TECHNICAL FIELD

[0001] The present invention provides mono- and dinucleoside 5'-phosphorothioate analogues as well as pharmaceutical compositions thereof. The compounds are useful for treatment of neurodegenerative diseases or disorders, e.g., Alzheimer's disease.

[0002] Abbreviations: AD, Alzheimer's disease; ADP, adenosine diphosphate; AMP, adenosine monophosphate; APCPP-y-S, adenosine 5'-[y-thio]-a,P-methylene triphosphate; APPCP-a-S, adenosine 5'-[a-thio]-P,y-methylene triphosphate; ATP, adenosine triphosphate; BBB, blood brain barrier; BCA, bicinchoninic acid; [Ca ²⁺ ]i, intracellular Ca ²⁺ concentration; CDI, carbodiimidazole; Clioquinol, 5-chloro-7- iodoquinolin-8-ol (CQ); CNS, central nervous system; DBU, l,8-diazabicyclo[5.4.0] undec-7-ene; DCM, dichloromethane; DLS, dynamic light scattering; DMAP, 4- dimethylaminopyridine; DMEM, Dulbecco's modified Eagles' medium; DMF, N,N- dimethylformamide; DMPO, 5,5'-dimethyl-l-pyrroline-N-oxide; DMSO, dimethyl sulfoxide; EDTA, ethylenediamine tetraacetic acid; ESI, electrospray ionization; ESR, electron spin resonance; GDP, gunosine diphosphate; GFP, green fluorescent protein; GLUT1, glucose transporter 1; GSH, glutathione; GTP, guanosine triphosphate; HPLC, high- pressure liquid chromatography; LC, liquid chromatography; MDPT, methylene diphosphonotetrathioic acid; MTT, dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NPP, nucleotide pyrophosphatase/ phosphodiesterase; NTPDase, nucleoside triphosphate diphosphohydrolase; PBS, phosphate buffered saline; pnp-TMP, thymidine 5' -monophosphate p-nitrophenyl ester; ROS, reactive oxygen species; RT, room temperature; TBA, tert-butyl alcohol; TEAA, triethylammonium acetate; TEAB, trietliylammonium bicarbonate: TEM, transition electron microscopy; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin layer chromatography.

BACKGROUND ART

[0003] Alzheimer's disease (AD) is a progressive neuronal disease characterized by an irreversible neuronal damage which causes memory loss, impaired cognitive functions and loss of speech. The main features of AD include amyloid plaques constituted of the amyloid beta (Αβ), a 39-43 amino acid peptide, neurofibrillary tangles, consisting mainly of paired helical filaments of abnormally hyper-phosphorylated micro tubule-associated τ protein (Iqbal et al, 2005), oxidative stress and ROS formation (Pratico, 2008), and neuroinflammatory processes (Akiyama et al, 2000).

[0004] Αβ forms oligomers which give rise to fibrils. The role of Αβ is still ambiguous, although several possibilities have been suggested such as an antioxidant (Baruch- Suchodolsky and Fischer, 2009) or oxidant role (White et al, 2004), regulation of synaptic vesicle release (Abramov et al, 2009), and antimicrobial activity (Soscia et al, 2010).

[0005] Numerous studies indicate that Αβ binds Cu ²⁺ , Fe ³⁺ and Zn ²⁺ ions with high affinity (Garzon-Rodriguez et al, 1999; Faller and Hureau, 2009). Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) data showed that Αβ peptide binds the metal-ion through three histidine residues, His6, Hisl3, and Hisl4 in a N ₃ 0 manner, while the oxygen atom origin is TyrlO, Glu5, or Aspl (Faller and Hureau, 2009; Curtain et a/., 2001; Karr ei al, 2005).

[0006] High concentrations of Zn ²⁺ , Cu ²⁺ and Fe ²⁺ ions have been found in senile plaques, in histological section of AD patients (Lovell et al, 1998). In vitro it was shown that with the addition of Zn ²⁺ at pH 7.4 and Cu ²⁺ at pH 6.6 Αβ ₄₀ /42 readily precipitated (Atwood et al., 1998). It was also demonstrated that these metal-ions can crosslink two Αβ peptides by His-M ²⁺ /M ⁺ -His intermolecular bridges (Faller, 2009; Miura et al., 2000) or lead to intermolecular cross-linked Αβ due to reaction of two TyrlO tyrosyl radicals (Atwood et al., 2004). Moreover, Αβ was shown to be neurotoxic when incubated with Cu ²⁺ or Fe ²⁺ (Dai et al, 2009; Salvador et al, 2010).

[0007] A correlation was found between the increase of Fe/Cu/Zn ions concentrations in AD brains, 3-5 times more than in brains of healthy individuals, and the formation of Αβ plaques (Bush, 2003). Furthermore, the high concentrations of Fe/Cu ions were related to enhanced oxidative stress in AD (Jomova et al, 2010). Current therapies are not able to stop AD progression but offer only symptomatic relief and can, in the best case, slow cognitive decline (Lau and Brodney, 2008). These therapies attempt to address neurotransmitter defects (Francis et al, 1999), slow neurodegeneration (Simons et al, 2002), or treat inflammation and oxidative stress (Lim et al, 2000).

[0008] Other treatment strategies target Αβ production, aggregation, toxicity, or enhancement of Αβ degradation. These strategies include γ-secretase inhibitors that reduce Αβ production (Panza et al, 2010), neprilysin that promote Αβ degradation (Selkoe, 2001), β-sheet breakers which prevent or slow oligomers/fibril formation (Bartolini et al, 2007), humanized antibodies against Αβ peptide which reduce Αβ load (Bombois et ah,

2007) , and metal-ion chelators that block Αβ aggregation (Scott and Orvig, 2009).

[0009] Several β-sheet inhibitors have been reported (Bartolini et ah, 2007); however, these inhibitors do not address the increasing age-related metal-ion concentration that is a key factor for Αβ oligomerization, fibril formation, and oxidative stress.

[0010] Metal-ion chelators such as clioquinol (PBT1) (Ritchie et al, 2003) and PBT2 (8- hydroxy quinoline analogue) (Cherny et al., 2008) are moderate affinity binding chelators considered to be ionophores. The mode of action of clioquinol and PBT2 is denoted as metal-protein attenuating compounds (MPAC) (Ritche et al., 2004). Clioquinol and PBT2 lowered Αβ load both in in vitro and in vivo studies (Adlar et ah, 2008; LeVine et ah, 2009); however, clioquinol failed phase II clinical trials. PBT2 showed better in vivo results than clioquinol in reducing insoluble Αβ in transgenic mice brain (Cherny et ah,

2008) . Other metal-ion chelators such as deferiprone (Green et ah, 2010) and Nl,N2- bis(pyridine-2-yl-methyl)-ethane-l,2-diamine (Lakatos et ah, 2010) have been recently shown to redissolve Aβ4o/42-metal-ion aggregates. Subsequent to metal-ion chelators disassembly of Αβ ₄₀ /42-Μ ²⁺ aggregates, the free Αβ peptide may be degraded by proteases (Selkoe, 2001) or cleared to the bloodstream (Zlokovic, 2004).

[0011] AD is a multi-parameter disease involving highly complex biochemical mechanisms. Therefore, an AD disease modifying drug is preferentially a multifunctional one simultaneously addressing several drug targets. An ideal drug candidate, for instance, lowers the Αβ load in the brain, and in addition serves as MPAC and an antioxidant (Doraiswamy and Finefrock, 2004).

[0012] Nucleotide analogues are natural metal-ion chelators (Sigel and Griesser, 2005). For instance, ATP forms stable complexes with various divalent metal ions (e.g., Fe ²⁺ , Mg ²⁺ , Zn ²⁺ and Cu ²⁺ ), of which the most stable is the Cu ²⁺ -ATP complex (log K 6.34) that is 1.2-2.5 orders of magnitude more stable than the other complexes (Sigel and Griesser, 2005). Furthermore, ATP and GTP were shown to be the dominant ligands affecting the chelation of iron and transferring it into the cell before it is incorporated into heme and ferritin (Weaver, 1989; Weaver et ah, 1993). Related observations were made for the Cu ²⁺ -ion (Barnea et al, 1991).

[0013] Previously, we investigated nucleotides and phosphate analogues as potential antioxidants. Specifically, we found that ΑΤΡ-γ-S proved a most potent antioxidant inhibiting OH radical production in the Fe ²⁺ /H ₂ 02 system with IC ₅₀ of 10 μΜ (being 100 and 20 times more active than ATP and the potent antioxidant Trolox, respectively). Likewise, nucleotides and phosphates (e.g., ATP, ADP, and thiophosphate) proved potent antioxidants in Cu ⁺ /Cu ²⁺ -H ₂ 0 ₂ systems (Richter and Fischer, 2006; Baruch-Suchodolsky and Fischer, 2008). Modification of a nucleotide by a terminal thiophosphate moiety (e.g. ΑΤΡ-γ-S and ΑϋΡ-β-S) resulted in significantly enhanced antioxidant activity as compared to that of the corresponding parent compound. Our previous findings demonstrating the antioxidant activity of nucleoside 5'-phosphorothioate analogues encouraged us to evaluate them as biocompatible and water-soluble agents for the dissolution of Αβ-Μ ²⁺ aggregates.

[0014] US 7,368,439 discloses diribo-, di-2'-deoxyribo, and ribo-2'-deoxyribo-nucleoside boranophosphate derivatives that can be useful for prevention or treatment of diseases or disorders modulated by P2Y receptors such as type 2 diabetes, cystic fibrosis and cancer. WO 2009/066298 discloses non-hydrolyzable adenosine and uridine polyphosphate derivatives, said to be useful for prevention or treatment of diseases modulated by P2Y- receptors such as type 2 diabetes. WO 2011/077435 discloses ophthalmic compositions for reducing intraocular pressure, comprising a non-hydrolyzable nucleoside di- or triphosphate analogue in which the α,β- or β,γ-bridging-oxygen, respectively, is replaced with, e.g., a methylene or dihalomethylene group. WO 2012/032513 discloses pharmaceutical compositions for treatment and management of osteoarthritis, comprising either a dinucleotide boranophosphate derivative or a nucleoside boranophosphate derivative, in which at least one of the bridging-oxygens in the dinucleoside boranophosphate derivative, preferably both the α,β- and δ,ε-bridging-oxygens, and at least one of the bridging-oxygens in the nucleoside boranophosphate derivative, each is replaced with a group selected from -NH- or -QRioRn)-, wherein R ₁₀ and Rn each independently is H or halogen. WO 2012/073237 discloses uridine nucleotides in which the carbon atom at position 5 of the uracil ring is substituted by -O-alkyl or -S-alkyl, and at least one of the non-bridging oxygen atoms of the di- or tri-phosphate is replaced by a borano group, which can be useful for treatment of diseases, disorders and conditions modulated by P2Y ₆ receptors, particularly for lowering intraocular pressure. All these publications, based on studies conducted in the laboratories of the present inventors, are herewith incorporated by reference in their entirety as if fully described herein. SUMMARY OF INVENTION

[0015] In one aspect, the present invention provides a compound, more particularly, a mono- or dinucleoside 5'-phosphorothioate of the general formula I:

(B ⁺ ), or a diastereomer or mixture of diastereomers thereof,

wherein

X is -O ^" , Nu', a glucose moiety linked through the oxygen atom linked to its 1- or 6-position, or a group of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH-(CHRi ₃ )-C(0)-OR ₁₃ ;

Nu and Nu' each independently is an adenosine residue of the formula la, linked through the oxygen atom linked to the 5'-position:

wherein

Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR ₄ R ₅ , heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 ₂ , -OR ₄ , -SR ₄ , -NR ₄ R ₅ or heteroaryl, wherein R ₄ and R ₅ each independently is H or hydrocarbyl, or R ₄ and R ₅ together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and

R ₂ and R ₃ each independently is H or hydrocarbyl;

or an uridine residue of the formula lb, linked through the oxygen atom linked to the 5'-position:

wherein

R ₆ is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NRgRg, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 ₂ , -OR ₈ , -SR ₈ , -NRgRg or heteroaryl, wherein R ₈ and R ₉ each independently is H or hydrocarbyl, or Rg and R9 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and

R ₇ is O or S;

Y and Y' each independently is H, -OH or -NH ₂ ;

Wi and W ₂ each independently is -0-, -NH- or -QRioRn)-, wherein R ₁₀ and Rn each independently is H or halogen;

Z _1; Z'i, Z ₂ , Z' ₂ and Z'3 each independently is O, -O ^" , S, -S ^" or -BH ₃ ^~ ;

Z ₃ is -0 , -S ^" , -BH ^" , or a group of the formula -0-CH ₂ -OC(0)-R ₁₂

(CHR ₁₃ )-C(0)-OR ₁₃ ;

Ri2 is (Ci-C ₄ )alkyl;

R ₁₃ each independently is (Ci-C ₄ )alkyl, (C ₆ -Cio)aryl or (C ₆ -C ₁ o)aryl-(C ₁ -C ₄ )alkyl; n is 0 or 1 ;

m is 2, 3 or 4; and

B ⁺ represents a pharmaceutically acceptable cation,

provided that (i) at least one of Wi and W ₂ is not -0-, and at least one of Z _1; Z' _1; Z ₂ , Z' ₂ , Z ₃ and Z' ₃ is S or -S ^" ; and (ii) when X is a glucose moiety, Z ₃ is -O ^" , -S ^" , or -BH ₃ ^" ; and when one of X and Z is a compound of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH- (CHR ₁ )-C(0)-OR ₁₃ , the other one of X and Z is a compound of the formula -0-CH ₂ - OC(0)-R ₁₂ or -NH-(CHRi ₃ )-C(0)-OR ₁₃ , respectively.

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, i.e., provided that (i) at least one of Wi and W ₂ is not -0-, and at least one of Z _{1 ;} Z'i, Z ₂ , Z' ₂ , Z ₃ and Z' ₃ is S or -S ^" ; and (ii) when X is a glucose moiety, Z ₃ is -O ^" , -S ^" , or -BH ^" ; and when one of X and Z is a compound of the formula -0-CH ₂ -OC(0)-R ₁₂ or - NH-(CHR ₁₃ )-C(0)-OR ₁₃ , the other one of X and Z ₃ is a compound of the formula -0-CH ₂ - OC(0)-R ₁₂ or -NH-(CHR ₁₃ )-C(0)-OR ₁₃ , respectively, or a diastereomer or mixture of diastereomers thereof, and a pharmaceutically acceptable carrier or diluent. The compounds and pharmaceutical compositions of the invention are useful in treatment of neurodegenerative diseases or disorders such as Alzheimer's disease.

[0017] In a further aspect, the present invention thus relates to a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, for use in treatment of a neurodegenerative disease or disorder such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease.

[0018] In still a further aspect, the presenty invention relates to use of a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, for the preparation of a pharmaceutical composition for treatment of a neurodegenerative disease or disorder such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease.

[0019] In yet another aspect, the present invention relates to a method for treatment of a neurodegenerative disease or disorder in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof.

BRIEF DESCRIPTION OF DRAWINGS

[0020] Fig. 1 shows Cu ⁺ titration of 1 mM Αβ ₂₈ solution (pD 7) monitored by 1H-NMR at 700 MHz.

[0021] Fig. 2 shows titration of Αβ ₂₈ -Οι ⁺ complex by various chelators monitored by 1H-NMR at 700 MHz, pD 7: (a) Αβ ₂₈ ; (b) Αβ ₂₈ -Οι ⁺ 1: 1 complex; (c) Αβ ₂₈ -Οι ⁺ complex titrated by 6 eq of clioquinol; (d) Αβ ₂₈ -Οι ⁺ complex titrated by 6 eq of triphosphate; (e) Αβ ₂₈ -Οι ⁺ complex titrated by 6 eq of thiophosphate; (f) Αβ ₂₈ -Οι ⁺ complex titrated by 6 eq of GDP-P-S; (g) Αβ ₂₈ -Οι ⁺ complex titrated by 5 eq of ΑϋΡ-β-S; and (h) Αβ ₂₈ -Οι ⁺ complex titrated by 3.2 eq of GTP-y-S.

[0022] Figs. 3A-3B show titration of 9 mM ΑϋΡ-β-S, pD 7.4, by Cu ⁺ monitored by 1H- NMR at 600 MHz (3A); and ³¹ P-NMR at 243 MHz (3B).

[0023] Figs. 4A-4B show detection of free thiophosphate by UV-Vis spectra using Ellmans' reagent (DTNB) in thiophosphate (4A); and GDP^-S (4B). Abs. - absorption.

[0024] Figs. 5A-5C show disaggregation of Αβ ₄₀ -Μ2 ⁺ by various chelators as measured by DLS: chelator-dependent changes in average _H of Αβ ₄₀ -^ ²⁺ aggregate (5A); EDTA- relative re- solubilization efficacy of chelators of Αβ ₄ ο-Ζη ²⁺ aggregates (5B); and EDTA- relative re- solubilization efficacy of chelators of Αβ ₄ ο-Οι ²⁺ aggregates (5C).

[0025] Figs. 6A-6D show TEM images of 25 μΜ nine-day-old aggregates: Αβ ₄₀ -Οι ²⁺ aggregate at pH 6.6 (6A); upon addition of APCPP-y-S (150 μΜ) to Αβ ₄₀ -Οι ²⁺ aggregate at pH 6.6 (6B); Αβ ₄₀ -Ζη ²⁺ aggregate at pH 7.4 (6C); upon addition of APCPP-y-S (150 μΜ) to Αβ ₄₀ -Ζη ²⁺ aggregate at pH 7.4 (6D).

[0026] Fig. 7 shows Αβ ₄₀ -Οι ⁺ titration by APCPP-y-S monitored by 1H-NMR at 700 MHz: (a) 0.25 mM Αβ ₄₀ , pD 11; (b) 0.25 mM Αβ ₄₀ , pD 7.8; (c) 0.25 mM Αβ ₄₀ -Οι ⁺ 1: 1, pD 7.8; and (d) Αβ ₂₈ -Οι ⁺ complex titrated by 6 eq of APCPP-y-S.

[0027] Figs. 8A-8B show disaggregation of Αβ ₄₂ -Μ ²⁺ by various chelators as measured by turbidity assay at 405 nm: chelator-dependent changes of Αβ ₄₂ -Ζη ²⁺ aggregation relative to EDTA (8A); and chelator-dependent changes of Αβ^-Cu ^2"1" aggregation relative to EDTA (8B).

[0028] Fig. 9 shows FeS0 ₄ induced toxicity in cultured cortical neurons. Neurons were exposed to FeS0 ₄ (0.8-6 μΜ) for 24 h and toxicity assessed by direct microscopic examination and by XTT assay.

[0029] Figs. 10A-10B show the neuroprotective effect of ΑΤΡ-γ-S and GDP^-S (0.2- 200 μΜ, t = 24 h) as evaluated by MTT production in cortical neurons exposed for 24 h to either FeS0 ₄ at final concentration of 3 μΜ (10A); or both FeS0 ₄ (3 μΜ) and H ₂ 0 ₂ (ΙΟΟμΜ) (10B). The results shown are the mean + SEM of three independent experiments in quadruplicate.

[0030] Fig. 11 shows application of Αβ ₄₂ to neuronal cell culture. Primary neurons cells were cultured in 96 wells plate (95x10 ⁴ per well). After 24 h the cells were treated with various concentrations (5-50 μΜ) of Αβ ₄₂ for 48 h. Cell viability was measured by dyeing the cells with trypan blue and counts of the vital cells. The results shown are the mean+S.D of three independent experiments in triplicate (* P<0.05, ** P<0.01 vs. control).

[0031] Fig. 12 shows that APCPP-y-S protects neuronal cell culture subjected to Αβ ₄₂ . Primary neuron cells were cultured in 96 wells plate (95x10 ⁴ per well). After 24 h the cells were treated with 50 μΜ Αβ ₄₂ and various concentrations of APCPP-y-S (0.04-25 μΜ) for 48 h. Cell viability was measured by dyeing the cells with trypan blue and counts of the vital cells. The results shown are the mean+S.D of three independent experiments in triplicate (* P<0.05, vs. Αβ ₄₂ treatment).

[0032] Fig. 13 shows the efficacy of ATP and ΑΤΡ-γ-S as neuroprotectants against Αβ ₄₂ toxcity. Primary neurons cells were cultured in 96 wells plate (95x10 ⁴ per well). After 24 h the cells were treated with 50 μΜ of Αβ ₄₂ and various concentration of ATP or ΑΤΡ-γ-S (0.04-25 μΜ) for 48 h. Cell viability was measured by dyeing the cells with trypan blue and counts the vital cells. The results shown are the mean+S.D of three independent experiments in triplicate (* P<0.05, **P<0.01 vs. Αβ ₄₂ treatment).

[0033] Fig. 14 shows the efficacy of APCPP-y-S (1 μΜ) at P2Y ₁ -R astrocytoma cells vs. natural ligands (ATP and ADP). Calcium response of astrocytoma cells transfected with plasmids encoding human P2Yi-GFP receptor fusion protein. Ratio of fluorescence values at 340 nm and 380 nm was calculated (R = AF340/380). Basal values were subtracted and the peak height for each cell was determined.

[0034] Fig. 15 shows that PC 12 cell viability after treatment with APCPP-y-S. PC 12 cells were treated with 1-1000 μΜ APCPP-y-S, and after 24 h cell viability was measured by the MTT assay, compared to non-treated cells. The results shown are the mean+S.D of three independent experiments in triplicate.

[0035] Fig. 16 demonstrates a kinetic profile showing the changes in the percentage of adenosine-5'-tetrathiobisphosphonate in acidic conditions (pD=1.5), as monitored by 31 P- NMR at 81 MHz, at 300 K.

[0036] Fig. 17 demonstrates a kinetic profile showing the changes in the percentage of adenosine-5'-tetrathiobisphosphonate subjected to air-oxidation, as monitored by 31 P-NMR at 81 MHz, at 300 K.

