaccharides

ΰΙοΑ 1 - 4 GlcNAC

Heparin oligomers Arixtra

dp10/do8/dp6 (heparin 5-mer)

Enoxaparin

-4-Su!phate CSC/Chondroitin-6-Sul hate

SAR - non-hits

Kappa-carageenan

Hyaluron disaccharide C1012

Conclusions

A 96-well plate screening assay was developed against novel neurotoxic factors and has identified high (Mw~500,000) and lower molecular weight (Hw <S000) GAG 'hits'.

The heparin derived 4-mer Enoxaparin (Mw ~1500) represents the smallest saccharide length hit identified.

O-siilphation and or N-sulphation are absolutely required to heparin polysaccharides; un-sulphated heparin 5 polysaccharide (K5001) was identified as a 'non-hit'.

A heparin backbone substituted with the epimer, iduronic acid, has similar activity to a uro.nic acid based polysaccharide; compare K50Q2 with EK5.

Non-heparin GAG 'hits' are preferably sulphated; e.g. fucoidan, mesoglycan, dextran sulphate (see exception below).

Un-sulphated GAGs were classified as ^! non-hits' e.g. un-sulphated heparin K5 backbone K5001; maltooligosaccharide. MD-6-1; hyaluronic acid oligomers Hya6-12; chitin; chitosan; dextran.

Heparin and chondroitin sulphate disaccharides were classified as ^v non-rtits'.

The GAG kappa-carageenan is O-sulphated, however this was classified as 'non-hit' and could be due to the 3,6 anhydro bridge which affects conformation and overall stereochemistry.

Low molecular weight heparins are recognized as neuroprotectants, ² ' ³ have known safety profiles and cross the blood brain barrier ^{4, s} . Heparin compounds are also known to reduce plaques in a mouse model of Alzheimer's disease* and to prevent both the production and aggregation of Αβ through inhibiting BACE-1 ^6"8 and fibrillisation respectively ⁹ .

PREPARATION OF COMPOUNDS

Described below are the methods used for preparing compounds ¹ C and C and the compounds related to compound H - the neutral trisaccharide (C), the monosulfated library (C) and the disulfated library (H) respectively.

Preparation of compounds C and C

3,4 isopropylidene alpha methyl L-fucose

1.0 gm of alpha methyl L-fucose was dissolved in 30 ml of DMF and 10 ml dimethoxypropane followed by 0.9 gm toiuenesulfonic acid added, The reaction mixture was stirred at ambient temperature for 24 hours. 0.48 gm of triethylamine was added and, the mixture partitioned between ethyl acetate and brine, the organic layer washed again with brine, dried over sodium sulfate then the solvent removed in vacuo. 1.2 gm

2 a!lyl, 3,4 isopropylidene alpha methyl L-fucose

The foregoing compound was stirred in 6 ml of DMF, 0.4 gm of 60% sodium hydride was gradually added. Stirred for 20 minutes then 0.7 ml of allyl bromide was added and the mixture stirred for a further 30 minutes, 0.5 ml of methanol was added then the mixture partitioned between ethyl acetate and brine, the organic layer washed again with brine, dried over sodium sulfate then the solvent removed in vacuo, 1,3 gm

2-allyl alpha methyl L-fucose The previous compound was stirred at 40C for 2 days with 15 ml of 80% acetic acid. 100 ml of toluene was added and the solvents removed in vacuo. Reaction mixture was again coevaporated twice with 50 ml of toluene then purified on a silica gel column eluting with 3:1 toluene: ethyl acetate to afford 0.72 gm of the 2-al!yl alpha methyl L-fucose.

2-a!lyi 3,4 dibenzyl alpha methyl L-fucose

The foregoing compound was stirred i 20 ml of DMF, 0.33 gm of 60% sodium hydride was gradually added Stirred for 20 minutes then 1.35 gm of benzyl bromide was added and the mixture stirred for a further 60 minutes. 0.5 ml of methanol was added then the mixture partitioned between ethyl acetate and brine, the organic layer washed again with brine, dried over sodium sulfate then the solvent removed in vacuo.