[0037] Fig. 18 shows 31 P-NMR spectra of di-adenosine-5',5"-tetrathiobisphosphonate at pD=1.5. [0038] Figs. 19A-19B show titration of 3 mM di-adenosine-5',5"-tetrathiobis phosphonate in D ₂ 0 at pD=7.38 with Zn 2+ . 31 P-NMR spectrum was measured at 160 MHz, 300K (19A); and 1H-NMR spectrum was measured at 400 MHz, 300K (19B).

[0039] Figs. 20A-20B show titration of 5 mM adenosine-5'-tetrathiobisphosphonate in D ₂ 0 at pD=7.40 with Zn ²⁺ . ³¹ P-NMR spectrum was measured at 160 MHz, 300K; (20A); and 1H-NMR spectrum was measured at 400 MHz, 300K (20B).

[0040] Figs. 21A-21C show inhibition of pnp-TMP and ATP hydrolysis with NPP1,3 and NTPDasel,2,3,8, respectively, by adenosine-5'-tetrathiobisphosphonate (21A); di- adenosine-5',5"-tetrathiobisphosphonate (21B); and ΑϋΡ-β-S (21C).

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention provides, in one aspect, a compound of the general formula I:

(B ⁺ ), m

or a diastereomer or mixture of diastereomers thereof,

wherein

X is -O ^" , Nu', a glucose moiety linked through the oxygen atom linked to its 1- or 6-position, or a group of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH-(CHRi ₃ )-C(0)-OR ₁₃ ;

Nu and Nu' each independently is an adenosine residue of the formula la, linked through the oxygen atom linked to the 5'-position:

wherein Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR ₄ R ₅ , heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 ₂ , -OR ₄ , -SR ₄ , -NR ₄ R ₅ or heteroaryl, wherein R ₄ and R ₅ each independently is H or hydrocarbyl, or R ₄ and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and

R ₂ and R ₃ each independently is H or hydrocarbyl;

or an uridine residue of the formula lb, linked through the oxygen atom linked to the 5'-position:

wherein

R ₆ is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR ₈ R ₉ , heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 ₂ , -OR ₈ , -SR ₈ , -NRsRg or heteroaryl, wherein R ₈ and R ₉ each independently is H or hydrocarbyl, or R ₈ and R ₉ together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and

R ₇ is O or S;

Y and Y' each independently is H, -OH or -NH ₂ ;

Wi and W ₂ each independently is -0-, -NH- or -QRioRn)-, wherein R^ and Rn each independently is H or halogen;

Z _1; Z'i, Z ₂ , Z' ₂ and Z' each independently is O, -O ^" , S, -S ^" or -BH ^" ;

Z ₃ is -0 , -S ^" , -BH ₃ ^" , or a group of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH- (CHR ₁₃ )-C(0)-ORi ₃ ;

Ri2 is (Ci-C ₄ )alkyl;

R ₁ each independently is (Ci-C ₄ )alkyl, (C ₆ -C ₁ o)aryl or (C ₆ -C ₁ o)aryl-(C ₁ -C ₄ )alkyl; n is 0 or 1 ;

m is 2, 3 or 4; and

B ⁺ represents a pharmaceutically acceptable cation,

provided that (i) at least one of W and W ₂ is not -0-, and at least one of Z _1; Z , Z ₂ , Z' ₂ , Z ₃ and Z' ₃ is S or -S ^" ; and (ii) when X is a glucose moiety, Z ₃ is -O ^" , -S ^" , or -BH ₃ ^" ; and when one of X and Z ₃ is a compound of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH- (CHR ₁ )-C(0)-OR ₁₃ , the other one of X and Z is a compound of the formula -0-CH ₂ - OC(0)-Ri ₂ or -NH-(CHR ₁₃ )-C(0)-ORi ₃ , respectively.

[0042] The compound of the present invention may be an adenosine- or uridine-5'-di- or tri-phosphorothioate derivative, as well as a dinucleoside 5'-di- or tri-phosphorothioate derivative in which each one of the two nucleosides may independently be an adenosine derivative or an uridine derivative, but preferably both nucleosides are identical. In a further configuration, the compound of the present invention is a mono-nucleoside 5'-di- or tri-phosphorothioate derivative in the form of a prodrug, wherein (i) one of the non- bridging oxygen atoms at position β of the diphosphorothioate, or at position γ of the triphosphorothioate, is replaced by a glucose moiety linked through the oxygen atom linked to its 1- or 6-position; or (ii) two of the non-bridging oxygen atoms at position β of the diphosphorothioate, or at position γ of the triphosphorothioate, are each replaced by a group of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH-(CHR ₁₃ )-C(0)-OR ₁₃ , wherein R ₁₂ is (C C ₄ )alkyl, and R ₁ each independently is (Ci-C ₄ )alkyl, (C ₆ -Cio)aryl or (C ₆ -C ₁ o)aryl-(C ₁ - C ₄ )alkyl. The common feature unifying all these compounds is the fact that at least one of the bridging oxygen atoms of the phosphorothioate, i.e., either or both the α,β- and β,γ- bridging-oxygen atoms, is replaced by a group selected from -NH- or -QRioRn)-, wherein R ₁₀ and Rn each independently is H or halogen, preferably by CH ₂ , CC1 ₂ or CF ₂ , and at least one, i.e., 1, 2, 3, 4, 5 or 6, of the non-bridging atoms or negatively-charged atoms of the phosphorothioate is either a sulfur atom (S) or a sulfur ion (S ).

[0043] As used herein, the term "halogen" includes fluoro, chloro, bromo, and iodo, and is preferably fluoro or chloro.

[0044] The term "hydrocarbyl" in any of the definitions of the different radicals Ri to R ₉ refers to a radical containing only carbon and hydrogen atoms that may be saturated or unsaturated, linear or branched, cyclic or acyclic, or aromatic, and includes (CrC^alkyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, (C ₃ -C ₁₀ )cycloalkyl, (C ₃ -C ₁₀ )cycloalkenyl, (C ₆ -C ₁₄ )aryl, (Ci-C ₈ )alkyl(C ₆ -Ci ₄ )aryl, and (C ₆ -Ci ₄ )aryl(Ci-C ₈ )alkyl. [0045] The term "C ₁ -C ₈ )alkyl" typically means a linear or branched hydrocarbon radical having 1-8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec -butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, n-octyl, and the like. Preferred are (C ₁ -C ₆ )alkyl groups, more preferably (C ₁ -C ₄ )alkyl groups, most preferably methyl and ethyl. The terms "(C ₂ -C ₈ )alkenyl" and "C ₂ -C ₈ )alkynyl" typically mean straight and branched hydrocarbon radicals having 2-8 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, 3-buten-l-yl, 2-ethenylbutyl, 3-octen-l-yl, and the like, and propynyl, 2-butyn-l-yl, 3-pentyn-l-yl, and the like. (C ₂ -C ₆ )alkenyl and (C ₂ -C ₆ )alkynyl radicals are preferred.

[0046] The term "(C3-C ₁ o)cycloalkyl" as used herein means a mono- or bicyclic saturated hydrocarbyl group having 3-10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1]octyl, bicyclo[2.2.1]heptyl, and the like, which may be substituted, e.g., with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N0 ₂ , -CN, -SCN, (Ci-C ₈ )alk l, -0-(C C ₈ )alkyl, -S-(Ci-C ₈ )alkyl, -NH ₂ , -NH-(C ₁ -C ₈ )alkyl, or -N-((C ₁ -C ₈ )alkyl) ₂ .

[0047] The term "(C ₆ -C ₁₄ )aryl" denotes an aromatic carbocyclic aromatic group having 6-14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bonf such as, but not limited to, phenyl, naphthyl, phenanthryl and biphenyl. Preferred are (C ₆ -Cio)aryl, more preferably phenyl. The aryl radical may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N0 ₂ , -CN, -SCN, (Ci-C ₈ )alkyl, -0-(Ci-C ₈ )alkyl, -S-(Ci-C ₈ )alkyl, -NH ₂ , -NH- (Ci-C ₈ )alkyl, or -N-((Ci-C ₈ )alkyl) ₂ . The term "ar(C ₁ -C ₈ )alkyl" denotes an arylalkyl radical such as benzyl and phenetyl.

[0048] The term "heteroaryl" refers to a radical derived from a mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1 or 2, heteroatoms selected from N, O or S. When the heteroaryl is a monocyclic ring, it is preferably a radical of a 5-6- membered ring such as, but not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1,2,3-triazinyl, 1,3,4-triazinyl, and 1,3,5-triazinyl. Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobenzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl, pyrido[l,2-a]pyrimidinyl and 1,3-benzodioxinyl. The heteroaryl ring may be substituted. It is to be understood that when a polycyclic heteroaromatic ring is substituted, the substitution may be in the heteroring or in the carbocyclic ring.

[0049] The term "heterocyclic ring" denotes a mono- or poly-cyclic non-aromatic ring of 4-12 atoms containing at least one carbon atom and one to three heteroatoms selected from sulfur, oxygen or nitrogen, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Preferred are 5- or 6-membered heterocyclic rings. Non-limiting examples of radicals -NR ₄ R ₅ and -NRgRg include amino, dimethylamino, diethylamino, ethylmethylamino, phenylmethyl- amino, pyrrolidino, piperidino, tetrahydropyridino, piperazino, ethylpiperazino, hydroxyethyl piperazino, morpholino, thiomorpholino, thiazolino, and the like.

[0050] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each independently is an adenosine residue of the formula la, wherein R is H, halogen, -O- hydrocarbyl, -S-hydrocarbyl, -NR ₄ R ₅ , heteroaryl, or hydrocarbyl; R ₄ and R5 each independently is H or hydrocarbyl, or R ₄ and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; said hydrocarbyl each independently is (C Cs kyl, (C ₂ -C ₈ )alkenyl, (C ₂ -C ₈ )alkynyl, or (C ₆ -C ₁₄ )aryl; and said heteroaryl is a 5 -6-membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S. In particular such embodiments, Ri is H, -O- hydrocarbyl, -S-hydrocarbyl, -NR ₄ R ₅ , or hydrocarbyl; R ₄ and R ₅ each independently is H or hydrocarbyl; and said hydrocarbyl each independently is (Ci-C ₄ )alkyl, preferably methyl or ethyl, (C ₂ -C ₄ )alkenyl, (C ₂ -C ₄ )alkynyl, or (C ₆ -C ₁ o)aryl, preferably phenyl. More particular such embodiments are those, wherein Ri is H, -O-hydrocarbyl, -S-hydrocarbyl, - NR ₁ R ₅ , or hydrocarbyl; R ₄ and R ₅ each independently is H or hydrocarbyl; and said hydrocarbyl each independently is methyl or ethyl.

[0051] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each independently is an adenosine residue of the formula la, wherein R ₂ and R ₃ each independently is H or hydrocarbyl; and said hydrocarbyl is (Ci-C ₄ )alkyl, preferably methyl or ethyl, (C ₂ -C ₄ )alkenyl, (C ₂ -C ₄ )alkynyl, or (C ₆ -Cio)aryl, preferably phenyl. [0052] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each is an adenosine residue of the formula la, wherein R _1; R ₂ and R ₃ are H, i.e., a purine nucleoside comprising a molecule of adenine linked to a moiety of either ribofuranose or a ribofuranose derivative via a β-Ncrglycosidic bond.

[0053] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each independently is an uridine residue of the formula lb, wherein R ₆ is H, halogen, -O- hydrocarbyl, -S-hydrocarbyl, -NRgRg, heteroaryl, or hydrocarbyl; R ₈ and R ₉ each independently is H or hydrocarbyl, or R ₈ and R ₉ together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1-2 further heteroatoms selected from N, O or S; said hydrocarbyl each independently is (C ₁ -Cg)alkyl, (C ₂ -Cg)alkenyl, (C ₂ -Cg)alkynyl, or (C ₆ -C ₁₄ )aryl; and said heteroaryl is a 5 -6-membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S. In particular such embodiments, R ₆ is H, -O- hydrocarbyl, -S-hydrocarbyl, -NRgRci, or hydrocarbyl; Rg and R9 each independently is H or hydrocarbyl; and said hydrocarbyl each independently is (Ci-C ₄ )alkyl, preferably methyl or ethyl, (C ₂ -C ₄ )alkenyl, (C ₂ -C ₄ )alkynyl, or (C ₆ -C ₁ o)aryl, preferably phenyl. More particular such embodiments are those, wherein R ₆ is H, -O-hydrocarbyl, -S-hydrocarbyl, - NRgRci, or hydrocarbyl; Rg and R9 each independently is H or hydrocarbyl; and said hydrocarbyl each independently is methyl or ethyl.

[0054] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each independently is an uridine residue of the formula lb, wherein R ₇ is O.

[0055] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Nu and Nu', if present, each is an uridine residue of the formula lb, wherein R ₆ is H; and R ₇ is O, i.e., a pyrimidine nucleoside comprising a molecule of uracil linked to a moiety of either ribofuranose or a ribofuranose derivative via a β-Nrglycosidic bond. [0056] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein Y' each independently is -OH; and Y is H or -OH.

[0057] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein W and W ₂ each independently is -O- or -CiRioRn)-, wherein R ₁₀ and Rn each independently is H, CI or F, preferably H.

[0058] In certain embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein n is 0, i.e., an adenosine- or uridine-5'-diphosphorothioate derivative, as well as a dinucleoside 5'-di-phosphorothioate derivative in which each one of the two nucleosides may independently be either an adenosine derivative or an uridine derivative in any one of the configurations defined above, but preferably both nucleosides are identical.

[0059] In other embodiments, the compound of the present invention is a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined above, or a diastereomer or mixture of diastereomers thereof, wherein n is 1, i.e., an adenosine- or uridine-5'-triphosphorothioate derivative, as well as a dinucleoside 5'-triphosphorothioate derivative in which each one of the two nucleosides may independently be either an adenosine derivative or an uridine derivative in any one of the configurations defined above, but preferably both nucleosides are identical.

[0060] In certain particular embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, wherein X is -O ^" , i.e., a mono-nucleoside 5'-di- or tri- phosphorothioate. In other particular embodiments, the compound of the invention is a dinucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, wherein X is Nu', i.e., a dinucleoside 5'-di- or tri-phosphorothioate. In further particular embodiments, the compound of the invention is a mononucleoside 5'- phosphorothioate of the general formula I as defined in any one of the configurations above, wherein X is a glucose moiety linked through the oxygen atom linked to its 1- opposition, i.e., a mononucleoside 5'-di- or tri-phosphorothioate in one particular form of a prodrug. According to the invention, at least one of the bridging oxygen atoms of the phosphorothioate in all these compounds is replaced by a group selected from -NH- or - C(RioRn)- as defined above, and one or more of the non-bridging atoms or negatively- charged atoms of the phosphorothioate is either a sulfur atom (S) or a sulfur ion (S ).

[0061] In certain more particular such embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined hereinabove, i.e., when X is -O ^" , Nu', or a glucose moiety, wherein n is 0; W ₂ is - QRioRn)-, preferably wherein R ₁₀ and Rn each is H, CI or F; and 1, 2, 3 or 4 of Z _1; Z , Z ₃ and Z' ₃ is S or -S ^" , i.e., (i) one of _\ and Z is -S ^" or S, and another of _\ and Z' _1; Z and Z' ₃ each independently is O or -O ^" ; or one of Z ₃ and Z' ₃ is -S ^" or S, and Z _1; Z' _1; and another of Z ₃ and Z' ₃ , each independently is O or -O ^" ; (ii) one of _\ and Z' _1; and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and the other of Z _1; Z' _1; Z and Z' each independently is O or -O ^" ; _\ and Z each independently is -S ^" or S, and Z and Z' each independently is O or -O ^" ; or Z ₃ and Z' ₃ each independently is -S ^" or S, and _\ and Z each independently is O or -O ^" ; (iii) Z _1; Z' _1; and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and another of Z ₃ and Z' is O or -O ^" ; or Z , Z , and one of _\ and Z' _1; each independently is -S ^" or S, and another of _\ and Z is O or -O ^" ; or (iv) Z _1; Z' _1; Z and Z' each independently is -S ^" or S.

[0062] Particular such compounds are those wherein Y and Y' are -OH; n is 0; W ₂ is - CH ₂ -, -CC1 ₂ - or -CF ₂ -; and Nu and Nu', if present, each is (i) an adenosine residue of the formula la, wherein R _1; R ₂ and R ₃ are H; or (ii) an uridine residue of the formula lb, wherein R ₆ is H; and R ₇ is O. Specific such compounds exemplified herein are those wherein (i) X is -O ^" ; Nu is an adenosine residue of the formula la, wherein R _1; R ₂ and R are H, or an uridine residue of the formula lb, wherein R ₆ is H, and R ₇ is O; Y and Y' are - OH; n is 0; W ₂ is -CH ₂ -; and Z Z , Z ₃ and Z' ₃ are -S ^" or S (herein also identified APCP- α,α',β,β'-tetra-S" and "UPCP-a,a',fi,fi'-tetra-S ", respectively); or (ii) X is Nu'; Nu and Nu' each is an adenosine residue of the formula la, wherein R _1; R ₂ and R are H, or an uridine residue of the formula lb, wherein R ₆ is H, and R ₇ is O; Y and Y' are -OH; n is 0; W ₂ is - CH ₂ -; and Z _u Z , Z ₃ and Z' ₃ are -S ^" or S (herein also identified "APCPA-a,a',fl,fl'-tetra-S" and "UPCPU-a,a',fi,fi'-tetra-S ", respectively).

[0063] In other more particular such embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined hereinabove, i.e., when X is -O ^" , Nu', or a glucose moiety, wherein n is 1; either one of Wi and W ₂ is -O- and another of Wi and W ₂ is -C(RioRn)-, or both Wi and W ₂ each independently is -C(RioRn)-, preferably wherein Ri ₀ and Rn each is H, CI or F; and 1, 2, 3, 4, 5 or 6 of Z _1; Z' _1; Z ₂ , Z' ₂ , Z ₃ and Z' ₃ is S or -S ^" , i.e., (i) one of X _\ and Z is -S ^" or S, and another of _\ and Z' _1; Z ₂ , Z' ₂ , Z ₃ and Z' ₃ each independently is O or -O ^" ; one of Z ₂ and Z' ₂ is -S ^" or S, and Z _1; Z' _1; another of Z ₂ and Z' ₂ , Z and Z' each independently is O or -O ^" ; or one of Z and Z' is -S ^" or S, and Z _1; Z' _1; Z ₂ , Z' ₂ , and another of Z and Z' , each independently is O or -O ^" ; (ii) one of _\ and Z' _1; and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and the other of Z _1; Z' _1; Z ₂ , Z' ₂ , and Z ₃ and Z' ₃ , each independently is O or -O ^" ; one of _\ and Z' _1; and one of Z and Z' , each independently is -S ^" or S, and the other of Z _1; Z'i, Z , Z' , and Z ₂ and Z' ₂ , each independently is O or -O ^" ; one of Z ₂ and Z' ₂ , and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and Z _1; Z' _1; and the other of Z ₂ , Z' ₂ , Z ₃ , Z' ₃ , each independently is O or -O ^" ; _\ and Z each independently is -S ^" or S, and Z ₂ , Z' ₂ , Z ₃ and Z' each independently is O or -O ^" ; Z ₂ and Z' ₂ each independently is -S ^" or S, and Z _1; Z'i, Z and Z' are O or -O ^" ; or Z and Z' each independently is -S ^" or S, and Z _1; Z' _1; Z ₂ and Z' ₂ are O or -O ^" ; (iii) one of _\ and Z' _1; one of Z ₂ and Z' ₂ , and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and the other of Z _1; Z' _1; Z ₂ , Z' ₂ , Z ₃ and Z' ₃ each independently is O or -O ^" ; _\ and Z' _1; and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and another of Z ₂ and Z' ₂ , Z and Z' each independently is O or -O ^" ; _\ and Z' _1; and one of Z and Z' , each independently is -S ^" or S, and Z ₂ , Z ₂ , and another of Z ₃ and Z' ₃ each independently is O or -O ^" ; Z ₂ and Z' ₂ , and one of _\ and Z' _1; each independently is -S ^" or S, and another of Zi and Z'i, Z ₃ and Z' ₃ each independently is O or -O ^" ; Z ₂ and Z' ₂ , and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and Z _1; Z' _1; and another of Z and Z' , each independently is O or -O ^" ; Z and Z' , and one of _\ and Z' _1; each independently is -S ^" or S, and another of Zi and Z'i, Z ₂ and Z' ₂ each independently is O or -O ^" ; or Z ₃ and Z' ₃ , and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and Z _1; Z' _1; and another of Z ₂ and Z' ₂ , each independently is O or -O ^" ; (iv) Z _1; Z' _1; one of Z ₂ and Z' ₂ , and one of Z and Z' , each independently is -S ^" or S, and the other of Z ₂ , Z' ₂ , Z and Z' each independently is O or -O ^" ; Z ₂ , Z' ₂ , one of _\ and Z'i, and one of Z ₃ and Z' ₃ , each independently is -S ^" or S, and the other of Z _1; Z' _1; Z ₃ and Z' ₃ each independently is O or -O ^" ; Z ₃ , Z' ₃ , one of _\ and Z' _1; and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and the other of Z _1; Z' _1; Z ₂ and Z' ₂ each independently is O or -O ^" ; Z _1; Z'i, Z ₂ and Z' ₂ each independently is -S ^" or S, and Z and Z' each independently is O or -O ^" ; Z _1; Z' _1; Z ₃ and Z' ₃ each independently is -S ^" or S, and Z ₂ and Z' ₂ each independently is O or -O ^" ; or Z ₂ , Z' ₂ , Z ₃ and Z' ₃ each independently is -S ^" or S, and _\ and Z each independently is O or -O ^" ; (v) Z _1; Z' _1; Z ₂ , Z' ₂ , and one of Z and Z' , each independently is -S ^" or S, and another of Z and Z' is O or -O ^" ; Z _1; Z' _1; Z , Z' , and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and another of Z ₂ and Z' ₂ is O or -O ^" ; or Z ₂ , Z' ₂ , Z ₃ , Z' ₃ , and one of _\ and Z' _{1 ;} each independently is -S ^" or S, and another of _\ and Z is O or -O ^" ; or (vi) Z _{1 ;} Z' _{1 ;} Z ₂ , Z' ₂ , Z ₃ and Z' ₃ each independently is -S ^" or S.

[0064] Particular such compounds are those wherein X is -O ^" , Nu', or a glucose moiety; Y and Y' are -OH; n is 1 ; either one of Wi and W ₂ is -O- and another of Wi and W ₂ is - CH ₂ -, -CCI ₂ - or -CF ₂ -, or both Wi and W ₂ are -CH ₂ -, -CC1 ₂ - or -CF ₂ -; and Nu and Nu', if present, each is (i) an adenosine residue of the formula la, wherein R _{1 ;} R ₂ and R are H; or (ii) an uridine residue of the formula lb, wherein R ₆ is H, and R ₇ is O. Specific such compounds exemplified herein are those wherein (i) X is -O ^" ; Nu is an adenosine residue of the formula la, wherein R _{1 ;} R ₂ and R ₃ are H; Y and Y' are -OH; n is 1 ; Wi is -CH ₂ -; W ₂ is -0-; and one of Z and Z' is -S ^" or S, and Zi, ΖΊ, Z ₂ , Z' ₂ , and another of Z and Z' , are O or -O ^" (herein also identified "APCPP- -S"); or (ii) X is -O ^" ; Nu is an adenosine residue of the formula la, wherein Ri, R ₂ and R ₃ are H; Y and Y' are -OH; n is 1 ; Wi is -0-; W ₂ is -CH ₂ -; and one of Zi and ΖΊ is -S ^" or S, and another of Zi and ΖΊ, Z ₂ , Z' ₂ , Z ₃ and Z' ₃ are O or -O ^" (herein also identified "APPCP-a-S"), or a prodrug of APCPP-y-S wherein X is a glucose moiety linked through the oxygen atom linked to its 1 -position; Nu is an adenosine residue of the formula la, wherein Ri, R ₂ and R ₃ are H; Y and Y' are -OH; n is 1 ; Wi is -CH ₂ -; W ₂ is -0-; and one of Z ₃ and Z' ₃ is -S ^" or S, and Zi, ΖΊ, Z ₂ , Z' ₂ , and another of Z ₃ and Z' ₃ , are O or -0 (herein also identified l-D-glucosyl-Py-APCPP-y-S").

[0065] In certain particular embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, wherein X is a group of the formula -0-CH ₂ -OC(0)-Ri ₂ or -NH- (CHR ₁₃ )-C(0)-OR ₁₃ ; R ₁₂ is (Ci-C ₄ )alkyl; and R ₁₃ each independently is (Ci-C ₄ )alkyl, (C ₆ - Cio)aryl or (C6-Cio)aryl-(Ci-C ₄ )alkyl, i.e., a mono- or dinucleoside 5'-phosphorothioate in one of two additional forms of a prodrug. According to the invention, at least one of the bridging oxygen atoms of the phosphorothioate in all these compounds is replaced by a group selected from -NH- or -C(RioRii)- as defined above, and one or more of the non- bridging atoms or negatively-charged atoms of the phosphorothioate is either a sulfur atom (S) or a sulfur ion (S ^~ ).

[0066] In certain more particular such embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined hereinabove, i.e., when X is a group of the formula -0-CH ₂ -OC(0)-R ₁₂ or -NH-(CHR ₁ )- C(0)-OR ₁₃ , wherein n is 0, W ₂ is -C(RioRn)-, preferably wherein R ₁₀ and Rn each is H, CI or F, and 1, 2 or 3 of Zi, ΖΊ and Z' ₃ is S or -S ^" , i.e., (i) one of Zi and ΖΊ is -S ^" or S, and another of Zi and ΖΊ, and Z' ₃ each independently is O or -O ^" ; or Z' ₃ is -S ^" or S, and Zi and Z each independently is O or -O ^" ; (ii) one of Z _\ and ΖΊ, and Z' , each independently is -S ^" or S, and the other of Z _\ and Z is O or -O ^" ; or Z _\ and Z each independently is -S ^" or S, and Z' ₃ is O or -O ^" ; and (iii) Zi, Z and Z' ₃ each independently is -S ^" or S.

[0067] In other more particular such embodiments, the compound of the present invention is a mononucleoside 5'-phosphorothioate of the general formula I as defined hereinabove, i.e., when X is group of the formula -0-CH ₂ -OC(0)-R ₁ 2 or -NH-(CHR ₁ )- C(0)-OR ₁₃ , wherein n is 1, either one of Wi and W ₂ is -O- and another of Wi and W ₂ is - C(RioRn)-, or both Wi and W ₂ each independently is -C(RioRii)-, preferably wherein Rio and Rn each is H, CI or F, and and 1, 2, 3, 4 or 5 of Z _1; ΖΊ, Z ₂ , Z' ₂ and Z' is S or -S ^" , i.e., (i) one of Zi and Z'i is -S ^" or S, and another of Zi and ΖΊ, Z ₂ , Z' ₂ and Z' each independently is O or -O ^" ; one of Z ₂ and Z' ₂ is -S ^" or S, and Zi, ΖΊ, another of Z ₂ and Z' ₂ and Z' ₃ each independently is O or -O ^" ; or Z' ₃ is -S ^" or S, and Zi, ΖΊ, Z ₂ and Z' ₂ each independently is O or -O ^" ; (ii) one of Zi and ΖΊ, and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and the other of Zi, ΖΊ, Z ₂ , Z' ₂ , and Z' , each independently is O or -O ^" ; one of Zi and Z'i, and Z' ₃ , each independently is -S ^" or S, and the other of Zi and ΖΊ, and Z ₂ and Z' ₂ , each independently is O or -O ^" ; one of Z ₂ and Z' ₂ , and Z' ₃ , each independently is -S ^" or S, and Zi, ΖΊ, and the other of Z ₂ and Z' ₂ , each independently is O or -O ^" ; Zi and ΖΊ each independently is -S ^" or S, and Z ₂ , Z' ₂ and Z' each independently is O or -O ^" ; or Z ₂ and Z' ₂ each independently is -S ^" or S, and Zi, ΖΊ and Z' each independently is O or -O ^" ; (iii) one of Zi and ΖΊ, one of Z ₂ and Z' ₂ , and Z' ₃ , each independently is -S ^" or S, and the other of Zi, Z'i, Z ₂ and Z' ₂ each independently is O or -O ^" ; Zi and ΖΊ, and one of Z ₂ and Z' ₂ , each independently is -S ^" or S, and another of Z ₂ and Z' ₂ , and Z' each independently is O or -O ^" ; Zi and ΖΊ, and Z' , each independently is -S ^" or S, and Z ₂ , Z' ₂ are O or -O ^" ; Z ₂ and Z' ₂ , and one of Zi and ΖΊ, each independently is -S ^" or S, and another of Zi and ΖΊ, and Z' ₃ each independently is O or -O ^" ; or Z ₂ and Z' ₂ , and Z' ₃ , each independently is -S ^" or S, and Zi and Z'i each independently is O or -O ^" ; (iv) Zi, ΖΊ, one of Z ₂ and Z' ₂ , and Z' , each independently is -S ^" or S, and the other of Z ₂ and Z' ₂ is O or -O ^" ; Z ₂ , Z' ₂ , one of Zi and ΖΊ, and Z' ₃ , each independently is -S ^" or S, and the other of Zi and ΖΊ is O or -O ^" ; or Zi, ΖΊ, Z ₂ and Z' ₂ each independently is -S ^" or S, and Z' ₃ is O or -O ^" ; or (v) Zi, ΖΊ, Z ₂ , Z' ₂ , and Z' ₃ each independently is -S ^" or S. [0068] The specific mononucleoside 5'-phosphorothioate of the general formula I exemplified in the specification are herein identified "APCP-a,a',P,P'-tetra-S", "UPCP- α,α',β,β'-tetra-S", APCPP-y-S , "APPCP-a-S" and l-D-glucosyl-Py-APCPP-y-S"; and the specific dinucleoside 5'-phosphorothioate of the general formula I exemplified are herein identified "APCPA-a,a',p,P'-tetra-S" and "UPCPU-a,a',p,P'-tetra-S". APCP- α,α',Ρ,Ρ'-tetra-S is also identified by the name adenosine-5'-tetrathiobisphosphonate; UPCP-a,a',P,P'-tetra-S is also identified by the name uridine-5'-tetrathiobisphosphonate; APCPP-y-S is also identified by the name adenosine 5'-[y-thio]-a,P-methylene triphosphate; APPCP-a-S is also identified by the name adenosine 5'-[α-ΐ1ιίο]-β,γ- methylene triphosphate; l-D-glucosyl-Py-APCPP-y-S is also identified by the name D- glucosyl-1 -adenosine 5'-[y-thio]-a,P-methylene triphosphate; APCPA-a,a',P,P'-tetra-S is also identified by the name di-adenosine-5',5"-tetrathiobisphosphonate; and UPCPU- α,α',Ρ,Ρ'-tetra-S is also identified by the name di-uridine-5',5"-tetrathiobisphosphonate. The chemical structures of all these compounds, as well as of other compounds mentioned in the specification, are depicted in Appendix A and Schemes 1-7 hereinafter.

[0069] The compounds of the general formula I may be synthesized according to any technology or procedure known in the art, e.g., as described in detail in the Examples section hereinafter.

[0070] The compounds of the general formula I may have one or more asymmetric centers, and may accordingly exist as pairs of diastereoisomers. In cases a pair of diastereoisomers exists, the separation and characterization of the different diastereomers may be accomplished using any technology known in the art, e.g., HPLC.

[0071] The compounds of the general formula I are in the form of pharmaceutically acceptable salts, wherein B represents a pharmaceutically acceptable cation.

[0072] In certain embodiments, the cation B is an inorganic cation of an alkali metal such as, but not limited to, Na ⁺ , K ⁺ and Li ⁺ .

[0073] In other embodiments, the cation B is ammonium (NH ₄ ⁺ ) or is an organic cation derived from an amine of the formula R ₄ N ⁺ , wherein each one of the Rs independently is selected from H, Q-C^, preferably C -C alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3- 7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine. [0074] In further embodiments, the cation B is a cationic lipid or a mixture of cationic lipids. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents. Neutral lipids include, but are not limited to, lecithins; phosphatidyl-ethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidyl-choline; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3β hydroxy- sterols. Other neutral lipids contemplated herein include phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidylglycerols.

[0075] Examples of cationic lipid compounds include, without being limited to, Lipofectin® (Life Technologies, Burlington, Ontario) (1 : 1 (w/w) formulation of the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine™ (Life Technologies, Burlington, Ontario) (3: 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl] -Ν,Ν-dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipid formulation), Metafectene (Biontex, Munich, Germany) (Polycationic lipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.) (ethanolic cationic lipids numbers 1 through 12: C ₅₂ Hio ₆ N ₆ 0 ₄ 4CF ₃ C0 ₂ H, C ₈₈ Hi ₇₈ N ₈ 0 ₄ S ₂ 4CF ₃ C0 ₂ H, C ₄₀ H ₈₄ NO ₃ P CF ₃ CO ₂ H, C ₅₀ H ₁₀₃ N ₇ O ₃ -4CF ₃ CO ₂ H,

C ₄₉ H ₁₀₂ N ₆ O ₃ -4CF ₃ CO ₂ H, C ₁₀ oH ₂ o ₆ N ₁₂ 0 ₄ S ₂ -8CF ₃ C0 ₂ H, Ci ₆₂ H ₃₃₀ N ₂₂ O ₉ 13CF ₃ CO ₂ H, C ₄₃ H ₈₈ N ₄ 0 ₂ 2CF ₃ C0 ₂ H, C ₄₃ H ₈₈ N ₄ 0 ₃ 2CF ₃ C0 ₂ H,

C ₄₁ H ₇₈ N0 ₈ P); Cytofectene (Bio-Rad, Hercules, Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER® (Gene Therapy Systems, San Diego, Calif.) (formulation of a neutral lipid (Dope) and a cationic lipid) and FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, Ind.) (Multi-component lipid based non-liposomal reagent). [0076] As shown in the Examples section hereinafter, mononucleoside 5'- phosphorothioate of the general formula I as defined above are capable of protecting primary cortical neuronal cells from damage caused by FeS0 ₄ and from Αβ ₄₂ insult. As particularly shown, APCPP-y-S protected primary cortical neuronal cells from damage caused by FeS0 ₄ with IC ₅₀ values of 40 nM as compared to ΑΤΡ-γ-S (IC ₅₀ 10 nM), and furthermore, protected primary neurons from Αβ ₄₂ insult with IC ₅₀ of 200 nM as compared to ΑΤΡ-γ-S (IC ₅₀ 800 nM). These results are consistent with our preliminary results in PC12 cells under oxidative stress (IC ₅₀ value obtained for APCPP-y-S was 0.16 μΜ). Interestingly, the neuroprotection activity of APCPP-y-S is due to neither P2YiR nor P2Y ₂ R activation, but may be due to P2Yn receptor activation (EC ₅₀ value of 1 μΜ). The studies described herein also show that APCPP-y-S is metabolically stable with no significant degradation after 3 h in mouse blood or brain and liver homogenates; and that upon IV administration to mice, 64% of the APCPP-y-S injected remained in blood after 90 min. APCPP-y-S was further found to be of minor toxicity and reduced PC12 cell viability by only 25% at 1000 μΜ.

[0077] It is therefore concluded that mononucleoside 5'-phosphorothioate of the general formula I as defined above, such as APCPP-y-S, are highly effective neuroprotectants protecting primary neurons from Αβ toxicity and oxidative stress, as well as highly effective agents for dissolution of Αβ aggregates, and effective chelators of Zn/Cu/Fe ions. As specifically shown with respect to APCPP-y-S, these compounds act not only as metal- ion chelators but also as radical scavengers protecting neurons also through the activation of P2Y receptors. It is expected that other mononucleoside 5'-phosphorothioate of the present invention, such as APCP-a,a'^^'-tetra-S and UPCP-a,a'^^'-tetra-S exemplified herein, as well as dinucleoside 5'-phosphorothioate of the present invention such as APCPA-a,a',p,P'-tetra-S and UPCPU-a,a',p,P'-tetra-S, will have a similar activity.

[0078] The activity of the mono- and di-nucleoside 5'-phosphorothioate of the present invention, and in particular that of APCPP-y-S, makes these compounds attractive candidates for treatment of neurodegenerative diseases or disorders such as Alzheimer's disease (AD), closely associated with the formation of Αβ aggregates, as well as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Creutzfeldt-Jakob disease. A protocol for testing the efficacy of APCPP-y-S in a mouse model of AD is provided in the Examples section. [0079] In another aspect, the present invention thus provides a pharmaceutical composition comprising a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, but excluding those compounds excluded by means of proviso, or a diastereomer or mixture of diastereomers thereof, and a pharmaceutically acceptable carrier or diluent. In particular embodiments, the pharmaceutical composition of the invention comprises, as an active agent, a mono- or dinucleoside 5'-phosphorothioate of the general formula I selected from those exemplified herein, preferably APCPP-y-S.

[0080] The pharmaceutical compositions provided by the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 ^th Ed., 1995. The compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the general formula I as defined above, into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation. The compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients. In one embodiment, the pharmaceutical composition of the present invention is formulated as nanoparticles.

[0081] The pharmaceutical compositions of the invention can be formulated for any suitable route of administration, but they are preferably formulated for parenteral, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intrapleural, intratracheal, subcutaneous, transdermal, inhalational, or oral administration. The dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.

[0082] The pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleagenous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limiting, water, Ringer's solution and isotonic sodium chloride solution.

[0083] The pharmaceutical compositions of the invention, when formulated for administration route other than parenteral administration, may be in a form suitable for oral use, e.g., as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, e.g., magnesium stearate, stearic acid, or talc. The tablets may be either uncoated or coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated using the techniques described in the US Patent Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for control release. The pharmaceutical composition of the invention may also be in the form of oil-in-water emulsion.

[0084] Pharmaceutical compositions according to the invention, when formulated for inhalation, may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like.

[0085] The pharmaceutical compositions of the invention may be formulated for controlled release of the active agent. Such compositions may be formulated as controlled- release matrix, e.g., as controlled-release matrix tablets in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. In other configurations, the compositions comprise the active agent formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters.

[0086] Another contemplated formulation is a depot system based on a biodegradable polymer, wherein as the polymer degrades, the active agent is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLG).

[0087] The pharmaceutical composition of the present invention may be used for treatment of a neurodegenerative disease or disorder. Particular such neurodegenerative diseases or disorders include, without limiting, AD, Parkinson's disease, Huntington's disease, ALS, and Creutzfeldt-Jakob disease. In a particular embodiment, the pharmaceutical composition of the invention is used for treatment of AD.

[0088] In a further aspect, the present invention thus relates to a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, but excluding those compounds excluded by means of proviso, or a diastereomer or mixture of diastereomers thereof, for use in treatment of a neurodegenerative disease or disorder.

[0089] In still a further aspect, the presenty invention relates to use of a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, but excluding those compounds excluded by means of proviso, or a diastereomer or mixture of diastereomers thereof, for the preparation of a pharmaceutical composition for treatment of a neurodegenerative disease or disorder.

[0090] In yet another aspect, the present invention relates to a method for treatment of a neurodegenerative disease or disorder in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a mono- or dinucleoside 5'-phosphorothioate of the general formula I as defined in any one of the configurations above, but excluding those compounds excluded by means of proviso, or a diastereomer or mixture of diastereomers thereof.

[0091] The invention will now be illustrated by the following non-limiting Examples. EXAMPLES

Experimental

Materials and Methods

[0092] Reactions were performed in oven dried flasks under Ar atmosphere. Tetrakis(acetonitrile)-copper(I) hexafluorophosphate (Cu(CH ₃ -CN) ₄ PF ₆ ), BCA disodium salt, trisodiumthiophosphate, DMPO, and sodium triphosphate were purchased from Sigma-Aldrich Chemical Co. DBU and 3-hydroxypropionitrile were purchased from Sigma-Aldrich and distilled under reduced pressure before use. Clioquinol was purchased from Fisher scientific. Adenosine 5'-[y-thio]-triphosphate, adenosine 5'-[β- thio]diphosphate, guanosine 5'-[P-thio]diphosphate, and guanosine 5'-[y-thio]triphosphate were synthesized according to literature (Kowalska et ah, 2007). Deuterated solvents - D ₂ 0, DMSO-d ₆ , Tris-dn, NaOD, and DC1 - were purchased from Cambridge Isotope Laboratories, Inc. CHCI ₃ was distilled over P ₂ 0 ₅ .

[0093] Cu(CH ₃ CN) ₄ PF ₆ was purified before use by dissolving the salt in acetonitrile (HPLC grade) and filtering the insoluble Cu ²⁺ salt by a nylon syringe 0.45 μιη filter. The filtrate was deaerated with argon stream. The concentration of the Cu ⁺ salt was determined by UV spectroscopy by the addition of the specific Cu ⁺ indicator, BCA (ε ₅₆₂ = 7700 M ^-1 ) (Brenner and Harris, 1995). Crude Αβ ₂ § was purchased from Sigma-Aldrich and was purified by HPLC over a Chromolith performance RP-18E column, 100x4.6 mm, applying alinear gradient of 13% to 45% B in 30 min (A is 0.1% TFA in H ₂ 0 and B is 3: 1 acetonitrile: A). Solution of the purified peptide was filtered over a PVDF 0.45 μιη filter and the peptide purity was determined by 1H-NMR, RP-HPLC, and MALDI-TOF MS. The concentration of soluble Αβ ₂ 8/Αβ ₄ ο was based on UV measurements using the extinction coefficient of tyrosine residue (ε = 1280 M ^-1 at 280 nm). >95% Pure Αβ ₄₀ / ₄₂ TFA salt was purchased from GL-Biochem (Shanghai) and kept at -20°C. Αβ ₄₀ was weighted and dissolved in 10 mM NaOH and then freeze-dried (Fezoui et ah, 2000). The freeze-dried peptide was dissolved in PBS (10 mM; 2.7 mM potassium chloride and 137 mM sodium chloride), and the concentration of the mixture was determined by UV. Αβ ₄₂ was weighted and dissolved in 10 mM NaOH sonicated for 3 min and then freeze-dried.

[0094] The concentration of the spin trap, DMPO, was determined by UV spectroscopy (ε ₂₂ 8 nm = 8000 M ^-1 ) after purification by active charcoal. Purified DMPO was stored at - 18°C subsequent to deaeration with argon stream. Analysis of OH radicals produced in Cu ⁺ and Fe ⁺² -H202/tested compound systems were performed by solution ESR spectroscopy using a Bruker ER lOOd X-band spectrophotometer.

[0095] UV spectra were measured using a Shimadzu UV-VIS2401pc instrument. 1H and ³¹ P-NMR spectra were measured using a Bruker AC-200 (200 and 81 MHz for ¹ H and ³¹ P NMR, respectively), DMX-600 (600 and 243 MHz for 1H and ³¹ P NMR, respectively), or Avance III-700 (700 MHz for 1H NMR) spectrometers. DLS measurements were performed using a Malvern Zetasizer Nano ZS Instrument (Worcestershire, UK) at 25°C. TEM images were obtained by Tecnai G2 microscope, FEI Co (Hillsboro, OR, USA).