3,4 dibenzyl alpha methyl L-fucose

The foregoing compound was refluxed in 100 ml of 80% methanol with 200 mg 10% palladium on charcoal and 120 mg of toluenesulfonic acid for 1 hour. A further 200 mg of catalyst was added and reflex continued for a further hour. The catalyst was removed by filtration rotary-evaporated then the mixture partitioned between ethyl acetate and brine, the organic layer washed again with brine, dried over sodium sulfate then the solvent removed in vacuo. Compound was purified by silica gel chromatography eluting with 4: 1 toluene: ethyl acetate. 450 mg

4',6' Benzylidene D- glucose beta 1-2 (3,4 dibenzyl alpha methyl L-fucose)

358 mg of 3,4 dibenzyl alpha methyl L-fucose, 557 mg of 4,6 benzylidene 2,3 dibenzoyl 1-thiomethyi D-glucose, 1.3 gm of 4A molecular sieves stirred at ambient temperature in 20 ml of dichforomethane. 254 mg of N- iodosuccinimide was added followed by 20 mg of trifluoromethanesulfonic acid. After stirring at ambient temperature for 30 minutes, 20 mg of triethylamine was added and the molecular sieves removed by filtration. The reaction mixture was next partitioned between ethyl acetate and aqueous sodium sulfite,the organic layer washed again with brine then dried over sodium sulfate. Finally the solvent removed in vacuo. The fully protected disaccnaride was dissolved in methanol and 0.5 ml of 25% sodium methhoxide in methanol was added. Stood at ambient temperature for 4 hours then sodium ions were removed with fowex H+ resin, resin was removed by filtration and product purified by crystallising from ethyl acetate- hexane. 2.40 mg

2',3'-dibenzyl D- glucose beta 1-2 (3,4 dibenzyl alpha methyl L-fucose)

The foregoing compound was stirred in 10 ml of DMF, 0.16 gm of 60% sodium hydride was gradually added. Stirred for 20 minutes then 0.7 gm of benzyl bromide was added and the mixture stirred for a further 60 minutes. 0.5 mi of methanol was added then the mixture partitioned between ethyl acetate and brine, the organic layer washed again with brine, dried over sodium sulfate then the solvent removed in vacuo. The reaction mixture was dissolved in 80% acetic acid and stood at ambient temperature for 24 hours. The acetic acid was removed by rotary-evaporation last traces were removed byco-evaporation with toluene. Finally the product was purified by crystallising from ethyl acetate-hexane. 201 mg.

2',3'-dibenzyl, 6'-acetyl D- glucose beta 1-2 (3,4 dibenzyl alpha methyl L- fucose)

100 mg of the foregoing compound was stirred at 70C in 10 ml of dichioroethane with 110 mg of acetyl imidazole for 24 hours. The major product was isolated by chromatography in 3;1 toiuene:ethyl acetate. 90 mg {2",3" dibenzyl 4"-acetyl L-fucose} alphal-4 [2',3' dibenzyl 6'-acetyl D- glucose] beta 1-2 (3,4 dibenzyl alpha methyl L-fucose)

90 mg of the foregoing compound was stirred at ambient temperature in 5 mi of dichloromethane with 0.5 gm of 4A molecular sieves and 120 mg of 1- thioethyl 2,3 dibenzyl 4-acetyl beta L-fucose. (Hasegawa A. et al Carbohydrate Research 274, {1995} 155-163) 0.8 ml of a solution of 0.5 molar dimethylthiomethyl sulfonium triflate in dichloromethane was affed and the reaction mixture stfrred at ambient temperature for 2 hours. The reaction mixture was filtered, diluted with ethyl acetate then extracted three times with brine. Purified by silica gel chromatography in 4:1 toluene:ethyl acetate.

{4"-acetyi L-fucose} alphal-4 } 6'-sulfo D- glucose] beta 1-2 (alpha methyl L-fucose) (COMPOUND C)

25 mg of the foregoing compound was dissolved in 10 mi of methanol and 0.2 ml of 25% sodium methoxode on methanol added. After 1 hour the major product was isolated by chromatography in 2:1 toluene. 'ethyl acetate. 20 mg monoacetate. This compound was taken up in 0.8 ml of DMF and 40 mg of sulfur trioxide-pyridine complex added. After 2 hours the reaction mixture was partitioned between ethyl acetate and 0.5 molar citric acid the organic layer washed with brine dried, the solvent removed by rotary evaporation. This product was stirred in methanol (5 ml) with 10 mg 10% palladium on carbon under an atmosphere of hydrogen for 24 hours. Then catalyst was removed by filtration, the methanol by evaporation to afford 9 mg of COMPOUND C