[0096] Flash chromatography (silica-gel and C ₁₈ reverse phase) was done using a

Biotage SP1 instrument. 1 H, 13 C, and 31 P-NMR spectra were measured using Bruker AC- 200 (200, 50 and 81 MHz for 1H, ¹³ C, and ³¹ P NMR), and Bruker DMX-600 (600, 150 and

243 MHz for 1 H, 13 C, and 31 P-NMR) machines. Mass spectra analyses were performed on an ESI Q-TOF micro instrument (Waters, UK) and a high resolution MS-MALDI-TOF spectra with autoflex TOF/TOF instrument (Bruker, Germany). Purification of the nucleotides was achieved on a liquid chromatography (LC) (Isco UA-6) system with a Sephadex DEAE-A25 column, which was swelled in 1 M NaHC0 ₃ in the cold for 1 day.

Titration of Afi ₂ s-Cu ⁺ complex by various chelators monitored by ¹ H-NMR

[0097] Stock solutions of 8 mM nucleotides were prepared in D ₂ 0 and pD was adjusted to 7 by DC1 or NaOD. Clioquinol was dissolved in DMSO-d ₆ (80 mM). Stock solutions were deaerated by a stream of Ar.

[0098] Pure Αβ ₂₈ TFA salt >95 (1.3 mg, 5.5xl0 ^"4 mmol) was dissolved in D ₂ 0 and freeze-dried for two times to exchange H ₂ 0 molecules with D ₂ 0. The dry substance was dissolved in 10 mM Tris-dn (400 μΐ) to obtain 1 mM solution at pD 7, pH adjustment was achieved with NaOD or DC1. This sample was transferred via a syringe to an argon flashed NMR tube, covered with a rubber septum. 4 mM Cu ⁺ (0.25 eq, 25 μΐ) stock solution was added to the Αβ ₂₈ solution until the ratio of Αβ ₂₈ -Οι ⁺ reached 1: 1 and the mixture became cloudy. The addition of Cu ⁺ solution to Αβ ₂₈ was monitored by 1H-NMR spectra 700 MHz (96 scans). The final Αβ ₂₈ -Οι ⁺ concentration was 0.8 mM in 500 μΐ then, 50 μΐ, 1 eq of the tested nucleotide solution was added each time via syringe. At the end of the titration, the acetonitrile concentration was 17.2% (v/v). ¹ H/ ³¹ P-NMR monitored Cu ⁺ titrations ofADP-fi-S

[0099] A solution of ΑϋΡ-β-S (9 mM, 400 μΐ, pD 8.2 in D ₂ 0) was injected to an Ar purged NMR tube and 1 H/ 31 P-NMR spectra were measured. Then, a solution of 6 mM Cu(CH ₃ CN) ₄ PF ₆ in CD ₃ CN was added. After each addition ¹ H/ ³¹ P-NMR spectra were measured. Overall, 80 μΐ of Cu ⁺ solution was added to the NMR tube (0.87 eq).

Determination of free thiol in thiophosphate and GDP-fi-S with Ellman 's reagent monitored by UV-Vis

[00100] A solution of 1 mM Cu(CH ₃ CN) ₄ PF ₆ (200 μΐ) was added to Αβ ₂₈ (0.118 mM, 1690 μΐ) in 1 mM Tris buffer (pH 7.4). After 30 min 10 mM thiophosphate or GDP^-S (100 μΐ) was added. After an additional 1 h, 10 mM Ellman 's reagent (10 μΐ) in methanol was added to give a total volume of 2 ml. The final concentrations of reaction compounds were: 0.1 mM Cu(CH ₃ CN) ₄ PF ₆ , 0.1 mM Αβ ₂₈ , 0.5 mM thiophosphate or GDP^-S and, 0.05 mM Ellman' s reagent. Oxidation of thiophosphate compounds was monitored by UV spectroscopy (at the wavelength range of 275-500 nm). The control solution contained only the thiophosphate compound and Ellman' s reagent in Tris buffer.

DLS measurements

[00101] PBS buffer, Αβ ₄₀ , chelator, Zn(N0 ₃ ) ₂ , and Cu(N0 ₃ ) ₂ solutions were filtered through a 0.45 μΜ PVDF syringe filter. 496 μΜ Αβ ₄₀ solution in 10 mM PBS (pH 7.4) and 2 mM Zn(N0 ₃ ) ₂ solution were mixed to obtain 200 μΜ Αβ ₄ ο-Ζη ²⁺ (1: 1 ratio) of a cloudy solution. Similarly, 2 mM Cu(N0 ₃ ) ₂ solution was added to 496 μΜ Αβ ₄₀ in 10 mM PBS (pH 6.6). Αβ ₄₀ -Ζη ²⁺ /Οι ²⁺ (10 μΐ) solution was transferred to Eppendorf tubes containing 10 mM PBS buffer (64 or 67 μΐ) followed by the addition of 3 and 6 eq of 2 mM nucleotide (6 or 3 μΐ) solution and incubation for 45 min at RT. The resulting mixture was incubated for another 30 min at RT and DLS data were then collected in a 70 μΐ disposable cuvette. The final sample concentrations were 25 μΜ Αβ ₄₀ , 25 μΜ Zn ²⁺ or Cu ²⁺ , and 150 or 75 μΜ nucleotide.

TEM measurements

[00102] TEM sample was prepared from 200 μΜ Αβ-Μ ²⁺ 1: 1 solution (pH 7.4 or 6.6 for Αβ ₄₀ -Ζη ²⁺ or Αβ ₄₀ -Οι ²⁺ , respectively) incubated in a rotary shaker for 9 days at RT. A cloudy mixture with massive sediments was obtained. TEM samples were prepared by diluting the stock solutions with PBS to a concentration of 25 μΜ with or without APCPP- γ-S. In this way four samples were obtained: (i) 25 μΜ Αβ ₄₀ -Οι ²⁺ ; (ii) 25 μΜ Αβ^-Cu ^2"1" containing 150 μΜ APCPP-y-S; (iii) 25 μΜ Αβ ₄₀ -Ζη ⁺ ; and (iv) 25 μΜ Αβ ₄₀ -Ζη ⁺ containing 150 μηι APCPP-y-S. The mixtures were incubated in a rotary shaker for 7 h and after vortexing, a sample was transferred to a gold grid, for Αβ ₄₀ -Οι ²⁺ , or a copper grid for Αβ ₄₀ -Ζη ²⁺ . The wet grid was left to dry at RT overnight.

Disaggregation of Αβ ₄₂ -ϋιι ²⁺ /Ζη ²⁺ complexes by nucleotides monitored by a turbidity assay

[00103] Αβ ₄₂ was weighted and dissolved in 10 mM NaOH, sonicated for 3 min and then freeze-dried. The freeze-dried Αβ ₄₂ was dissolved in 50 mM Tris-HCl (pH 7.4). The mixture was split and the pH of one of the samples was adjusted to 7.4 and the second sample to 6.6 by addition of 200 μΜ HC1. Two 300 μΜ Αβ ₄₂ mixtures were obtained. From these mixtures, controls and Αβ ₄₂ -Μ ²⁺ aggregates were prepared by the addition of 1 mM Zn(N0 ₃ ) ₂ or Cu(N0 ₃ ) ₂ in DDW: 1. 200 μΜ Αβ42 (pH 7.4); 2. 200 μΜ Αβ ₄₂ (pH 6.6); 3.4x200 μΜ Αβ ₄₂ -Ζη ²⁺ (pH 7.4); 4.4x200 μΜ Αβ ₄₂ -Οι ²⁺ (ρΗ 6.6). The mixtures were left at RT for 2 h to form aggregates. 1 mM EDTA, ΑϋΡ-β-S, and APCPP-y-S in DDW were added to Αβ ₄₂ -Ζη ²⁺ /Οι ²⁺ aggregates, then the mixtures were diluted with buffer to give the following mixtures: (i) 50 μΜ Αβ ₄₂ -Οι ²⁺ /Ζη ²⁺ ; (ii) 50 μΜ containing 150 μΜ chelator (3 eq or) 300 μΜ chelator (6 eq) final volume 100 μΐ. The mixtures left at RT for 30 min before measurements, done in duplicate. 80 μΐ sample was taken for measurements in a quartz cuvette.

Titration of Afi ₄₀ -Cu ⁺ complex by nucleotides monitored by ¹ H-NMR

[00104] 1H-NMR spectrum of 0.25 mM Αβ ₄₀ (concentration determined by UV) in 10 mM TRIS-dn (500 μΐ, pD 11) was measured at 278 K. The pD was adjusted to 7.8 by the addition of 0.1 N DC1 (33 μΐ), and ¹ H-NMR spectrum was measured (700 MHz, 80 scans), as well as after each of the following additions: (A) 8.3 mM Cu ⁺ (15 μΐ, 1 eq); (B) 6 mM compound 7 (21 μΐ, 1 eq); (C) 6 mM compound 7 (42 μί, 3 eq); (D) of 6 mM APCPP-y-S (62.4 μΐ, 6 eq); (E) of 6 mM APCPP-y-S (62.4 μΐ, 9 eq). At the end of the titration, the acetonitrile concentration was 2% (v/v) in 735.8 μΐ, pD 8.4.

ESR OH radical assay

[00105] ESR settings for OH radicals detection were as follows: microwave frequency, 9.76 GHz; modulation frequency, 100 KHz; microwave power, 6.35 mW; modulation amplitude, 1.2 G; time constant, 655.36 ms; sweep time 83.89 s; and receiver gain 2xl0 ⁵ in experiments with Cu ⁺ and Fe ²⁺ . [00106] 1 mM Cu(CH ₃ CN) ₄ PF ₆ in acetonitrile (10 μΐ) or 1 Mm FeS0 ₄ (10 μΐ) were added to 5-500 μΜ tested compound (10 μΐ) solutions. All final solutions of Cu(CH ₃ CN) ₄ PF ₆ contained 10% v/v acetonitrile. Afterwards, 1 mM Tris buffer, pH 7.4, (10 μΐ) was added to the mixture. After mixing for two seconds, 100 mM DMPO (10 μΐ) were quickly added followed by the addition of 100 mM Η ₂ 0 ₂ (10 μΐ). The final sample pH values for the Cu ⁺ and Fe ²⁺ systems ranges between 7.2-7.4. Each ESR measurement was performed 150 sec after the addition of H ₂ 0 ₂ . All experiments were performed at RT, in a final volume of 100 μΐ.

Evaluation of the resistance of particular analogues to hydrolysis by NPP1,3

[00107] The percentage of hydrolysis of the analogues tested by human NPP1,3 was evaluated as follows: 67 μg or 115 μg of human NPP1 or NPP3 extract, respectively, was added to 0.579 ml the incubation mixture (1 mM CaCl ₂ , 200 mM NaCl, 10 mM KC1 and 100 mM Tris, pH 8.5) and pre-incubated at 37°C for 3 min. Reaction was initiated by the addition of 0.015 ml of 4 mM analogue; and was stopped after 30 min or 1 h for NPP1 or NPP3, respectively, by adding 0.350 ml ice-cold 1 M perchloric acid. These samples were centrifuged for 1 min at 10,000 g. Supernatants were neutralized with 140 μΐ 2 M KOH in 4°C and centrifuged for 1 min at 10,000 g. The reaction mixture was filtered and freeze- dried.

[00108] Each sample was dissolved in 200 μΐ HPLC-grade water and 20 μΐ sample was injected onto an analytical HPLC column (Gemini analytical column (5μ C-18 557 11 OA; 150 mm x 4.60 mm)), using isocratic elution with 85%-97% 100 mM TEAA (pH 7) and 15%-3% AcN, flow rate 1 ml/min. The percentage of the buffer and AcN depended on the chemical structure of the substrate.

[00109] The hydrolysis rates of all analogues by NPP1 or NPP3 were determined by measuring the change in the integration of the HPLC peaks for each analogue over time vs. control. The percentage of compound degradation was calculated versus control, to take into consideration the degradation of the compounds due to the addition of acid to stop the enzymatic reaction. Therefore, each of the samples was compared to a control which was transferred to acid, but to which no enzyme was added. The percentage of degradation was calculated from the area under the curve of the nucleoside monophosphate peak, after subtraction of the control, which is the amount of the nucleoside monophosphate peak formed due to chemical acidic hydrolysis. Evaluation of the resistance of particular analogues to hydrolysis by NTPDasel,2,3,8

[00110] The percentage of hydrolysis of the analogues tested by human NTPDasel,2,3,8 was evaluated as follows: 2.8 μg or 4.3 μg of human NTPasel or NTPDase2 extract, respectively, was added to 0.579 ml the incubation mixture (10 mM CaCl ₂ and 160 mM Tris, pH 7.4) and pre-incubated at 37°C for 3 min. The reaction was initiated by the addition of 0.012 ml of 4.24 mM analogue solution,; and was stopped after 1 h for NTPasel, 2,3, 8, by adding 0.350 ml ice-cold 1 M perchloric acid. These samples were centrifuged for 1 min at 10,000 g. Supernatants were neutralized with 140 μΐ 2 M KOH in 4°C and centrifuged for 1 min at 10,000 g. The reaction mixture was filtered and freeze- dried.

[00111] Each sample was dissolved in 200 μΐ HPLC-grade water and a 20 μΐ sample was injected to an analytical HPLC column (Gemini analytical column (5μ C-18 557 11 OA; 150 mm x 4.60 mm)), and eluted using isocratic elution with 78 -97 100 mM TEAA (pH 7) and 22 -3 AcN, flow rate 1 ml/min. The percentage of the buffer and AcN depended on the chemical structure of the substrate.

[00112] The hydrolysis rates of all analogues by NTPDasel,2,3,8 were determined by measuring the change in the integration of the HPLC peaks for each analogue over time vs. control. The percentage of compound degradation was calculated vs. control, to take into consideration the degradation of the compounds due to the addition of acid to stop the enzymatic reaction. Therefore, each of the samples was compared to a control which was transferred to acid, but to which no enzyme was added. The percentage of degradation was calculated from the area under the curve of the nucleoside monophosphate peak, after subtraction of the control, which is the amount of the nucleoside monophosphate peak formed due to chemical acidic hydrolysis.

Inhibition of NTPDase activity assays

[00113] Activity was measured as previously described (Kukulski et ah, 2005) in 0.2 ml of incubation medium Tris-Ringer buffer (in mM, 120 NaCl, 5 KC1, 2.5 CaCl ₂ , 1.2 MgS0 ₄ , 25 NaHC0 ₃ , 5 glucose, 80 Tris, pH 7.3) at 37°C with or without the analogue tested (final concentration 100 μΜ), and with or without 100 μΜ ATP (for NTPDases) or AMP (for ecto-5' -nucleotidase) as a substrates. The analogues were added alone when tested as potential substrate, and with ATP when tested for their effect on nucleotide hydrolysis. NTPDases protein extracts were added to the incubation mixture and pre- incubated at 37°C for 3 min. The reaction was initiated by the addition of substrate (ATP and/or the analogue tested); and was stopped after 15 min with 50 μΐ of malachite green reagent. The released inorganic phosphate (Pi) was measured at 630 nm according to Baykov et al. (1988).

Inhibition ofNPP activity assays

[00114] Evaluation of the effect of adenosine-5'-tetrathio bisphosphonate, di-adenosine 5',5"-tetrathiobisphosphonate, and ΑϋΡ-β-S on human NPPl and 3 activity was carried out either with pnp-TMP or ATP as the substrate (Belli and Goding, 1994). Pnp-TMP hydrolyses were carried out at 37°C in 0.2 ml of the following incubation mixture: in mM, 1 CaCl ₂ , 130 NaCl, 5 KC1 and 50 Tris, pH 8.5, with or without the analogue tested and/or substrates. Substrates and analogues were all used at the final concentration of 100 μΜ. Recombinant human NPPl or NPP3 cell lysates were added to the incubation mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by the addition of the substrate. For pnp-TMP hydrolysis, the production of para-nitro-phenol was measured at 310 nm, 15 min after the initiation of the reaction.

[00115] Evaluation of the activity of human NPPl and NPP3 with ATP (Sigma-Aldrich, Oakville, ON, Canada) and each one of the analogues tested was carried out at 37 °C in 0.2 ml of the following mixture: (in mM) 1 CaCl ₂ , 140 NaCl, 5 KC1, and 50 Tris, pH 8.5; (Sigma-Aldrich, Oakville, ON, Canada). Human NPPl or NPP3 extract was added to the reaction mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by addition of ATP or one of the analogues at a final concentration of 100 μΜ; and was stopped after 20 min by transferring a 0.1 ml aliquot of the reaction mixture to 0.125 ml ice-cold 1 M perchloric acid (Fisher Scientific, Ottawa, ON, Canada). The samples were centrifuged for 5 min at 13,000 x g. Supematants were neutralized with 1 M KOH (Fisher Scientific, Ottawa, ON, Canada) at 4°C and centrifuged for 5 min at 13,000 x g. An aliquot of 20 ml was separated by reverse-phase HPLC to evaluate the degradation of ATP and the the level of the analogue tested using a SUPELCOSIL™ LC-18-T column (15 cm x 4.6 mm; 3 mm Supelco; Bellefonte, Pennsylvania, USA) with a mobile phase composed of 25 mM TBA, 5 mM EDTA, 100 mM KH ₂ P0 ₄ /K ₂ HP0 ₄ , pH 7.0 and 2% methanol at a flow rate of 1 ml/min. Example 1. Synthesis of adenosine 5'-[y-thio]-a,P-methylene triphosphate, APCPP-γ- S

[00116] As depicted in Scheme 1, CDI (143 mg, 0.88 mmol) was added at RT to a solution of ADP-a,P-methylene (75 mg, 0.17 mmol) in dry DMF (2 ml) in a flamed-dried, nitrogen-flushed two-necked round bottom flask and stirred for 3 h. TLC on a silica gel plate (isopropanol:NH ₄ 0H:H ₂ 0 11:2:7) indicated the disappearance of the starting material and the formation of a less polar product. Dry MeOH (28 μΐ) was added and the reaction was stirred for 8 min, ZnCl ₂ (354 mg, 2.65 mmol) was added followed by thiophosphate (Bu ₃ NH ⁺ ) ₂ salt and tri-n-octyl amine and ADP-a,P-methylene trioctylammonium salt (190 mg, 1.06 mmol) in dry DMF (1 ml). The reaction was stirred for 3 h, and EDTA (1.18 g, 3.17 mmol) in distilled water (15 ml) was then added to the solution at RT. After a few minutes 1 M TEAB was added until the pH of the solution changed to pH~8. The colorless clear solution was freeze-dried overnight. The resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.4 M NH ₄ HCO ₃ ; total volume 700 ml). The relevant fractions were collected, freeze-dried, and excess NH ₄ HCO ₃ was removed by repeated freeze-drying with deionized water to yield the product as a yellow powder. Analogue APCPP-y-S was separated on a semipreparative reverse phase Gemini 5u column and isocratic elution with 96:4 (TEAA buffer pH 7:CH ₃ CN) over 20 min at a flow rate of 5 ml/min. Retention time: 9.0 min (20 mg, 19%). Finally, the purified analogue was passed through a Sephadex-CM C-25 Na ⁺ form column to exchange triethylammonium ions for Na ⁺ . APCPP-Y-S TEAA salt: 1H-NMR: 8.58 (s, H8), 8.26 (s, H2), 6.09 (J=5.8 Hz, HI'), 4.90 (m, H2'), 4.56 (m, H3'), 4.36 (m, H4'), 4.20 (m, H5'), 3.20 (m, Et N), 2.48 (t, J=20 Hz, CH ₂ ), 2.00 (s, CH ₃ C0 ₂ H), 1.30 ppm (m) Et N. ³¹ P-NMR: 39.02 (d, J=32 Hz, Ργ), 18.15 (d, J=9 Hz, Pa), 6.88 (dd, J=9 Hz, J=32 Hz, Ρβ) ppm. MS-ES m/z: 519 (M-H)-. HRMS-FAB (negative) m/z: calculated for CnHnNsOnPsS ^2" : 519.9853; found: 519.982.

Example 2. Synthesis of adenosine 5'-[a-thio]-P,y-methylene triphosphate, APPCP-a- S

[00117] As depicted in Scheme 2, a freshly prepared solution of 2-chloro-4H- 1,3,2- benzodioxaphosphorin-4-one (78 mg, 0.38 mmol) in anhydrous DMF (0.9 ml) was added via syringe to a solution of 2',3'-methoxymethylidene adenosine (100 mg, 0.323 mmol) and anhydrous pyridine (170 μΐ) in 0.9 ml of anhydrous DMF at 0°C under nitrogen. After stirring at RT for 1 h, bis(tributylammonium)methylenediphosphonate (62 mg, 0.35 mmol) in anhydrous DMF (1.5 ml) followed by diisopropylethylamine (600 μΐ) was added. The reaction mixture was stirred at RT for 1 h, then sulfur (21 mg, 0.64 mmol) was added at 0°C, the color changed to yellow, then after 0.5 h it changed to brown and then to red. After stirring at RT for 1 h, the mixture was cooled in ice, treated with lithium sulfide (74 mg, 1.61 mmol), and stirred for 1.5 h at RT, the color changed to blue. Water (7 ml) was added, the color changed to yellow. The resulting mixture was extracted with diethyl ether (3x10 ml). The aqueous phase was freeze-dried in the lyophilizer. The resulting residue was separated on an activated Sephadex DEAE-A25 column (0-0.4 M NH ₄ HCO ₃ ; total volume 700 ml). The relevant fractions were collected, freeze-dried, and excess NH ₄ HCO ₃ was removed by repeated freeze-drying with deionized water to yield the product as a yellow powder. The separation of APPCP-a-S diastereomers: APPCP-a-S isomer A and APPCP-a-S isomer B, was accomplished using a semipreparative reverse phase Gemini 5u column and with a gradient from 97:3 to 92:8 B:A (TEAA buffer pH 7:CH ₃ CN) over 20 min at a flow rate of 5 ml/min. Retention time: 8.8 min (AMP thiophosphate, 10 mg, 1%), 15.4 min (diastereomer A, 40 mg, 4%).

[00118] The separation of APPCP-a-S diastereomers: APPCP-a-S isomer A and APPCP- a-S isomer B was accomplished using a analytical reverse phase column with a gradient from 98:2 to 97:3 B:A (TEAA buffer: CH ₃ CN with 0.01% MgCl ₂ ) over 20 min at a flow rate of 1.2 ml/min. Retention time: 13.8 min (isomer A, 20 mg, 7%), 21.0 min (isomer B, 20 mg, 5%).

[00119] The separation APPCP-a-S isomer B diastereomer was accomplished using a analytical reverse phase column with a gradient from 98:2 to 96:4 B:A (TEAA buffer: CH ₃ CN with 0.01% MgCl ₂ ) over 20 min at a flow rate of 1 ml/min. Retention time: t _R 6.0 min (AMP thiophosphate), 18.0 min (isomer B, 30 mg, 7%). Finally, the purified diasteroisomers were passed through a Sephadex-CM C-25 Na ⁺ form column to exchange triethylammonium ions for Na ⁺ .

[00120] APPCP-a-S isomer B: ¹ H-NMR(D ₂ 0; 200 MHz): δ 8.67 (s,H8), 8.25 (s, H2), 6.15 (J=6 Hz, HI'), 5.00 (m, H2'), 4.59 (m, H3'), 4.41 (m, H4'), 4.28 (m, H5'), 2.31 (t, J=21 Hz, CH2) ppm. ³¹ P-NMR (D20; 243 MHz): δ 43.16 (d, J=32.8 Hz, Pa), 13.17 (br s, Ργ), 11.87 (br d J=27 Hz, Ρβ) ppm. MS-ES m/z: 542 (M+Na).

[00121] APPCP-a-S isomer A: ¹ H-NMR(D ₂ 0; 200 MHz): δ 8.62 (s,H8), 8.27 (s, H2), 6.15 (J=6 Hz, HI'), 5.00 (m, H2'), 4.60 (m, H3'), 4.42 (m, H4'), 4.28 (m, H5'), 2.28 (t, J=20 Hz, CH2) ppm. ³¹ P-NMR (D ₂ 0; 43 MHz): δ 42.90 (d, J=33.5 Hz, Pa), 13.00 (br m, Ργ and Ρβ) ppm.

[00122] APPCP-a-S isomer B: ¹ H-NMR(D ₂ 0; 200 MHz): δ 8.70 (s,H8), 8.27 (s, H2), 6.16 (J=6 Hz, HI'), 5.00 (m, H2'), 4.70 (m, H3'), 4.43 (m, H4'), 4.28 (m, H5'), 2.58 (t, J=21 Hz, CH2), 2.23 (t, J=20 Hz, CH ₂ ) ppm. ³¹ P-NMR (D20; 243 MHz): δ 43.20 (d, J=32.0 Hz, Pa), 13.32 (br s, Ργ), 8.48 (br d J=32.0 Hz, Ρβ) ppm.

[00123] APPCP-a-S isomer B TEAA salt: ¹ H-NMR(D ₂ 0; 200 MHz): δ 8.62 (s,H8), 8.26 (s, H2), 6.15 (J=6.4 Hz, ΗΓ), 5.00 (m, H2'), 4.60 (m, H3'), 4.43 (m, H4'), 4.29 (m, H5'), 3.22 (m) Et ₃ N, 2.58 (t, J=21 Hz, CH ₂ ), 2.30 (t, J=21 Hz, CH2), 1.30 (m) Et N ppm. ³¹ P- NMR (D ₂ 0; 243 MHz): δ 42.95 (d, J=32.0 Hz, Pa), 15.11 (br s, Ργ), 8.51 (m, J=8.42 Hz, J=32.0 Hz, Ρβ) ppm.

Example 3. Titration of Ap ₂₈ -Cu ⁺ complex by various phosphate-based chelators monitored by ¹ H-NMR

[00124] 1H-NMR is a sensitive analytical tool that can be utilized, by means of signal width and peak shifts, to observe Αβ-metal-ion coordination, Αβ precipitation, and Αβ resolvation. Since Αβ ₄₀ can readily form aggregates at physiological pH (Atwood et ah, 1998), we first studied Αβ ₂₈ , as a more soluble fragment of Αβ ₄ ο, to evaluate the possibility of application of Cu ^+/2+ chelators for resolvation of Αβ-Cu ^"1"72"1" oligomers and aggregates. Cu ⁺ was selected to induce aggregation due to its diamagnetic properties which enable NMR monitoring of the resolvation process. Moreover, Αβ-Cu ^"1" as a reduced form of Αβ-Οι ²⁺ aggregates, is of interest since it was suggested to promote initiation of ROS production leading to neuronal apoptosis (Shearer and Szalai, 2008).

[00125] We first conducted 1H-NMR monitored titrations of Αβ ₂₈ in tris-dn at pD 7 with Cu ⁺ to obtain a 1: 1 Αβ ₂₈ -Οι ⁺ complex (Fig. 1). Several measures were taken to ensure that Cu ⁺ will not oxidize to Cu ²⁺ : The NMR tube was flushed with argon, oxygen was excluded from the deuterated solvents by bubbling argon, Cu(CH ₃ CN) ₄ PF ₆ was used as the Cu ⁺ source and the concentration of acetonitrile, as stabilizing ligand for Cu ⁺ , was not less than 15%. The 0.8 mM Αβ ₂₈ -Οι ⁺ complex was titrated by one of the following chelators: thiophosphate, triphosphate, ΑϋΡ-β-S, GDP^-S, GTP-y-S and clioquinol (Fig. 2). By addition of 0.2 eq Cu ⁺ the signals of Αβ ₂₈ were broadened, and after addition of 1 eq Cu ⁺ the peaks were shifted downfield and aromatic Αβ ₂₈ signals merged into one very broad signal. To this solution, clioquinol was then added as a standard chelator known for its ability to redissolve metal-ion induced Αβ aggregates (Ritchie et al., 2003). After addition of 6 eq clioquinol the Αβ ₂₈ -Οι ⁺ spectrum slightly sharpened, yet, no signal pattern emerged, and a yellow solid was observed in the NMR tube (Fig. 2c vs. 2b). When 6 eq of triphosphate were added, the Αβ ₂₈ -Οι ⁺ spectrum showed only Phe peaks reappearances without any His signals (Fig. 2d). Thiophosphate (6 eq) was apparently a superior Cu ⁺ chelator, the addition of which resulted in a significant sharpening of the Αβ ₂₈ spectrum due to the removal of Cu ⁺ from Αβ ₂₈ (Fig. 2e). Yet, a black solid formed in the NMR tube, possibly a thiophosphate-Cu" ⁺ complex. Addition of 6 eq of GDP-P-S resulted in partial recovery of Αβ ₂₈ aromatic signals (Fig. 2f). However, unlike the case of thiophosphate, upon addition of GDP-P-S a clear solution was obtained. ΑϋΡ-β-S proved to be a better chelator (Fig. 2g) and upon addition of 5 eq the Αβ ₂₈ spectrum resembled that of pure Αβ ₂₈ . Furthermore, with ΑϋΡ-β-S the turbid solution of Αβ ₂₈ -Οι ⁺ turned clear. GTP-y-S was found to be the best chelator in this series. At only 3.2 eq of GTP-y-S the Αβ ₂₈ -Οι ⁺ complex spectrum sharpened (Fig. 2h) and looked as that of pure Αβ ₂₈ (Fig. 2a). Furthermore, a clear solution was obtained.

[00126] Since Cu ⁺ is a soft metal-ion it prefers soft ligands such as thiophosphate and nucleoside-5 '-phosphorothioate analogues, resulting in a significantly better dissolution of Αβ ₂₈ -Οι ⁺ oligomers as compared to hard ligands such as clioquinol and triphosphate.

[00127] GTP-y-S was found to be the most promising Cu ⁺ -chelator decomposing Αβ-Οι ⁺ oligomers and dissolving Αβ ₂₈ -Οι ⁺ aggregates better than ΑϋΡ-β-S and GDP^-S. Namely, a longer phosphate chain binds Cu ⁺ ion tighter. Surprisingly, ΑϋΡ-β-S performed better than GDP^-S implying the adenine moiety coordinates Cu ⁺ better than guanine. Furthermore, we found that ΑϋΡ-β-S is more stable than GDP^-S and GTP-y-S as observed in 31 P-NMR spectra measured at the end the titration (data not shown).

Example 4. Elucidation of the mode of chelation of Cu ⁺ by phosphorothioate compounds based on ¹ H/ ³¹ P-NMR and UV measurements

[00128] To investigate the mode of Cu ⁺ chelation by nucleoside-5 '-phosphorothioate analogues, we monitored Cu ⁺ titration of ΑϋΡ-β-S by 1H/ ³¹ P-NMR (Fig. 3). Changes in the 1H/ ³¹ P-NMR spectrum were observed upon titration of 9 mM ΑϋΡ-β-S with up to 0.87 eq of Cu ⁺ . With the addition of Cu ⁺ 1H-NMR spectra (Fig. 3A) exhibited downfield shift of the adenine H8 signal. Specifically, H8 signal shifted from 8.6 to 9.1 ppm and broadened upon addition of 0.53 eq Cu ⁺ . However, by the addition of 0.87 eq Cu ⁺ H8 signal sharpened and shifted to 9.4 ppm. In 31 P-NMR spectra we observed an upfield shift of Ρβ upon the addition of Cu ⁺ (Fig. 3B). Ρ _β broadened at 0.53 eq Cu ⁺ and after the addition of 0.87 eq Cu ⁺ , Ρ _β reappeared and shifted upfield by 16 ppm. The changes in the

1 H/ 31 P-NMR spectrum, broadening and shifting of Ρ _β and H8 signals of compound ADP- β-S indicated that the phosphate chain particularly Ρ _β phosphorothioate and N7 coordinate with Cu ⁺ .

[00129] The presence of free thiol in thiophosphate analogues-Cu ⁺ solution was determined by Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). Ellman's reagent reacts with free thiol to give new disulfide and free 2-nitro-5-thiobenzoate (NTB ^" ) resulting in a yellow mixture absorbing at 412 nm (Goody and Eckstein, 1971). Thiophosphate reacts with Ellman's reagent in the presence or absence of Cu ⁺ to give the disulfide (Fig. 4A). However, upon the addition of Ap ₂₈ -Cu ⁺ to 5 in 1:5 ratio (respectively), no disulfide product was formed even after 24 h (Fig. 4A). BCA, a specific Cu ⁺ indicator (Kd 2.16xl0 ^"15 ) (Yatsunyk and Rosenzweig, 2007), was used to quantify Cu ⁺ in the sample which was found to be 95% of the starting Cu ⁺ amount. Like thiophosphate, GDP-P-S reacts with Ellman's reagent to give the disulfide product; however, upon the addition of Cu ⁺ or Ap ₂₈ -Cu ⁺ and monitoring by UV spectrum, no NTB ^2" product could be detected (Fig. 4B). Addition of BCA to the sample showed that Cu ⁺ remained unchanged. In both cases of thiophosphate and GDP-P-S, a disulfide was formed when reacted with Ellman's reagent. However, unlike disulfide formation with 5- Cu ⁺ system, in the case of GDP-P-S-Cu ⁺ , a disulfide product was not formed. Apparently, the tight complex formed between GDP-P-S and Cu ⁺ does not allow the formation of NTB ^2" upon reaction of GDP-P-S with Ellman's reagent. Interestingly, in both cases of Ap complexes, Ap ₂₈ -Cu ⁺ -thiophosphate/GDP-P-S, the disulfide product was not observed. This finding possibly indicates that Cu ⁺ -ion in Ap ₂₈ -Cu ⁺ complex is still capable of tight interaction with phosphorothioate GDP-P-S. In this way reaction of the thiol with Ellman's reagent is avoided.

Example 5. Reduction of Αβ ₄₀ -Μ ²⁺ aggregate size by ΑΤΡ-γ-S, ΑϋΡ-β-S, APCPP-y-S, and GDP-p-S as monitored by DLS

[00130] DLS is used to measure the size of very small particles (0.6 nm to 6 μιη) in solution, and it is a well-established technique that had been utilized to measure the hydrodynamic diameter ( _H ) of monomer or aggregated Ap ₄₀ (Lomakin et ah, 1997). However, in the DLS technique a particle is considered as a sphere. Since Αβ is non- spherical by nature, the calculated dn is subject to changes in Αβ conformation.

[00131] Monomeric Αβ ₄₀ was incubated with either Cu ²⁺ or Zn ²⁺ for 45 min to form a 1: 1 peptide metal-ion complex. A precipitate was observed in both cases. We found that for Ap ₄₀ -Cu ²⁺ the particle size distribution reached a constant value at ~ 1836 nm after 45 min from 1 eq metal-ion addition to Αβ ₄ ο monomer. For the Αβ ₄ ο-Ζη ²⁺ particles the instrument readings indicated a different size distribution. Next, the chelators ATP, ΑΤΡ-γ-S, ADP, ΑϋΡ-β-S, APCPP-y-S, or GDP^-S were added and incubated for 30 min, followed by measurement of the _H of the resulting particles (Fig. 5). We evaluated the capacity of the phosphorothioate analogues to dissolve Αβ ₄₀ -Οι ²⁺ or Zn ²⁺ aggregates, as compared to EDTA and clioquinol known for their ability to dissolve Αβ-Μ ²⁺ aggregates (Ritchie et ah, 2003; Huang et ah, 1997). The reduction of dn of Αβ ₄ ο-Οι ²⁺ aggregates by EDTA, clioquinol, or tested compounds was compared to the _H of Αβ-Cu ^2"1" aggregate itself (Fig. 5A). In addition, the efficacy of the chelators ΑΤΡ-γ-S, ΑϋΡ-β-S, APCPP-y-S and GDP-β- S to resolubilize Αβ^-Cu ^2"1" and Αβ ₄ ο-Ζη ²⁺ is described in Fig. 5B and 5C relative to EDTA and clioquinol. EDTA efficiency in reducing Αβ particle size is considered as 100%.

[00132] The _H of Αβ ₄₀ -Οι ²⁺ in the presence of phosphorothioate compounds was smaller compared to that of ADP and ATP (Fig. 5A). Consistent with 1H-NMR data, ΑϋΡ-β-S performed 5.4-fold better than GDP^-S in reducing dn of Αβ ₄ ο-Οι ²⁺ aggregates. Surprisingly, ΑΤΡ-γ-S did not reduce aggregate _H better than ΑϋΡ-β-S, possibly because ΑϋΡ-β-S is more stable in the experimental system. For this reason, it was decided to study more stable derivatives of ΑΤΡ-γ-S such as APCPP-y-S and APPCP-a-S. We anticipated that APCPP-y-S would perform better than APPCP-a-S due to the presence of a terminal thiophosphate moiety. Indeed, compound APCPP-y-S was highly efficient in reducing _H , more than any of the studied chelators. Although both APCPP-y-S and ATP- y-S are nucleoside-5 '-triphosphate analogues bearing a terminal thiophosphate moiety, APCPP-y-S reduced Αβ ₄ ο-Οι ²⁺ particle size to 64 nm vs. 110 nm with compound ATP-y- S. Clioquinol was less effective than ATP-y-S, ΑϋΡ-β-S, and APCPP-y-S, decreasing the dn of Αβ ₄₀ -Οι ²⁺ to 127 nm. Surprisingly, APCPP-y-S was more effective even than EDTA in resolubilizing Αβ ₄₀ -Οι ²⁺ (Fig. 5C). ΑϋΡ-β-S and APCPP-y-S were equi-efficacious in resolubilizing Αβ ₄ ο-Ζη ²⁺ aggregates showing 88-90% of EDTA efficacy (Fig. 5B). Yet, both compounds performed better than clioquinol (ca. 75% of EDTA efficacy) (Fig. 5B, 5C). Interestingly, APCPP-y-S dissolved Αβ ₄₀ -Οι better than EDTA although the latter is a chelator with high affinity to Cu ²⁺ and Zn ²⁺ (Log K values for Cu ²⁺ and Zn ²⁺ EDTA complexes are 18.8 and 16.5, respectively) (Furia, 1980).

Example 6. Monitoring size reduction of Αβ ₄₀ -Μ ²⁺ aggregates by nucleoside-5'- phosphorothioate analogues using TEM

[00133] To validate the DLS data indicating the efficiency of APCPP-y-S, we monitored the dissolution of Αβ ₄₀ -Μ ²⁺ aggregates in the presence of APCPP-y-S by TEM. Αβ ₄₀ -Οι ²⁺ and Αβ ₄₀ -Ζη ²⁺ 1: 1 complexes were incubated for 9 days at RT, resulting in a significant sediment. Αβ ₄ ο-Οι ²⁺ aggregates of 2.5 μιη in diameter (Fig. 6A) and Αβ ₄ ο-Ζη ²⁺ aggregates of different sizes from 100 nm to 2.5 μιη (Fig. 6C) were observed by TEM measurements. APCPP-y-S (6 eq) was then added and the mixture was incubated for 7 h. Addition of APCPP-y-S to Αβ^-Cu ^2"1" and Αβ ₄₀ -Ζη ²⁺ aggregates resulted in a significant reduction in the aggregate size to less than 250 nm for Αβ ₄ ο-Οι ²⁺ and less than 500 nm for Αβ ₄ ο-Ζη ²⁺ aggregates, Fig. 6B and 6D, respectively.

Example 7. Re-solubilization of Ap ₄₀ -Cu ⁺ aggregates by APCPP-y-S monitored by 1H-NMR

[00134] APCPP-y-S, the most promising chelator identified here, was used also to resolubilize Αβ^-Cu ^"1" aggregates. This process was monitored by ¹ H-NMR similar to that described above for Αβ ₂₈ · First, the 1H-NMR spectrum of 0.25 mM Αβ ₄ ο monomer at 278 K, pD 11, was measured (Fig. 7a), after adjustment of Αβ ₄₀ solution pD to 7.8 the signals shifted and broadened (Fig. 7b). Upon addition of 1 eq 8.3 mM Cu(CH ₃ CN) ₄ PF ₆ the signals dramatically further broadened (Fig. 7c). When APCPP-y-S was added (6 eq) all signals reappeared (Fig. 7d), as seen for Αβ ₄ ο at pD 7.8 (Fig. 7b), indicating Cu ⁺ - coordination by APCPP-y-S and its removal from Αβ ₄₀ -Οι ⁺ complex. Addition of 9 eq of APCPP-y-S did not sharpen the spectrum any further.

Example 8. Re-solubilization of Ap ₄₂ -Zn ²⁺ /Cu ²⁺ aggregates by ΑϋΡ-β-S and APCPP- y-S monitored by turbidity assay

[00135] Aggregation of Αβ-metal ion complexes results in a turbid mixture that increases the light scattering in solution, leading to a higher absorbance at 405 nm (Storr et ah, 2009). [00136] Aggregation of monomeric Αβ ₄₂ was achieved by adding Zn(NO ₃ ) ₂ to a Αβ ₄₂ solution at pH 7.4, and Cu(N0 ₃ ) ₂ to a Αβ ₄₂ solution at pH 6.6. Those 200 μΜ Αβ ₄₂ -Μ ²⁺ mixtures, left at RT for 2 h, became turbid, and exhibited an increase of absorbance at 405 nm. The resultant Αβ ₄₂ mixtures were assayed for the resolubilization capacity of ADP-β- S and APCPP-y-S in comparison to EDTA. EDTA was highly effective in decreasing the absorbance of Αβ ₄₂ -Ζη ²⁺ mixtures (Fig. 8A). APCPP-y-S was found to be less effective than EDTA by 21% and 12% at 3 and 6 eq, respectively, and ΑϋΡ-β-S was less effective than EDTA by 63% and 27% at 3 and 6 eq, respectively (Fig. 8A). However, in the case of Αβ ₄₂ -Οι ²⁺ aggregates, ΑϋΡ-β-S and APCPP-y-S were more effective than EDTA at aggregate re- solubilization up to 28% and 12% at 6 and 3 eq, respectively. At 15 eq chelator, EDTA, ΑϋΡ-β-S and APCPP-y-S were almost equi-efficacious (Fig. 8B). The turbidity assay confirmed the DLS data that although EDTA has a higher affinity for Zn ²⁺ and Cu ²⁺ than APCPP-y-S, the latter was highly effective in decreasing the turbidity of Αβ ₄₂ -Μ ²⁺ mixtures. Moreover, consistent with the DLS data, APCPP-y-S was more effective than EDTA in the re- solubilization Αβ ₄₂ -Οι ²⁺ aggregates.

Example 9. ESR OH radical assay

[00137] To study the antioxidant effect of ΑϋΡ-β-S and APCPP-y-S, ESR was used to monitor the modulation of ΌΗ formation from H ₂ 0 ₂ by the Cu ⁺ or Fe ²⁺ induced Fenton reaction. For this purpose we applied DMPO as a spin trap. The OH radical formed by the reaction of Fe ²⁺ /Cu ⁺ with H ₂ O ₂ is trapped by DMPO, and the DMPO-OH adduct is then detected by ESR. The addition of chelators to Fe ²⁺ /Cu ⁺ -H ₂ O ₂ mixture lowers the DMPO- OH signal due to metal-ion chelation and radical scavenging (Richter and Fischer, 2006).