500 MHz proton NM in D20 key peaks, δ 1.05 {d, 3H, fucose' CH3CH) 1.18 (d, 3H, fucose'" CH3CH) 2.12 <s, 3H, fucose'" CH3CO) 3.32 (s, 3H _r fucose' CH30) 4.50 (d, 1H, Glucose" H"-l) 5.16 (d, 1H, fucose' H'-l) 5.24 <d, 1H, fucose'" H"'-l) <4"-5Ulfo L-fucose} alphal-4 } 6'-sulfo D- glucose] beta 1-2 (alpha methyl L-fucose) (COMPOUND C)

40 mg of {2",3" dibenzyl 4"-acetyl L-fucose> alphal-4 [2',3' diben2yl 6'- acetyl D- glucose] beta 1-2 (3,4 dibenzyl alpha methyl L-fucose) was dissolved in 10 ml of methanol and 0 ^' .8 ml of 25% sodium methoxode on methanol added. After 24 hours the major product was isolated by chromatography in 1: 1 toluene: ethyl acetate. This compound was taken up in 0.8 ml of DMF and 800 mg of sulfur trioxide-pyridine complex added. After 4 hours the reaction mixture was treated exactly as for the isolation of compound C".

COMPOUND C 12 mg

500 MHz proton NM in D20 key peaks, δ 1.14 (d, 3H, fucose' CH3CH) 1.24 (d, 3H, fucose'" CH3CH) 3.32 (s, 3H, fucose' CH30) 4.48 (d, 1H, Glucose" H"-l) 4.88 (d, 1H, fucose' H'-l) 5.18 (d, 1H, fucose'" H'"-l)

Compound H Core, Monosulfate And Disulfate Libraries

The following compounds were prepared:

1) Glc betal-4 Glc betal-4 GlcNAc beta 1-O-methyl (Compound H core trisaccharide)

2) Focussed monosulfate library based on Compound H core trisaccharide

3) Focussed disulfate library based on Compound H core trisaccharide

Glc beta 1-4 Glc beta 1-4 (3,6-dibenzyl) N-acetylglucosamine 300 mg of Methyl 3,6 dibenzyl beta N-acetyiglucosamine, 2.0 gm of hepta-O- acety) l-bromo alpha cellobiose, 2 gm of 4A molecular sieves stirred at ambient temperature in 1: 1 toluene: nitromethane then 750 mg solver carbonate followed by 770 mg silver triflate were added. The reaction mixture was stirred at ambient temperature for 1 hour. Filtered, washed twice with distilled water then purified by chromatography in 1: 1 toluene: ethyl acetate. This purified trisaccharide was de-O-acetylated by dissolving in 20 ml methanol, adding 0.6 ml 25% sodium methoxtde in methanol and standing at ambient temperature for 24 hours. Sodium johs were removed with dowex H= resin and the product purified by chromatography on silica gel in 10% methanol in dich!oromethane. 190 mg Glc beta 1-4 61c betal-4 GlcNAc beta - 1-O-methyl (Compound H core trisaccharide)

15 mg of the foregoing compound was stirred at ambient temperature with 15 mg of 10% palladium on charcoal under an atmosphere of hydrogen for 24 hours. The catalyst was removed by filtration and the product isolated by evaporation of the methanol. 6 mg

500 MHz proton NMR in D20 key peaks. 5 1.94 (s, 3H, GlcNAc CH3CO) 3.40 (s, 3H, GlcNAc CH30) 4.36 (d, 1H, GlcNAc H-l) 4.40 (d, 1H, Glucose' H'-l) 4.46 (d, 1H, Glucose" H"-l)

Focussed monosulfate library based on Compound H core trisaccharide

20 mg of Glc beta 1-4 Glc beta 1-4 (3,6-dibenzyl) N-acety!glucosamine was stirred in 2 ml of 1: 1 DMF:pyridine and sulfur trioxide-trimethylamine complex added gradually with frequent monitoring if the reaction mixture by thin layer chromatography. Reagent was added just to the point at which all starting material had reacted, then the reaction was quenched by adding water. DMF and pyridine were removed by coevaporating the mixture 4 times with water then the mono sulfated components isolated by silica gel chromatography in 40% methanol in DCM. Finally benzyl groups were cleaved by palladium-carbon catalysed hydrogenation as previously carried out. 15 mg

ESI-MS accurate mass analysis gave a spectrum identical with the theoretical model for library monosuifated components with molecular formula C21H36N019S (Accurate Mass 638.16)

Focussed disulfate library based on Compound H core trisaccharide

10 mg of the monosuifated library was subjected to a further degree of sulfation in a similar way. The purification and workup procedure was also similar to that described for the monosulfared library. 6 mg

ESI-MS accurate mass analysis gave a spectrum identical with the theoretical model for library disulfated components with molecular formula C21H35N022S2 {Accurate Mass 717.11).