[00138] The inhibition of radical production by the chelators ΑϋΡ-β-S and APCPP-y-S (expressed in IC ₅₀ and IC ₉₀ values, Table 1) was compared to the inhibitory effect of common antioxidants including ascorbic acid, GSH and the metal-ion chelator EDTA. Based on our previous reports (Richter and Fischer, 2006; Baruch-Suchodolsky and Fischer, 2008) we expected ΑϋΡ-β-S and APCPP-y-S to be more potent inhibitors than ADP and ATP since the formers bear a sulfur substitution at the Ρ _β /Ρ _γ position. Unlike phosphate, thiophosphate is a soft ligand that binds preferably soft and borderline metal- ions. Indeed, this prediction was found to be true for ΑϋΡ-β-S and APCPP-y-S in the Fe ²⁺ /H ₂ O ₂ -system, with IC ₅₀ values in the range of 86-100 μΜ (Table 1). Yet, in the Cu+/H ₂ O ₂ - system, the IC ₅₀ values for ΑϋΡ-β-S and APCPP-y-S were in the range of 300- 400 μΜ, whereas the parent compounds were mediocre inhibitors of the Cu ⁺ -induced Fenton reaction (IC ₅₀ values of 226 and 183 μΜ for ADP and ATP). ΑϋΡ-β-S and APCPP-y-S were better antioxidants than ascorbic acid and glutathione in the Fe ²⁺ /H ₂ 0 ₂ - system, the IC ₅₀ values of which were 93 and 216 μΜ, respectively. However, in the Cu ⁺ /H ₂ 0 ₂ -system GSH was highly efficient with IC ₅₀ value of 63 μΜ, while ascorbic acid was a poor OH radical inhibitor with IC ₅₀ > 500 μΜ. EDTA, as a better metal-ion chelator, was indeed more efficient than ΑϋΡ-β-S and APCPP-y-S in reducing OH radicals production in both systems with IC ₅₀ values of 64 and 62 μΜ, respectively.

Table 1: Inhibition of OH radical production in Fenton system by adenine nucleotides, phosphate, and control antioxidants, as monitored by ESR

Antioxidant IC ₅₀ /IC ₉₀ values represent the compound' s concentration that inhibits 50% / 90% of the OH radical amount produced, respectively. N/A=not available, the minimal amount of radical production exceeds 50% (IC ₅₀ ) or 10% (IC ₉₀ ).

[00139] ΑϋΡ-β-S and APCPP-y-S were less potent antioxidants in Cu ⁺ /H ₂ 0 ₂ system than in Fe ²⁺ /H ₂ 0 ₂ system, probably due to oxidation of the phosphorothioate moiety to form a disulfide product in the presence of H ₂ 0 ₂ (Richter and Fischer, 2006), thus concealing the terminal sulfur which might be required for binding Cu ⁺ ion. However, the disulfide product apparently binds Fe ²⁺ -ion better than ADP and ATP, probably by creating a full- coordination sphere as proposed before (Richter and Fischer, 2006). The full coordination sphere provided by the disulfide dimer of ΑϋΡ-β-S and APCPP-y-S prevents an electron transfer from Fe ²⁺ , thus making both ΑϋΡ-β-S and APCPP-y-S potent antioxidants.

Example 10. Nucleoside 5 '-phosphorothioate analogues are potent antioxidants at PC12 cells

[00140] In this study, nucleoside 5'-phosphorothioate analogues were explored as inhibitors of Fe(II)-induced oxidative stress in PC 12 cells used as a neuronal model. Reduction of ROS production in PC 12 cells by each one of the tested analogues was measured by DCFH-DA, a radical sensitive indicator. After DCFH-DA was removed, the tested analogue was added to the cells at a final concentration of 0.2-200 μΜ. Oxidation was initiated by the addition of FeS0 ₄ (0.16 μΜ) to the wells. The plates were incubated for 1 h at 37°C, during which the absorbance was read by a Tecan fluorometer at 485/530 nm.

[00141] ΑϋΡ-β-S, GDP-P-S, GTP-y-S, ΑΤΡ-γ-S and APCPP-y-S inhibited ROS formation with IC ₅₀ values of 26, 10, 5, 0.18 and 0.16 μΜ, respectively (values represent mean+S.D of three experiments, P<0.05; data not shown). It should be noted that GDP-β- S and ΑϋΡ-β-S were 4.5- and 3-fold more stable in PC12 cells than GDP and ADP, respectively. In addition, all the phosphorothioate analogues tested were nontoxic up to 200 μΜ, and did not harm the basal level of ROS production in cells.

Example 11. Nucleoside 5 '-phosphorothioate analogues are neuro-protectants of primary neurons exposed to oxidative damages by FeS0 ₄ or FeS0 ₄ /H ₂ 0 ₂

[00142] Cultured cortical neurons in 96-well plates were treated with different concentrations of FeS0 ₄ , or hydrogen peroxide and FeS0 ₄ , for 24 h at 37°C. Following exposure to various insults, the cells were treated with ΑΤΡ-γ-S and GDP-P-S as described. Cells were subsequently incubated for a further 18-24 h as indicated before being assessed for viability measures. Neurons were treated with ΑΤΡ-γ-S and GDP-P-S at three concentrations (25, 100 or 200 Mm) simultaneously with 1.5, 3 or 6 μΜ FeS0 ₄ for 24 h. Following 24 h incubation in the presence of both FeS0 ₄ and ΑΤΡ-γ-S and GDP-P-S cell viability measures were assessed by XTT assay. All experiments were performed in triplicate.

Application ofFeS0 ₄ and H2O2 to cultured neurons cells

[00143] FeS0 ₄ induced a concentration-dependent decrease in cell viability as assessed by XTT assay and morphological assessment following 24 h of exposure (Fig. 9). The neuroprotective effect of ΑΤΡ-γ-S and GDP-P-S (due to iron chelation) was evaluated in cortical neurons exposed to FeS0 ₄ for 24 h (Fig. 10). Co-application of FeS0 ₄ (3 μΜ) with ΑΤΡ-γ-S and GDP-P-S (Fig. 10A) resulted in 100 and 130% protection, respectively. Their IC ₅₀ values of ΑΤΡ-γ-S and GDP-P-S were 0.01 and 0.008 μΜ, respectively. When the cells were treated with co-application of FeS0 ₄ (3 μΜ) and Η ₂ 0 ₂ (ΙΟΟμΜ) the IC ₅₀ values of ΑΤΡ-γ-S and GDP-P-S were 1 and 4 μΜ, respectively (Fig. 10B). [00144] The IC ₅₀ values of the nucleoside-5'-phosphorothioate analogues tested were compared to those of the natural nucleotides (Table 2). On the average IC ₅₀ values of the synthetic compounds is -0.01 μΜ compared to the natural with IC ₅₀ of ~25μΜ, indicating that ΑΤΡ-γ-S, GTP-y-S, and ΑϋΡ-β-S are highly potent neuroprotectants active at the low nanomolar concentrations.

Table 2: IC ₅₀ values of various nucleoside-5'-phosphorothioate analogues vs. natural nucleotides

Example 12. APCPP-y-S protects primary neuron culture against Αβ_ι ₂ insult

[00145] In this study, the neuroprotective activity of APCPP-y-S in neuronal cells exposed to toxic Αβ ₄₂ was evaluated. At first we measured the number of the viable neuronal cells after treatment with Αβ ₄₂ (5-50 μΜ) (Fig. 11). At 50 μΜ Αβ ₄₂ , 50% of the neuronal culture remained vital. Next, we measured the protective effect of APCPP-y-S: primary neurons were treated with APCPP-y-S (0.04-25 μΜ) and 50 μΜ Αβ ₄₂ for 48 h. Fig. 12 shows the viability of primary neuronal cells due to the treatment with APCPP-y- S, in a dose-dependent manner. APCPP-y-S maintained 50% of neuronal cells at 0.2 μΜ, while at a similar experiment ATP-y-S maintained 50% of neuronal cells only at 0.8 μΜ, and ATP maintained 45% of neuronal cells at 25 μΜ (Fig. 13).

Example 13. Evaluation of APCPP-y-S as P2Yi _{/ /2} receptor agonist

[00146] Antioxidant, antiapoptotic and anti-inflammatory activities were described to be mediated by P2Y receptors. Promising subtypes are the P2Yi and P2Yn receptors (Fujita et ah, 2009; Shinozaki et ah, 2005). High potency at P2Y receptors enables nucleotides to evoke signals at low concentration which is advantageous from a pharmacological point of view. Modifications at the phosphate groups may change the preference of the receptor for the nucleotide analogues. In this study, the potency of APCPP-y-S, in which the Ρ _α /Ρ _β position was modified by a methylene group to improve the chemical and metabolic stability, and by sulfur atom at Ρ _γ position to improve the antioxidant activity, to activate P2Yi and P2Yn receptor was evaluated. APCPP-y-S showed very weak potency at the P2Yi receptor (no activity up to 10 μΜ). Interestingly, APCPP-y-S showed potency at the P2Yn receptor (ECso=l μΜ) being 6.7 times more potent than the natural ligand ATP. APCPP-y-S was found to be neither agonist nor antagonist at the P2Y ₂ receptor (Fig. 14).

Methods

[00147] Cell culture and transfection. GFP constructs of human P2Y ₂ -R, P2YrR and P2Yn-R were stably expressed in 1321N1 astrocytoma cells, which lack endogenous expression of P2X- and P2Y-receptors. The respective cDNA of the receptor gene was cloned into a pEGFPNl vector and after transfection, using FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany), cells were selected with 0.5 mg/ml G418 (geneticine; Merck Chemicals, Darmstadt, Germany) and grown in DMEM supplemented with 10% serum (FCS), 100 U/ml penicillin and 100 U/ml streptomycin at 37°C and 5% C0 ₂ . The expression and cell membrane localization of the respective P2Y receptors was confirmed through the analysis of the GFP fluorescence. The functionality of the expressed GFP-labeled receptor in cells was verified by recording a change of [Ca ²⁺ ]; after stimulation with the appropriate receptor agonist.

[00148] Single cell calcium measurements. 1321N1 astrocytoma cells transfected with the respective plasmid for P2Y-R-GFP expression plated on coverslips (22 mm diameter) and grown to approximately 80% density, were incubated with 2 μΜ fura 2/AM and 0.02% pluronic acid in Na-HBS buffer (Hepes buffered saline: 145 mM NaCl, 5.4 mM KC1, 1.8 mM CaCl ₂ , 1 mM MgCl ₂ , 25 mM glucose, 20 mM Hepes/Tris pH 7.4) for 30 min at 37°C. The cells were superfused (1 ml/min, 37°C) with different concentrations of nucleotide in Na-HBS buffer. The nucleotide-induced change of [Ca ²⁺ ] was monitored by detecting the fluorescence intensities with excitations at 340 nm and 380 nm. Only GFP- labeled cells were analyzed. Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and SigmaPlot (SPSS Inc., Chicago, IL, USA) were used to derive the concentration-response curves and EC ₅₀ values from the average response amplitudes obtained in at least three independent experiments (Ecke et ah, 2006; Ecke et ah, 2008). Only cells with a clear GFP-signal and with the typical calcium response kinetics upon agonist pulse application were included in the data analysis. The nucleotide induced change of [Ca ²⁺ ]i was monitored by detecting the respective emission intensity of fura 2/AM at 510 nm with 340 nm and 380 nm excitations (Ubl et ah, 1998). The average maximal amplitude of the responses and the respective standard errors were calculated from ratio of the fura 2/ AM. The GFP-tagged P2Y receptors are suitable for pharmacological and physiological studies, as previously reported (Tulapurkar et ah, 2004; Tulapurkar et ah, 2006; Zylberg et ah, 2007).

Example 14. APCPP-y-S is metabolically stable

[00149] In order to use APCPP-y-S as a neuroprotectant agent and test its activity in an animal model, we first evaluated the metabolic stability of APCPP-y-S in several tissues such as liver, brain and blood. In this assay we took samples from mice brain, liver and blood. The tissue samples were homogenated by sonication and split to 0.5 ml aliquots. Each sample was mixed with 0.1 mg of APCPP-y-S and incubated at 37°C for 10, 30, 90 and 180 min. After incubation, the samples were collected and extracted with chloroform at 1: 1 v/v ratio. The aqueous layer was removed and freeze-dried. Samples were loaded onto an activated Starta X-AW weak anion exchange cartridge, washed with H ₂ 0 (1 ml) and eluted with MeOH:H ₂ 0 (1: 1, 1 ml) followed by NH ₄ OH:MeOH:H ₂ 0 (2:25:73, 1 ml), and then freeze-dried. The resulting residue was analyzed by HPLC. All chromatographic analyses were performed at 30°C using SUPELCOSIL™ LC-18-S HPLC Column (5 μπι particle size, LxI.D. 25 cm x 2.1 mm), flow rate 0.2 ml/min under isocratic elution conditions with the following buffer composition: [50 mM potassium phosphate, 100 mM triethylamine, 0.1 mM MgCl ₂ , pH 6.5 (adjusted with phosphoric acid)]:Acetonitrile (98.5: 1.5). Each analysis cycle was set to 30 minutes. The chromatographic flow was monitored at 260 nm and integrated using EZChrom Elite Software.

[00150] As shown from the chromato grams (data not shown), APCPP-y-S could be detected even after 180 min in brain, liver and blood.

Example 15. APCPP-y-S is of limited toxicity at PC12 cells up to 1000 μΜ

[00151] In this study, the toxicity of APCPP-y-S, at 1-1000 μΜ, at PC12 cells was tested by MTT assay. Fig. 15 shows that APCPP-y-S was not toxic to PC 12 cells after 24 h of incubation up to 100 μΜ. At 1000 μΜ, 75% of the cells were still viable.

Example 16. Pharmacokinetics and BBB permeation of APCPP-y-S

[00152] As shown in Example 14, APCPP-y-S is stable in human blood serum as well as in brain, liver and blood from mice. In this study, we evaluated APCPP-y-S blood-brain barrier (BBB) permeability and pharmacokinetics in Fl (C57BxS29) mice. APCPP-y-S was injected intravenously (IV) at 40 mg/Kg into four mice (each mouse was injected with 1.5 mg of APCPP-y-S). The mice were sacrificed after 30 minutes (2 of the 4) or 90 minutes (thr other 2 of the 4), and samples from brain and blood were taken to analysis. Each sample was collected into 0.5 ml saline, sonicated and extracted with chloroform, applied onto an anion exchange cartridge, and freeze-dried. The resulting residue was analyzed by HPLC. We detected the presence of 67.5 and 64% of the injected amount of APCPP-y-S in the blood samples after 30 and 90 minutes, repectively. In the brain sample we obtained ca. 2% permeability (data not shown).

Example 17. Synthesis of nucleoside 5'-phosphorothioate prodrugs

[00153] In order to achieve oral bioavailability and intracellular delivery of the nucleotide analogues exemplified herein, e.g., APCPP-y-S and ΑϋΡ-β-S, various prodrug strategies have been explored, mainly focusing on the partial masking of these analogues' negatively charged backbone by bioreversible, lipophilic groups, that provide the prodrug permeability through different membrane tissues. The prodrug is then decomposed, either spontaneously or enzymatically, in the brain, and releases the biologically active compound.

[00154] The BBB forms an interface between the circulating blood and the brain, and possesses various carrier-mediated transport systems for small molecules such as glucose and amino acids to support and protect CNS function (Ohtsuki and Terasaki, 2007). Hence, the development of drugs that structurally mimic substrates of influx transport is an effective strategy to increase BBB permeability.

Synthesis of ADP-β-Ξ analogue based on conjugation with D-glucose

[00155] It was assumed that by coupling these nucleoside-5'-phosphorothioate analogues identified herein as promising neuroprotectants, i.e., ΑϋΡ-β-S and APCPP-y-S, with D- glucose, which is the main energy source for the brain and transported by GLUT1, it will be possible to increase the permeability of these nucleoside-5'-phosphorothioate analogues through glucose transporters. As depicted in Scheme 3, D-glucopyranoside-1- - thiophosphate, synthesized according to the literature (Singh et ah, 1988), was coupled with AMP in the presence of CDI and ZnCl ₂ to yield l-D-glucosyl-Pp-ADP-P-S. Synthesis of APCPP-y-S analogue based on conjugation with D-glucose (Scheme 4)

Adenosine-2',3'-methylidene

[00156] Adenosine (2 g, 7.49 mmol) was dissolved in dry DMF (13.1 ml) under N ₂ atmosphere. TsOH (2.85 g, 15 mmol) was added to the reaction flask as solid. Then, trimethylorthoformate (41 ml, 37.5 mmol) was introduced into the flask. After 3 days of reaction a DOWEX (free base form) was added with cooling of the reaction flask in ice water bath. The mixture was stirred for 2 h and filtered. The filtrate was evaporated to get yellow oil. Three extractions with CHC1 ₃ (70 ml)/NaHC0 ₃ (70 ml) were performed followed by extraction with brine (70 ml). An organic phase was dried with Na ₂ S0 ₄ , filtered and evaporated. Crystallization from acetone with cooling in ice water bath was performed to get 1.15 g (49.6 %) of the compound. 1H-NMR (CDC1 ₃ , 200 MHz): 8.50 (s, 1H), 8.14 (s, 1H), 7.2 (bs, 2H), 6.25 (s, 1H), 6.15 (d, 1H), 4.65 (m, 1H), 4.45 (m, 1H), 4.40 (m, 1H), 4.02 (m, 2H), 3.47 (s, 1H), 3.41 (s, 1H) ppm, ¹³ C-NMR (CDC1 ₃ , 50 MHz): 155.7,

152.2, 150.0, 139.8, 125, 120, 97.5, 86.5, 74.5, 73.0, 70.1, 52.1 ppm, MS (ESI, negative mode): 309.

Adenosine-2',3'-methylidene-5'-tosyl

[00157] Adenosine-2',3'-methylidene (523 mg, 1.7 mmol) was dissolved in dry DCM (30 ml) under N ₂ atmosphere at RT. 4-dimethylaminopyridine (837 mg, 6.9 mmol) was dissolved in dry DCM (3 ml) and introduced dropwise into the reaction flask. Tosyl chloride (795 mg, 4 mmol) after crystallization and drying was dissolved in dry DCM (5 ml) and dropped into the reaction flask. After 3 h the reaction was monitored by TLC (DCM : MeOH 9: 1) and almost complete consumption of the reagent was observed. Three extractions with saturated sodium bicarbonate (40 ml) were performed. The organic phase was evaporated to get 380 mg (47%) of the product. 1H-NMR (CDC1 ₃ , 200 MHz): 8.50 (s, 1H), 8.14 (s, 1H), 7.56 (dd, J=6 Hz, 2 Hz, 2H), 7.2 (bs, 2H), 7.12 (dd, J=6 Hz, 2 Hz, 2H), 6.25 (s, 1H), 6.15 (d, 1H), 4.65 (m, 1H), 4.45 (m, 1H), 4.40 (m, 1H), 4.02 (m, 2H), 3.47 (s, 1H), 3.41 (s, 1H), 2.31 (s, 3H) ppm, ¹³ C-NMR (CDC1 ₃ , 50 MHz): 155.7, 152.2, 150.0,

144.3, 140.7, 139.8, 130.5, 128.3, 125, 120, 97.5, 86.5, 74.5, 73.0, 70.1, 52.1, 24 ppm, MS (ESI, negative mode): 463.

HPLC purification for Adenosine-2',3'-methylidene-5'-tosyl

[00158] The purification was performed on silica column of Biotage apparatus with ethanol (strong solvent) and dichloromethane (weak solvent) elution. The gradient of the strong solvent was: 0%-3% - 3 CV; 0%-8% - 10 CV; 8%-10% - 3 CV; 10%-10% - 3 CV; 10%-90% - 1 CV; and 90%-90% - 2 CV. The first of a two peaks was collected and evaporated to get mixture of two stereoisomers of the product.

Adenosine-5'-methylene-diphosphate

[00159] Adenosine-2',3'-methylidene-5'-tosyl (285 mg, 0.6 mmol) was dissolved in dry DMF (1 ml) in a two-neck flask under N ₂ atmosphere. Methylene-diphosphate (265 mg, 1.5 mmol) tetrabutylammonium salt (obtained by passing methylene-diphosphonic acid through Sephadex CM resin (tetrabutylammonium form)) was evaporated for 3 times with dry DMF (1 ml each time). After 2 days, the reaction was monitored by 31 P-NMR and signals of both methylene-diphosphate and the product were observed (16.4 ppm (s), 14.8 (d, J=19.4 Hz), 18.9 (d, J=19.4 Hz)). The solvent was evaporated and the product was deprotected by 10% HC1 (0.5 ml, pH 2.3) treatment with stirring for 2.5 h. 10% Ammonium hydroxide solution (0.3 ml, pH 9) was added and the solution was stirred for 45 min. The solution was freeze-dried and the residue was applied to DEAE-Sephadex anion-exchange column for LC purification with ammonium bicarbonate buffer (pH 7.5, 600 ml+600 ml of water) with 0.0 M - 0.3 M gradient. Final purification was achieved by reverse phase HPLC separation with TEAA (0.0 M - 0.4 M) eluent to get 58 mg (21%) of the product. 1H-NMR (D ₂ 0, 200 MHz): 8.50 (s, 1H), 8.14 (s, 1H), 7.2 (bs, 2H), 6.25 (s, 1H), 6.15 (d, 1H), 4.65 (m, 1H), 4.45 (m, 1H), 4.40 (m, 1H), 4.15 (m, 2H), 3.47 (s, 1H), 3.41 (s, 1H) ppm, ¹³ C-NMR (D ₂ 0, 50 MHz): 155.0, 153.0, 150.0, 139.1, 123, 120, 97.5, 86.5, 74.5, 72.0, 70.1, 52.1, 41 ppm, ³¹ P-NMR (D ₂ 0, 83 MHz): 16.4 (s), 14.8 (d, J=19.4 Hz), 18.9 (d, J=19.4 Hz) ppm, MS (ESI, negative mode): 464.

[00160] The D-glucose was added to a HEPES buffer solution (20 mmol, 4.76 g) containing sucrose phosphorylase (60 U), sodium thiophosphate (0.30 mmol, 54 mg), sucrose (342.3 mg, 1.0 mmol), and magnesium sulfate (0.30 mmol, 73.9 mg) at 25°C. The solution was stirred for 24 h at RT. The reaction progress was monitored by TLC chromatography (H ₂ 0 : isopropanol : NH ₄ OH 6: 12:2) with the Ellman's reagent development which stained phosphorothioate compounds. Two spots were observed on the TLC plate: R _f =0.355 (product), R _f =0.067 (reagent). At the end of the reaction (determined by TLC) the solution was filtered through Amicon PM-30 filter and freeze dried. The product was separated by LC (DEAE-Sephadex anion-exchange column, from 0.0 M to 0.3 M of TEAB buffer) monitored by TLC and Ellman's reagent staining. The fraction containing product was freeze dried to get 126.5 mg of product (46% yield). 1H-NMR (D ₂ 0, 200 MHz): 5.6 (d, 1H, J=3.4 Hz), 3.7-3.85 (m, 3H), 3.35-3.45 (m, 2H), 3.5-3.6 (m, 1H) ppm. ¹³ C-NMR (D ₂ 0, 50 MHz): 92, 73, 72, 71, 69, 60 ppm. ³¹ P-NMR (D ₂ 0, 83 MHz) : 43.9 ppm (s). MS (ESI, negative mode): 275.

Glucose- l-a-ATP-a,p-methylene-Y-S

[00161] Adenosine-methylenediphosphate and glucose- 1-a-thiophosphate were converted to corresponding trioctylammonium and tributylammonium salts by passing them through Sephadex CM resin (trioctylammonium and tributylammonium form, respectively). Adenosine-methylenediphosphate (114 mg, 0.093 mmol) was evaporated for 3 times with dry DMF (1 ml each time) and introduced into a two-neck dry flask under N ₂ atmosphere in dry DMF (1 ml). CDI (Ι,Γ-carbonyldiimidazole, 151 mg, 0.93 mmol) was introduced into the reaction flask as solid. After 6 h the reaction was monitored by TLC (water : isopropanol : NH ₄ OH 7: 11:2) indicating almost complete consumption of the adenosine- methylenediphosphate. Dry methanol (38 ul, 0.93 mmol) was added and after 8 min ZnCl ₂ (59 mg, 0.43 mmol) was introduced into reaction flask. After 2 min glucose- la- thiophosphate (227 mg, 0.279 mmol) was added. TLC (water : isopropanol : NH ₄ OH 7:11:2) was performed after 21 h showing no further progress of the reaction. After 22 h the reaction was stopped by addition of EDTA (192 mg, 0.516 mmol), water (5 ml and TEAB (1 M, 0.5 ml) till pH 7.5 was attained. The mixture was freeze-dried, and the residue was purified by LC on DEAE-Sephadex anion-exchange column eluting with 0.0 M - 0.3 M gradient ammonium bicarbonate buffer (pH 7.5, 600 ml of each). Fractions containing product were freeze- dried several times to get 4.1 mg of the product (4.5% yield). The final purification was performed by HPLC separation with TEAA buffer and acetonitrile with gradient of 3% - 20% of acetonitrile. Two diastereomers were collected separately at the 8% of acetonitrile. 1H-NMR (D ₂ 0, 200 MHz): 8.71 (s), 8.47 (s), 8.23 (s), 7.99 (d, J=8.5 Hz), 7.74 (d, 8.5 Hz), 7.63 (s), 7.43-7.28 (m), 6.93-6.86 (m), 6.07 (d, J=5.6 Hz), 4.3-4.47 (m), 4.1-4.16 (m), 3.6-3.75 (m) ppm. ¹³ C-NMR (D ₂ 0, 50 MHz): 155.0, 153.0, 150.0, 139.1, 120.3, 103.5, 97.5, 86.5, 78.1, 74.5, 72.0, 71.9, 71.5, 70.1, 69.3, 60.5, 20.3 ppm, ³¹ P-NMR (D ₂ 0, 83 MHz): 18.35 (m), 19.38 (m), 34.25 (m) ppm. Example 18. Synthesis of uridine/adenosine-5'-tetrathiobisphosphonate and di- uridine/di-adenosine 5 ' ,5 " -tetrathiobisphosphonate

[00162] As depicted in Scheme 5, in order to synthesize uridine/adenosine-5'-tetrathio bisphosphonate we applied methylene-bis(l,3,2-dithiaphospholane-2-sulfide), 5b, prepared from bis-methylene(phosphonicdichloride) that was treated with 1,2- ethanedithiol and 10 mol% A1C1 ₃ in CHBr ₃ As has been shown, primary alcohols can successfully react with 5b to yield Ο,Ο'-diester-methylenediphosphonotetrathioate analogues (Amir et ah, 2013). Compounds 5c-u and 5c-a were obtained from 5a-u and 5a- a, respectively, in a one -pot reaction. First, 2',3'-methoxymethylidene uridine, 5a-u (or 2',3'-methoxymethylidene adenosine, 5a-a) was treated with 5b in the presence of molecular sieves in DCM for 24 h, and then, a mixture of 1 eq. of DBU in DCM was added dropwise over 1 h period. The reaction progress was monitored by 31 P-NMR, the formation of doublets at 100.1 and 90.4 ppm indicated the formation of intermediate 5c-u (or 5c-a), also indicated by the cloudy reaction mixture turning immediately clear. Without isolating the product, 3-hydroxypropionitrile (6 eq.) and DBU (1 eq.) were added to the reaction flask at 45°C. ³¹ P-NMR indicated the formation of 5d-u (doublets at 104.8 and 104.5 ppm). The work-up of the reaction included filtration of the molecular sieves and evaporation of the solvent. This one-pot synthesis is highly moisture sensitive, thus the use of molecular sieves in this process is necessary. Steps a and b were performed in a one-pot reaction because attempts to isolate products 5c-u and 5c-a on a reverse phase column resulted in hydrolytic ring opening of the thiophospholane ring, as indicated by MS analysis and 31 P-NMR (peaks at 105 and 67 ppm).

[00163] Products 5d-u and 5d-a were separated on a silica gel column applying CHCl ₃ :MeOH (85: 15) eluent. Further purification was performed on medium pressure chromatography on a reverse phase column using 1M TEAA (pH=7):CH CN (78:22) eluent. Products 5d-u and 5d-a were obtained in 37% and 28% yield, respectively. Products 5e-u and 5e-a were obtained after treatment with tBuO ^" Na ⁺ in THF resulting in β-elimination. The formed acrylonitrile was scavenged with ethanethiol while products 5e- u and 5e-a precipitated from the reaction mixture together with EtSNa salt. The solvent was removed by decantation and the solid residue was dissolved in water and titrated with 10% HC1 followed by the addition of 40% NH ₄ OH, for the removal of the methoxymethylidene protecting group. The residue was subjected to Sephadex DEAE ion- exchange chromatography to yield the desired products UPCP-a,a',P,P'-tetra-S and APCP-a,a',P,P'-tetra-S in 45% and 55% yield, respectively (from 5d-u and 5d-a, respectively).

[00164] As depicted in Scheme 6, this new synthetic route was expanded further to obtain the corresponding di-uridine/di- adenosine 5',5"-tetrathiobisphosphonate. Intermediates 6c- u and 6c-a were synthesized from reaction of 6b with 6a-u and 6a-a, respectively. Due to the high moisture sensitivity of this reaction step, compounds 6a-u (or 6a-a) and 6b were stirred together with molecular sieves in dry acetonitrile overnight. Then, DBU (2.1 eq.) was added at 60°C, and the completion of the reaction was monitored by 31 P-NMR. After 2 h, the desired intermediate 6c-u (or 6c-a) was obtained. The work-up and the removal of the protecting group were performed as mentioned above for uridine/adenosine 5'- tetrathiobisphosphonate. Final purification of the di-uridine/di-adenosine 5',5"-tetrathio bisphosphonate obtained was carried out by HPLC, on a reverse-phase column, applying 1M TEAA (pH=7):CH ₃ CN eluent. Di-uridine 5',5"-tetrathio bisphosphonate and di- adenosine 5',5"-tetrathio bisphosphonate were obtained in 30% and 36% yield, respectively.

UDP-a,fi-tetrathiobisphosphonate tris-ammonium salt, UPCP-a,a',fl,fl'-tetra-S

[00165] To a two necked round bottom flask containing molecular sieves, 5b (150 mg, 0.462 mmol), 5a-u (264.65 mg, 0.924 mmol) and DCM (4.5 ml) were added. The mixture was stirred overnight under nitrogen atmosphere, and then, a mixture of DBU (0.462 mmol, 0.07 ml) in DCM (4.5 ml) was added dropwise, over a period of lh. 3 ^J 1 ¹ P-NMR showed the formation of 5c-u (doublets at 100.10 and 90.45 ppm). 3-Hydroxypropionitrile (2.772 mmol, 0.19 ml) and DBU (0.462 mmol, 0.07 ml) were then added. The reaction mixture was stirred under nitrogen at 45°C for 30 min. 31 P-NMR showed the formation of 5d-u (doublets at 104.8 and 104.5 ppm). The mixture was filtered and the molecular sieves were washed with DCM. After evaporation of the solvent, 5d-u was separated on silica column using CHCl ₃ :MeOH (85: 15). This fraction was further purified on a reverse phase column using TEAA 1M (pH=7):CH ₃ CN (78:22) eluent, to give 5d-u in 37% yield (130 mg). ¹ H NMR (acetone-d ₆ ; 600 MHz): δ 8.42 (d; J = 7.8 Hz; 1H), 6.06-6.07 (m; 2H), 5.77 (d; J = 7.8 Hz; 1H), 5.48 (dd; J = 6.0 Hz; J = 1.8 Hz; 1H), 5.19 (dd; J = 6.0 Hz; J 3.6 = Hz; 1H), 4.43-4.46 (m; 2H), 4.30-4.32 (m; 3H), 3.51 (td; J = 13.2 Hz; J = 1.8 Hz; 2H), 3.27 (s; 3H), 2.88 (td; J = 7.5 Hz; J = 1.8 Hz) ppm. ³¹ P NMR (acetone-d ₆ ; 81 MHz): δ 105.28 (d; J = 25.8 Hz; P„), 104.18 (d; J = 25.8 Hz; P _p ) ppm. ¹³ C NMR (acetone-d ₆ ; 151 MHz): δ 163.6, 151.5, 143.6, 119.2, 118.1, 103.3, 90.9, 85.4 (d; J = 9.8 Hz), 84.9, 81.8, 64.6 (d; J = 6.6 Hz), 62.7 (t; J = 60.6 Hz), 60.1 (d; J = 6.5 Hz), 50.9, 19.9 (d; J = 8.6 Hz) ppm. HR MALDI (negative): Calcd for C ₁₅ H ₂ oN308P ₂ S4, 559.960; found, 559.957.

[00166] Product 5d-u (130 mg, 0.17 mmol) was dissolved in THF (3 ml) and ethylmercaptane (3 ml). Then potassium tert-butoxide (57.3 mg, 0.51 mmol) was added in portions. After 2 h, 31 P-NMR indicated the presence of only the starting material in the solution and a mixture of the starting material and the product in the precipitate obtained in the reaction. The solution was treated with an additional portion of potassium tert- butoxide (57.3 mg, 0.51 mmol). The solid residue was dissolved again in THF (3 ml) and ethylmercaptane (3 ml). After 1.5 h, 31 P-NMR showed no starting material in the solution and the desired product, 5e-u, was observed in the precipitate. The solvent was removed by decantation and the solid was dissolved in water and freeze-dried. Product 5e-u was dissolved in water and then titrated with 10% HC1 until pH=2.4 was achieved. The mixture was stirred at RT for 3 h. Then 40% NH ₄ OH was added until pH=9 and stirred for 45 min. The mixture was freeze-dried. The residue (100 mg, yield: 90%) was subjected to ion-exchange chromatography (on a DEAE Sephadex column, swollen overnight in 1 M NaHC0 ₃ at 4 °C). The product was eluted applying a gradient of 0-0.5 M (800 ml each) of ammonium bicarbonate solution, pH 7.6, to obtain UPCP-a,a',P,P'-tetra-S in 45% yield (40 mg). 1H NMR (D ₂ 0; 600 MHz): δ 8.14 (d; J = 7.8 Hz; 1H), 5.95 (d; J = 8.4 Hz; 1H), 5.91 (d; J = 5.4 Hz; 1H), 4.50 (dd; J = 4.8 Hz; J = 4.2; 1H), 4.44 (t; J = 5.4 Hz; 1H), 4.25- 4.26 (m; 3H), 3.43 (t, J = 13.2 Hz, PCH ₂ P, 2H) ppm. ³¹ P NMR (D ₂ 0; 81 MHz): δ 106.26 (d; J = 22.5 Hz; P„), 78.06 (d; J = 22.5 Hz; P _p ) ppm. ¹³ C NMR (D ₂ 0; 151 MHz): δ 166.1, 151.8, 142.4, 102.5, 87.9, 83.5, 73.7, 69.9, 62.9, 60.3 (t; J = 56.3 Hz) ppm. HR MALDI (negative): Calcd for C ₁₀ Hi ₅ N ₂ O ₇ P ₂ S ₄ , 464.923; found, 464.920.

ADP-a,fi-tetrathiobisphosphonate tris-ammonium salt, APCP-α,α ',β,β '-tetra-S

[00167] Product APCP-a,a',P,P'-tetra-S was prepared according to the above procedure for the preparation of UPCP-a,a',P,P'-tetra-S. Compound 5c-a was obtained from 5a-a (285.80 mg, 0.924 mmol) and 5b (150 mg, 0.462 mmol) in 28% yield (100 mg). 1H NMR (acetone-d _6; 600 MHz): δ 9.09 (s; 1H), 9.03 (s; 1H), 8.19 (s; 1H), 8.18 (s; 1H), 6.46 (d; J = 3.6 Hz), 6.22 (d; J = 3.6 Hz), 6.16 (s; 1H), 5.98 (s; 1H), 5.45-5.63 (m; 3H), 5.41-5.42 (m; 1H), 4.59-4.62 (m; 1H), 4.53-4.58 (m; 1H), 4.26-4.30 (m; 4H), 3.55-3.62 (m; 4H), 3.42 (s; 3H), 3.27 (s; 3H), 2.87-2.89 (m; 4H) ppm. ³¹ P NMR (acetone-d ₆ ; 81 MHz): δ 105.35 (d; J = 25.9 Hz; P„), 104.02 (d; J = 25.9 Hz; P _p ) ppm. ¹³ C NMR (acetone-d _6; 151 MHz): δ 156.5, 156.4, 153.5, 150.3, 141.6, 141.5, 119.6, 119.5, 119.1, 117.8, 90.4, 89.9, 87.1 (d; J = 9.9 Hz), 86.1, 85.5 (d; J = 9.7 Hz), 85.4, 82.8, 82.4, 65.0 (d; J = 6.9 Hz), 64.5 (d; J = 6.8 Hz), 61.5-62.3 (m; PCP), 59.7-59.9 (m; CH ₂ -0), 52.4, 50.8 ppm. HR MALDI (negative): Calcd for C ₁₆ H ₂ iN ₆ 0 ₆ P ₂ S4, 582.987; found, 582.987.

[00168] After LC separation, APCP-a,a',p,P'-tetra-S was obtained in 55% yield (38 mg). 1H NMR (D ₂ 0; 600 MHz): δ 8.74 (s; 1H), 8.27 (s; 1H), 6.12 (d; J = 5.4 Hz; 1H), 4.90 (t; J = 5.4 Hz; 1H), 4.68 (dd; J = 4.2 Hz; J = 4.8 Hz; 1H), 4.28-4.44 (m; 3H), 3.47 (t, J = 13.2 Hz, PCH ₂ P, 2H) ppm. ³¹ P NMR (D ₂ 0; 81 MHz): δ 104.99 (d; J = 21.1 Hz; P„), 90.59 (d; J = 21.1 Hz; Pp) ppm. ¹³ C NMR (D ₂ 0; 150 MHz): δ 154.4, 151.1, 148.8, 140.9, 118.5, 87.1, 84.0, 74.3, 70.5, 62.9, 57.8 (t; J = 59.0 Hz) ppm. HR MALDI (negative): Calcd for CnHieNsOsP^, 487.950; found, 487.952.

Di-uridine-S^S^-diphosphate-ayP-methylene-ayP-tetra-thiop hosphate-bis- triethylammonium salt, UPCPU-α,α',β,β '-tetra-S

[00169] To a two necked round bottom flask containing molecular sieves, 6a-u (281.60 mg, 0.984 mmol), 6b (80 mg, 0.246 mmol) and dry acetonitrile (7 ml) were added. The mixture was stirred overnight under nitrogen atmosphere. Then DBU (0.520 mmol, 0.08 ml) was added and the mixture was stirred at 60°C for 2h. 31 P-NMR showed the formation of the desired product 6c-u (singlet at 105.1 ppm). The mixture was filtered and the molecular sieves were washed with CHC1 ₃ . After evaporation of the solvent, 6c-u was separated on a silica-gel column using CHCl ₃ :MeOH (90: 10).

[00170] Most of product 6c-u (73 mg) was dissolved in water and then titrated with 10% HCl until pH=2.4 was achieved. The mixture was stirred at RT for 3 h. Then 40% NH ₄ OH was added until pH=9 and stirred for 45 min. The mixture was freeze-dried. The residue was purified on a reverse phase column using 1M TEAA (pH=7):CH ₃ CN (92:8) eluent, to give UPCPU-a,a',P,P'-tetra-S in 30% yield (65 mg). Final purification was carried out by HPLC, using a semipreparative reverse-phase column, applying an isocratic TEAA/CH ₃ CN 92:8 in 15 min (4 ml/min): t _R 9.36 min.

[00171] ³¹ P NMR (D ₂ 0; 81 MHz): δ 104.02 (s; 2P) ppm. 1H NMR (D ₂ 0; 600 MHz): δ 8.15 (d; J = 8.4 Hz; 1H), 5.95-5.98 (m; 2 Hz), 4.49 (dd; J = 4.8 Hz; J = 4.2 Hz; 1H), 4.44 (t; J = 4.8 Hz; 1H), 4.28-4.32 (m; 3H), 3.51 (t; J = 13.8 Hz; PCH ₂ P; 2H), 3.19 (q; J = 7.2 Hz; 5H), 1.27 (t; J = 7.2 Hz; 8H) ppm. ¹³ C NMR (D ₂ 0; 150 MHz): δ 166.4, 151.9, 142.2, 102.4, 88.4, 83.2 (t; J = 4.8 Hz), 73.9, 69.8, 62.3, 57.4 (t; J = 65 Hz), 46.5, 8.1 ppm.

Di-adenosine-5 5''-diphosphate-a,fi-methylene-a,fi-tetra-thiophosphate-bis- triethylammonium salt, APCPA-α,α ',β,β '-tetra-S

[00172] Compound APCPA-a,a',P,P'-tetra-S was prepared according to the same procedure as for UPCPU-a,a',p,P'-tetra-S. Compound 6c-a was obtained from 6a-a (371 mg, 1.19 mmol) and 6b (100 mg, 0.308 mmol). Compound APCPA-α,α',ρ,ρ '-tetra-S was obtained after the removal of the methoxymetylidene protecting group from the intermediate 6c-a and purified on a reverse phase column using 1M TEAA (pH=7):CH ₃ CN (93:7) eluent, to give APCPA-a,a',p,P'-tetra-S in 36 % yield (100 mg). Final purification was carried out by HPLC, using a semipreparative reverse-phase column, applying an isocratic elution with TEAA/CH ₃ CN 90: 10 in 15 min (4 ml/min): t _R 8.35 min.

[0001] 1H NMR (D ₂ 0; 600 MHz): δ 8.50 (s; 1H), 8.06 (s: 1H), 6.01 (d; J = 5.4 Hz), 4.61 (t; J = 4.2 Hz; 1H), 4.43-4.47 (m; 1H), 4.28-4.35 (m; 2H), 3.59 (t; J = 14.4 Hz; PCH ₂ P; 2H), 3.19 (q; J = 7.2 Hz; 5H), 1.27 (t; J = 1.2 Hz; 8H) ppm. ³¹ P NMR (D ₂ 0; 81 MHz): δ 104.44 (s; 2P) ppm. ¹³ C NMR (D ₂ 0; 150 MHz): δ 154.7, 152.5, 148.2, 139.6, 117.6, 87.2, 84.0 (t; J = 5.1 Hz), 75.6, 70.7, 62.2, 57.9 (t; J = 68.7 Hz), 46.5, 8.1 ppm.

Example 19. Evaluation of chemical properties of adenosine-5'-tetrathio

bisphosphonate and di-adenosine 5 ',5 "-tetrathiobisphosphonate

[00173] In order to study and evaluate the chemical stability of adenosine-5'-tetrathio bisphosphonate and di-adenosine 5',5"-tetrathiobisphosphonate to basic and acidic condition, as well as to air-oxidation, kinetic measurements were performed by monitoring the changes in the percentage of these amalogues, using 31 P-NMR (Figs. 16-18).

[00174] The evaluation of the stability of adenosine-5'-tetrathio bisphosphonate by 31 P- NMR was first conducted at pD=1.5 for four days. In the course of the experiment, new signals emerged in ³¹ P-NMR spectra at 104.4, 92.3, 89.2, 86.1, 67.8 ppm and the percentage of starting material was obtained from the ratio of integration between the starting material and the total peaks in the spectrum. As shown in Fig. 16, adenosine-5'- tetrathio bisphosphonate was relatively stable under these conditions with calculated half- life of 44 h. Mass spectrum (ESI-QTOF negative) analysis of freeze-dried adenosine-5'- tetrathio bisphosphonate after four days at pD 1.5 revealed the fragmentation products shown in Scheme 7.

[00175] In the mass spectrum, we observed the signal that can be correlated to 7b, m/z 488, and the fragmentations of the hydrolysis products. The combination of mass analysis with 31 P-NMR data for 7b subjected to acidic media for 4 days reveals that the signals at 104.4 and 67.8 ppm are correlated to the asymmetric hydrolysis product 7a m/z 472.