Preparation Of Fucoidan Mimetic Library

191 mg of 4-0-acetyl, 2,3 dibenzyl 1-ethylthio-L-fucose {Carb. Res. 274 (1995) 155-163} was stirred in 5 ml of diehloromethane with 150 mg of 4A molecular sieves and 50 mg of methanol. 0.8 ml of a 1M solution of dimethyl thiomethyl sulfonium triflate was added and the mixture stirred at ambient temperature for 20 minutes.

2 ml of methanol was added followed by 0.6 mi of 25% methanolic sodium methoxide and the mixture stirred for a further 2 hours. It was then neutralised with acetic acid, filtered then partitioned between ethyl acetate (50 ml) and water (50 ml). Then organic layer was separated, dried over sodium sulfate then rotary evaporated to afford 2,3 dibenzyl 1-O-methyl-L- fucose (160 mg). The foregoing compound was stirred in 5 ml of dichloromethane with 230 mg of 2-benzyl, 3,4 di-O-acetyl, 1- ethylthio-L-fucose {Carb, Res, 274 (1995) 155-163} and 100 mg of 4A molecular sieves then 1-0 ml of dimethyl thiomethyl sulfonium inflate was added.

After 20 minutes stirring at ambient temperature 2 ml of methanol was added followed by 0.5 ml of 25% methanolic sodium methoxide and the mixture stirred for a further 2 hours. It was then neutralised with acetic acid, filtered then partitioned between ethyl acetate (50 ^;' ml) and water (50 ml).

Then organic layer was separated, dried over sodium sulfate then purified on a short chromatography column eluting with 2 :1 toluene:ethyl acetate to afford 73 mg of a disaccharide mixture.

The disaccharide mixture (73 mg) plus 66 mg of 2-benzyl, 3,4 di-O-acetyl, 1- ethy!thio-L-fucose and 50 mg of 4A molecular sieves was stirred at ambient .temperature in 2 ml of dichloromethane then 0.25 ml of dimethyl thiomethyl sulfonium triflate was added.

After 20 minutes, the reaction mixture was de-0 acetylated and worked up as for the disaccharide mixture then purified on a short column eluting with 2:1 toluene : ethyl acetate to afford 60 mg of a trisaccharide mixture.

The foregoing mixture (60 mg) was stirred in 2 ml of dichloromethane with 50 mg of 4A molecular sieves and 50 mg of 2-benzyl, 3,4 di-O-acetyl, 1- ethylthio-L-fucose. 0.2 ml of dimethyl thiomethyl sulfonium triflate was added and the reaction mixture stirred at ambient temperature for 20 minutes. The reaction mixture was de-0 acetylated, worked- υρ and purified as for the trisaccharide mixture to afford 30 mg of a tetrasaccharide mixture.

The tetrasaccharide mixture (30 rng) was stirred at ambient temperature overnight in 1 ml of 1: 1 dimethylformamide: yridine with 50 mg of sulfur trioxidetrimethylamine complex, the reaction mixture was diluted with 50 ml of ethyl acetate then washed successively with 10 ml 1M hydrochloric acid, then twice with 10 ml of brine, It was dried then rotary-evaporated to an oil.

The foregoing compound mixture was stirred at .ambient temperature with- 30 mg of 10% palladium oh charcoal under an atmosphere of hydrogen for 24 hours. The catalyst was removed by filtration and the product isolated by evaporation of the methanol and finally freeze-drying to afford 12 mg of a sulfated tetrafucosyl library. (Compounds shown in Table 2).

Preparation Of A Partially Sulfated Pentasaccharide Mixture

4 mg of mannosyl pentasaccharide:

3,6 Di- (IM-acetyl Glucosamine β1-2 Mannose) annose 3,6 Di-(GlcNAcpl-2 Man) Man was stirred at ambient temperature in 0.2 m! of 1: 1 dimethylformamide: pyridine with 4 mg of sulfur trioxidetrimethylamine complex. The reaction mixture was diluted with water then applied to a short column of Dowex 50Wx8 hydrogen form. The purified product was eluted with water to afford a mixture of partially sulfated mannosyl pentasaccharide components.

The solution was freeze-dried to afford 4 mg of mannosyl pentasaccharide sulfated library. (Compounds shown in Table 3).

ASSAY

In vitro assays were carried out by growing dissociated neurons on coverslips for 48 hrs and then treated for 48hrs with synthesised carbohydrates according to the invention ^" (micomolar) In the presence or absence of histone Hi (100 nanomolar). Untreated controls were also grown for four days. The total population was estimated by counting the number of nuclei positive for DAPI (da) and the number of dead cells by counting the number of nuclei positive for propidium iodide (pi). Results are expressed as % dead/total and with mean ceil counts. In these results histone H i is referred to as ^W F".

The connpounds according to the invention tested in the assays were as follows:

C

wherein X=X'=su!fo, _t =Me and R ₂ =H e

wherein 12 000468

70

L701

wherein R^Me, R _Z =H , one of X=suiphate, the other two X are H _.

L702

wherein the other X is H

wherein R ₁ =Me, R ₂ =X=H

DATA

Compound Without Histone Hi Compound With Histone HI mean mean % % mean mean dead(d) total (t) dt d/t % dead total control 8.7 105.8 8.2 37.5 28.9 77.1 HI lOOnM+cpd

Cpd Cpd

( M) (uM)

C30 6.2 120.1 5.2 10.1 10.9 107.5 C30

CIOO 6.3 152.9 4.1 8.1 10 122.8 CIOO

C 30 6.6 143.2 4.6 13J 15 109 C30

CIOO 6.3 125.2 12,9 12,4 95.6 C 100

H30 7.5 !05.2 7.3 14,6 11.6 79.3 H30

H 100 30.5 171.2 1» 9,8 .9.1 93.3 H100

L70130 5.3 141.9 3.7 tbe 70130

1701 9.2 123.2 7.5 15,9 12.9 81,1 701100 100

1,70230 9.7 Π .1 8.1 16.4 18.9 US 70230

L702 7.1 121.1 5.7 14.1 13.9 98.9 702100 100 Rank order of compounds by % cells dead in presence of lOOnM historic HI

Control

HI lOOnM cpd (uM)

CI 00

moo

C30

CI 00

C'30

L702 100

H30

L701 100

L702 30

Summary Of

Results 1 mean mean control da mean 146.3182 F100 85.19048

SD - 41.16678 24.7055 control pi mean 9.666667 27.68182

SD 2.957898 9.270467

C 30 da mean 120.1364 C30 F100 107.5238

SD 14.16332 29.60848

C 30 pi mean 6.2 10.95238

SD 2.345208 2.80136

C 300 da mean 152.9091 C100F100 122.8077

SD 39.01027 34.86204

C I 00 pi mean 6.285714 10.03704

SD 3.068271 4.484859 30 da mean 143.1739 C30 F10G 109.0385 SD 46.40107 42.30601 30 pi mean 6.565217 C30F100 15 SD 2.67694 3.438908

C' lOO da mean 125.2083 C'lOOnO 95.62963

O SD 27.39562 17.57872

C' OO i mean 6.291667 CIOOFIO 12.40909

0

SD 2.136213 3.58 Π56 701 30 da mean 141.8947 TBD

SD 24.4538

703 30pi mean 5.263158

SD 2.077448

701 lOOda mean 123.2353 701 iOOFl 81.09524

00

SD 38.88369 18.98395

70] JOOpi mean 9.157895 701100F1 12.85

00

SD 3.905312 3.528456

702 30da mean 119.125 702 US

30P10O

SD . 14.29294 29.52523 702 30pi mean 9.666667 702 18.86957

30F100

SD 2.987898 4.20286

702 lOOda mean 121.05 702100F1 98.9

00

SD 31.16421 30.45946 702 lOOpi mean 7.1 70210 F1 13.9

00

SD 2.125039 3.878415 Summary Of

Results 2

control da mean 78.18182 F100 mean 84.27273

SD 17.58305 SD 31.16758 control pi mean 5.5 F100 mean 39.09091

SD 2.645751 SD 14.35587 control da raean 74.14286

SD 12.2396? F100 mean 61.7 control pi mean 10.42857 SD 26.35252

SD 2.992053 F100 mean 20

SD 5.887841

H30 da mean 76.76923 Η30 1 00 mean 81.75 da

SD 10.40155 SD 16.13086

H30 pi mean 5.923077 H30 F100 mean ^' 11.25

P ^' i

SD 4,15254 SD 6.326352

Summary Of H30 F100 Mean 76.66667 Results 2 da

(continued)

SD 12.04788

H30 F100 Mean 10.75 P ^'1

SD 4.070403

H100 Mean 93.25 FlOOda

SD 15.93239

H100 Mean 9.0909β9 FiOOpi

SD 4.158234 .

H100 Mean 89.08333 FlOOda

SD 20.34457

HI 00 Mean 8.25 FiOOpi

SD 5.594234

Summary Of

Results 3

control da mean 124.4737

SD 37.26537

control pi mean 9.055556

SD 3.17105

H30 da mean 133.5263 H30 FlOO mean 112.9 da

SD 33.94664 SD 35.7151

H30 pi mean 9.052632 H30 F100 mean 28.3

P ^' »

SD 3.170589 SD 10.26542

H30 F100' mean 306.4286 da

SD 19.63793

H30 FJ 00 mean 39.36364 P ^' l

SD 14.65109

H100 da mean 171.2143 HI 00 mean 115,1765

FlOOda

SD 34.06143 SD 34.79985

HI 00 pi mean 30.9 HI 00 mean 25.91667

FlOOpi

SD 5.915141 SD 9.15051 Cortical Neurons and Luciferase Assay for Tetrasaccharide (table 2) and Pentasaccharide Libraries (table 3)

Primary cortical neurons were prepared in lOOui aliquots and seeded onto poly-D-!ysine and iaminin (both lOug/mi, Gibco) coated black 96 well plates (Greiner) at 3 x 104 ceils per well. Cells were incubated for 48hrs in Neurobasai medium supplemented with Glutamax, B27 and 1% Antibiotic- Antimycotic (all Gibco) and then treated with histones (Upstate) at 0, 100, 200 and 400nM for a further 24hrs. Histone-induced ceil death was determined by the CytoTox-GloTM Cytotoxicity Assay (Promega). Briefly, degenerating ceils release intracellular proteases which cleave acetylaminofluorene (AAF) to liberate aminoluc ^' iferin which is a substrate for luciferase. This reaction generates luminescence proportional to the number of dead cells (Ming-Hsuang Cho et al 2008) and was measured using a Promega GloMax 96 luminometer. The read-out (RLU) is the relative luminosity unit and this is linearly related to the number of dead cells,

The tetrasaccharide (table 2) and pentasaccharide (table 3) libraries were dissolved in culture medium and applied for 24hrs at concentrations of 30- 400uM.

Results are shown below;

Summary results for tetrasaccharide

library

RLU H1 nM 0 100 200 400

H1 17668 66053 136633 179009

SO 1362.5 5123,5 6665.9 5441.3

ST30u 17860 67161 142414 183573

SD 773.6 6597.8 7202,7 4175.8

ST OOuM 17978 56866 132136 180472

SD 666.0 8161.3 7116,4 8460.0

ST 200u 16495 50432 117577 173097

SD 7399.7 9190.5 12987,5 8949.7

ST 00uM 14068 29503 104184 170029

SD 6270.4 2159.8 4591 ,4 5 15.3

Summary results for Pentasaccharide library

H1 nM 0 100 200 400

H1 176S8 66053 136633 1 9009

SD 1362.5 5123.5 6865,9 5441.3

PS30uM ^• 17964 37476 132746 178001

SD 786.4 2096.3 5270.2 4688.0

PS OOuM 17903 28810 125249 176555

SD 1129.9 3245.5 3829.1 7548.7

PS 2Q0uM 17081 19504 95588 167972

SD - 1290,6 1498.2 9293.6 6886.2

it will be appreciated by those skilled in the art that the foregoing description is exemplary and explanatory in nature, and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, an artisan will recognise apparent modifications and variations that may t e made without departing from the spirit as scope of the invention as defined in the appended claims. ' . Bibliography

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