Moreover, the mass spectroscopic analysis and the 31 P-NMR shift at 92.3 ppm revealed the presence of MDPT, 239 m/z. In addition, the shift at 86.1 ppm in ³¹ P-NMR can be correlated with the formation of oxidized MDPT product 7c (237 m/z). The singlet at 89.2 ppm can be correlated with compound 7d that formed by an intramolecular nucleophilic attack and the loss of water. Moreover, four-membered ring heterocyclic compounds such as 7d were reported before, and the typical 31 P-NMR signal at -90 ppm we found here for 7d is in accordance with previous findings (Toyota et ah, 1993).

[00176] Next, we studied the stability of adenosine-5'-tetrathio bisphosphonate under basic conditions, pD=l l. We found that compound 7b is highly stable under these conditions. After two weeks, the 31 P-NMR spectrum was identical to the starting material, without any indication of decomposition. We associate this with the repulsion between the negative charges of the tetrathio-bisphosphonate moiety in adenosine-5'-tetrathio bisphosphonate and OH ^" ions. In addition, intramolecular nucleophilic attack and formation of disulfide bond are less likely to occur under these conditions.

[00177] Furthermore, we tested the stability of adenosine-5'-tetrathio bisphosphonate under air-oxidizing conditions, by performing 31 P-NMR measurements in an open rotating NMR tube. The half-life of adenosine-5'-tetrathio bisphosphonate under these conditions was 14 h. The MS and 31 P-NMR analysis of the freeze-dried sample of adenosine-5'- tetrathio bisphosphonate after 3 days indicated the formation of an intramolecularly oxidized product. The new asymmetric centers that formed after the oxidation of adenosine-5'-tetrathio bisphosphonate resulted in complex multiplets signals in the 31 P- NMR spectrum (-105 and -65 ppm).

[00178] Di-adenosine 5',5"-tetrathiobisphosphonate exhibited half-life of 9 h under pD

1.5. The combination of mass analysis with 31 P-NMR data for di-adenosine 5',5"- tetrathiobisphosphonate subjected to acidic media for 2 days revealed that di-adenosine 5',5"-tetrathiobisphosphonate undergoes decomposition, first to mono-nucleotide, adenosine 5'-tetrathiobisphosphonate. 31 P-NMR showed two indicative doublets at 105 and 91 ppm (Fig. 18) that correspond to the chemical shifts of adenosine 5'- tetrathiobisphosphonate. Then, MDPT, was formed as indicated by a singlet at 92 ppm. These observations are supported by MS analysis of the freeze-dried sample.

[00179] Di-adenosine 5',5"-tetrathiobisphosphonate was highly stable under air-oxidizing conditions in an open NMR tube for 3 days. No change in di-adenosine 5',5"- tetrathiobisphosphonate was observed. The dinucleotide scaffold increased the resistance to oxidation and formation of a disulfide bond.

[00180] At pD=l l, adenosine 5'-tetrathiobisphosphonate was completely stable even after two weeks. The stability of di-adenosine 5',5"-tetrathiobisphosphonate was identical to that of adenosine 5'-tetrathiobisphosphonate under these conditions. Moreover, 1H-NMR indicated that the bridging methylene hydrogen atoms are exchangeable, since the methylene typical triplet signal had broadened and the integration of this peak decreased. The exchange of the hydrogen atoms with deuterium at pD 11 implies on the acidity of the phosphonate methylene group.

[00181] In order to determine the Zn ²⁺ -coordination to adenosine 5'-tetrathiobis phosphonate and di-adenosine 5',5"-tetrathiobisphosphonate, we preformed Zn ²⁺ -titration monitored by 1H- and ³¹ P-NMR spectroscopy (Figs. 19-20). The shift of NMR signals as well as their line-broadening indicates Zn ²⁺ -coordination to several atoms in both analogues tested. Solutions of the analogues tested were titrated by 0.1-10 eq Zn ²⁺ and monitored by 1H- and ³¹ P-NMR at 400 and 160 MHz, respectively. Relatively low nucleotide concentrations were used (3-5 mM) to avoid inter-molecular base stacking. The titration was performed with 0.2-0.35 M Zn ²⁺ solutions in D ₂ 0 at pD 7.4 and 300K. Chemical shifts (δπ, δ _ρ ) were measured at different Zn ²⁺ concentrations.

[00182] Addition of 0.1 eq Zn ²⁺ to di-adenosine 5',5"-tetrathiobisphosphonate caused line- broadening and an upfield shift. Line-broadening is a result of dynamic equilibrium between the free ligand, e.g. said analogue, and the Zn ²⁺ -ligand complex. The singlet at 103 ppm corresponds to the free ligand, and the emerging singlet at 100.5 ppm corresponds to the Zn ²⁺ -ligand complex. After addition of 0.5 eq Zn ²⁺ only one singlet at 100.5 ppm is observed. Line- sharpening indicates that there is no free ligand, i.e., all molecules of the analogue are engaged in Zn ²⁺ complex. These results are consistent with common tetrahedral geometry of zinc complexes, in which two ligands of di-adenosine 5',5"-tetrathiobisphosphonate form a complex with one zinc ion. The Δδ value of di- adenosine 5',5"-tetrathiobisphosphonate due to metal-ion binding is 3 ppm. The addition of up to 10 eq of Zn 2+ resulted in no change in 31 P-NMR spectrum.

[00183] Data of 1H-NMR monitored Zn ²⁺ -titrations for di-adenosine 5',5"-tetrathiobis phosphonate are presented in Fig. 20A and the results show a similar trend. After addition of only 0.1/0.2 eq of Zn ²⁺ , shift of signals and line-broadening was evident for di- adenosine 5',5"-tetrathiobisphosphonate. H8 was shifted upfield by 0.5 ppm while H2 was shifted by 0.2 ppm. The shifts of H8 imply that N7 is a coordination site of Zn ²⁺ . Whereas the upfield shifts of H2 in the presence of zinc ions possibly result from stacking interactions (Stern et ah, 2010). Addition of Zn ²⁺ to a solution of adenosine 5'- tetrathiobisphosphonate resulted in upfield shifts, both in 31 P- and 1 H-NMR monitored titrations. Upon the addition of 0.2 eq Zn ²⁺ there are two types of species, free adenosine 5'-tetrathiobisphosphonate and Zn ²⁺ - adenosine 5'-tetrathiobisphosphonate complex. When 0.5 eq Zn ²⁺ were added all adenosine 5'-tetrathiobisphosphonate molecules were engaged in zinc complex and Ρ _β signal shifted 38 ppm upfield. The tremendous shift of -40 ppm indicates that the sulfur modification has a high affinity to Zn ²⁺ and the terminal phosphate is involved in metal-ion binding, as was shown for the terminal thiophosphate analogues ΑΤΡ-γ-S, ΑϋΡ-β-S and GDP- -S. Data of ¹ H-NMR monitored Zn ²⁺ -titrations for adenosine 5'-tetrathiobisphosphonate show line-broadening and upfield shifts of H2 and H8. The upfield shift of H8 by 0.3 ppm implies that N7 is a coordination site of Zn ²⁺ , as also found for di-adenosine 5',5 "-tetrathiobisphosphonate.

Example 20. Adenosine-5' -tetrathiobisphosphonate and di-adenosine-5',5"- tetrathiobisphosphonate are highly stable to hydrolysis by human ectonucleotidases compared to ΑϋΡ-β-S and GDP-P-S, and AP ₂ A, respectively

[00184] Since NPP hydrolyzes the Ρα-β bond, adenosine-5'-tetrathiobisphosphonate was compared with ΑϋΡ-β-S to examine the effect of methylene group and extra thiophosphonate groups on the stability and inhibition of NPPl. In addition, the effect of the nucleobase was examined by comparing ΑϋΡ-β-S with GDP- -S.

[00185] NPPl activity was measured at pH 8.5. Human NPPl preparation was added to the incubation buffer at 37°C, and the reaction was started by the addition of a particular nucleotide analogue, and terminated after 1-2 h by addition of perchloric acid. The nucleotide degradation products were separated and quantified by HPLC, and the concentrations of reactants and products were determined from the relative areas for their absorbance maxima peaks. The acid used to terminate the enzymatic reaction can cause partial degradation of the nucleotide analogues. Therefore, the percentage of degradation for each analogue due to the acidic treatment was assessed in the absence of enzyme, and this value was substracted from the percentage of analogue degradation in the presence of enzyme. Human NPP1 hydrolyzed all analogues tested to NMP and either pyrophosphate or inorganic phosphate, wherein the identity of the degradation products was determined by comparing their retention times to those of controls.

Table 3: Hydrolysis of adenosine-5'-tetrathiobisphosphonate, di-adenosine-5',5"- tetrathiobisphosphonate, ΑϋΡ-β-S and GDP- -S by human ectonucleotidases

[00186] As shown in Table 3, over 2 h period, adenosine-5'-tetrathiobisphosphonate, di- adenosine-5',5"-tetrathiobisphosphonate and ΑΟΡ-β-S were not metabolized at all by NPP1, but the latter was significantly hydrolyzed by NPP3 at 25%. However, GDP- -S was significantly metabolized by both NPP1 and 3 at 21% and 32%, respectively, indicating higher rate of hydrolysis for the guanine nucleotide compared with that for the adenine nucleotide. Adenosine-5'-tetrathiobisphosphonate, di-adenosine-5',5"-tetrathio bisphosphonate, and ΑΟΡ-β-S modified with dithiophosphonate and thiophosphate groups were both stable towards NPP1, NPP3 and NTPDases hydrolysis. The terminal thiophosphate group in ΑΟΡ-β-S and the methylene group in adenosine-5'- tetrathiobisphosphonate and di-adenosine-5',5"-tetrathiobisphosphonate conferred stability to NPP and NTPDase hydrolysis, since the latter bond, Ρ _α _ _β , is cleaved in ADP analogue.

[00187] In the next study, adenosine-5'-tetrathiobisphosphonate, di-adenosine-5',5"- tetrathiobisphosphonate and ΑΟΡ-β-S were evaluated as inhibitors of ectonucleotidases, using the protocol described in Experimental.

[00188] As shown in Figs. 21A-21C, at 100 μΜ, adenosine-5'-tetrathiobisphosphonate inhibited NPP1 and NPP3 by 4% and 7%, respectively. In contrast, NTPDase 1, 2 and 8 were inhibited by 54%, 42%, and 49%, respectively. Adenosine-5'-tetrathiobis phosphonate thus does not inhibit NPPl, and it is also not an NTPDasel selective inhibitor. Di-adenosine-5',5"-tetrathiobisphosphonate inhibited the pnp-TMP hydrolysis by NPPl and NPP3 at -60% and 20%, respectively. Likewise, this analogue inhibited the hydrolysis of ATP by NTPDasel by -60%; however, NTPDase2, 3 and 8 were inhibited by 5-20% only, indicating that di-adenosine-5',5"-tetrathiobisphosphonate is neither a potent nor selective NPPl inhibitor, as it inhibits both NPPl and NTPDasel by ca. -60%. It is thus concluded that although di-adenosine-5',5"-tetrathiobisphosphonate was found to be a highly chemically and metabolically stable analogue, possibly due the methylene group replacing the phosphate bridging oxygen, it cannot be applied as a NPPl inhibitor due to lack of protein selectivity. At 100 μΜ, ΑϋΡ-β-S inhibited NPPl by 95%, while NPP3 and NTPDasel, 2, 3 and 8 were inhibited by less than 50% . However, ΑϋΡ-β-S is also a very good agonist of Ρ2Υ _{1 >12} ,ΐ3 receptors. This protein-inselectivity precludes the use of ΑϋΡ-β-S as a NPPl inhibitor.

Example 21. The efficacy of APCPP-y-S in a mouse model for AD

[00189] Six-month old homozygous 3xTg-AD mice (known as a mouse model of Alzheimer's disease) are daily injected (LP.) during three months with either APCPP-y-S or its prodrug l-D-glucosyl-Ργ- APCPP-y-S (2 mg/kg and 20 mg/kg), wherein mice treated with Menamtine (30 mg/kg) are used as positive controls. Age-matched mice injected with PBS, as well as age-matched non-Tg mice, are used as controls.

[00190] Animal Behavioral Testing: Morris Water Maze. The Morris Water Maze (MWM) is used, and the parameters measured during the probe trial include (1) initial latency to cross the platform location; (2) number of platform location crosses; and (3) time spent in the quadrant opposite to the target quadrant. Novel object recognition. This task measures the time spent exploring the familiar object and the novel object is calculated. Time spent with the novel object as compared to time spent with both objects is used as memory index.

[00191] Protein analysis, Immunohistochemistry . Mice are sacrificed and their brains are tested with the following antibodies: Anti-Αβ (6E10), anti-APP (22C11), and Αβ ( ₄ o/ ₄₂ ) anti-Tau HT7, anti-GSK3P-p, anti-P-actin, anti-p38, and anti-CDK5.

[00192] PKC and GSK3fl activities. PKC activity is measured using a kit from Streegen (Victoria, Canada) and the GSK3P activity is measured using a kit from SIGMA.

Thiophosphate Triphosphate Scheme 1: Synthesis of adenosine 5'-[y-thio]-a,P-methylene triphosphate, APCPP-y-S

1 d

APCPP-y-S

Reaction conditions

(a) DCM, DMAP, TsCl, RT, 3 h; (b) DMF, tribis-(tetrabutylammonium)methylene diphosphonate, 24 h (50% yield); (c)10% HCl, pH 2.5, 3 h, 10% NH ₄ OH, pH 7, RT, 45 min; (d) DMF, CDI, 5 h, ZnCl ₂ , MeOH, 8 min, PS0 ₃ ^3~ , 3 h, EDTA, H ₂ 0, 10 min (29% yield).

Scheme 2: Synthesis of adenosine 5'-[a-thio]-P,y-methylene triphosphate, APPCP-

2a 2b

APPCP-a-S

2c

Reaction conditions

(a) 2-chloro-4H-l,3,2-benzodioxaphosphorin-4-one, DMF, pyridine, RT, lh; (b) bis- (tetrabutylammonium)methylenediphosphonate, DMF, tributylamine, RT, 2 h; (c) 1. S ₈ , 0 C, 1.5 h 2. 1M TEAB, 0.5 h, 3. 10% HCl, pH 2.5, 3 h, 4. 10% NH ₄ OH, pH 7, RT, 45 min. APPCP-a-S was obtained in 9% yield.

Scheme 3: Synthesis of l-D-glucosyl-P -ADP-P-S

3a 3b 3c

1 -D-glucosyl-P -ADP- -S

Reaction conditions

(a) sucrose phosphorylase, thiophosphate, HEPES buffer, 25 h, RT, 55% LC purification; (b) counter ion exchange (c) l.AMP-imidazolide, DMF, RT, dry conditions, 2 h, 37%, 2. MeOH, ZnCl ₂ , dry conditions, 3 d, LC purification, 11.2%, HPLC purification

2

65

Scheme 4: Synthesis of l-D-glucosyl-Py-APCPP-y-S

3HN(Oct) ₃

1 -D-glucosy l-Py-APCPP-y-S

Reaction conditions

ia) 1. trimethylorthoformate, TsOH, 3 d, RT, 55%, 2. DOWEX base form, 2 h; (b) Tosyl- chloride, DMAP, DCM, 3h, RT, dry conditions, 50% (c) methylene-diphosphonic acid, DMF, 2 d, dry conditions (d) 1.HC1 10%, 2.5 h, 2. NH ₄ OH, 45 min, 21%; (e) l.CDI, dry DMF, 6 h, RT, 2. MeOH, ZnCl ₂ , dry conditions, 4 d, 4.5%. Scheme 5: Synthesis of uridine/adenosine-5'-tetrathiobisphosphonate analogues APCP- α,α',β,β'-tetra-S and UPCP-a,a',p,P'-tetra-S

5a-u B=uracil 5b 5c-u B=uracil

5a-a B=adenine 5c-a B=adenine

5d-u B=uracil 5e-u B=uracil

5d-a B=adenine 5e-a B=adenine

UPCP-a,a',p, '-tetra-S B=uracil

APCP-a. '. . '-tetra-S B=adenine

Reaction conditions

(a) DBU, DCM, RT, 1 h; (b) DBU, 3-hydroxypropionitrile, 45°C, 30 min; (c) ethylmercaptane:THF (v:v) 1: 1, potassium tert butoxide, RT, 2 h; (d) (1) 10% HCl, pH=2.3, RT, 3 h; (2) 40% NH ₄ OH, pH=9, RT, 45 min.

Scheme 6: Synthesis of di-uridine/di-adenosine 5',5"-tetrathiobisphosphonate analogues APCPA-a a',p,P'-tetra-S and UPCPU-a,a',p,P'-tetra-S

6c-u B=uracil

6c-a B=adenine

UPCPU-a.a'. . '-tetra-S B=uracil

APCPA-a,a'^^'-tetra-S B=adenine

Reaction conditions

(a) DBU, CH ₃ CN, 60°C, 2 h; (b) (1) 10% HCl, pH=2.3, RT, 3 h; (2) 40% NH ₄ OH, pH=9, RT, 45 min.

Scheme 7: Fragmentation of adenosine-5'-tetrathiobisphosphonate after 4 days hydrolysis at pD=1.5 as observed by mass spectroscopy

7a 7b

m/z 472 m/z 488

MDPT 7c 7(1

m z 239 m/z 237 m/z 221 REFERENCES

Abramov, E., Dolev, L, Fogel, H., Ciccotosto, G.D., Ruff, E., Slutsky, L, Nat. NeuwscL, 2009, 12, 1567-1576

Adlar, P.A., Cherny, R.A., Finkelstein, D.L, Gautier, E., Robb, E., Cortes, M., Volitakis, L, Liu, X., Smith, J.P., Perez, K., Laughton, K., Li, Q.X., Charman, S.A., Nicolazzo, J.A., Wilkins, S., Deleva, K., Lynch, T., Kok, G., Ritchie, C.W., Tanzi, R.E., Cappai, R., Masters, C.L., Barnham, K.J., Bush, A.L, Neuron, 2008, 59, 43-55

Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S.T., Hampel, H., Hull, M., Landreth, G., Lue, L.F., Mrak, R., Mackenzie, I.R., McGeer, P.L., O'Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C, Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, L, Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-Coray, T., Neurobiol. Aging, 2000, 21, 383-421

Amir, A., Sayer, A.H., Zagalsky, R., Shimon, L.J., Fischer, B., / Org Chem., 2013, 78(2), 270-277

Atwood, C.S., Moir, R.D., Huang, X., Scarpa, R.C., Bacarra, N.M.E., Romano,

D.M., Hartshorn, M.A., Tanzi, R.E., Bush, A.L, J. Biol. Chem., 1998, 273, 12817-12826 Atwood, C.S., Perry, G., Zeng, H., Kato, Y., Jones, W.D., Ling, K.Q., Huang, X.,

Moir, R.D., Wang, D., Sayre, L.M., Smith, M.A., Chen, S.G., Bush, A.L, Biochemistry,

2004, 43, 560-568

Barnea, A., Cho, G., Katz, B.M., Brain Res., 1991, 541, 93-97

Bartolini, M., Bertucci, C, Bolognesi, M.L., Cavalli, A., Melchiorre, C,

Andrisano, V., ChemBioChem, 2007, 8, 2152-2161

Baruch-Suchodolsky, R., Fischer, B., /. Inorg. Biochem., 2008, 102, 862-881 Baruch-Suchodolsky, R., Fischer, B., Biochemistry, 2009, 48, 4354-4370

Baykov, A.A., Evtushenko, O.A., Avaeva, S.M., Analytical Biochemistry, 1988,

171, 266-270

Belli, S.I., Goding, J.W., European Journal of Biochemistry, 1994, 226, 433-443 Bombois, S., Maurage, C.A., Gompel, M., Deramecourt, V., Mackowiak-

Cordoliani, M.A., Black, R.S., Lavielle, R., Delacourte, A., Pasquier, F., Arch. Neurol.,

2007, 64, 583-587

Brenner, A.J., Harris, Ε.Ό., ΑηαΙ. Biochem., 1995, 226, 80-84 Bush, A.L, Trends NeuroscL, 2003, 26, 207-214

Cherny, R.A., Adlard, P.A., Finkelstein, D.L, Gautier, E., Robb, E., Cortes, M., Volitakis, L, Liu, X., Smith, J.P., Perez, K., Laughton, K., Li, Q.X., Charman, S.A., Nicolazzo, J.A., Wilkins, S., Deleva, K., Lynch, T., Kok, G., Ritchie, C.W., Tanzi, R.E., Cappai, R., Masters, C.L., Barnham, K.J., Bush, A.L, Neuron, 2008, 59, 43-55

Curtain, C.C., Ali, F., Volitakisi, L, Chernyi, R.A., Norton, R.S., Beyreuther, K., Barrow, C.J., Mastersi, C.L., Bushi, A.L, Barnham, K.J., /. Biol. Chem., 2001, 276, 20466-20473

Dai, X., Sun, Y., Gao, Z., Jiang, Z., /. Mol. NeuroscL, 2009, 41, 66-73

Doraiswamy, P.M., Finefrock, A.E., Lancet Neurol, 2004, 3, 431-434

Ecke, D., Tulapurkar, E.M., Nahum, V., Fischer, B., Reiser, G., British Journal of

Pharmacology, 2006, 149(4), 416-423

Ecke, D., Hanck,T., Tulapurkar, E.M., Schafer, R., Kassack, M., Strieker, R,

Reiser, G., Biochemical Journal, 2008, 409(1), 107-116

Faller, P., Hureau, C, Dalton Trans., 2009, 1080-1094

Faller, P., ChemBioChem, 2009, 10, 2837-2845

Fezoui, Y., Hartley, D.M., Harper, J.D., Khurana, R., Walsh, D.M., Condron,

M.M., Selkoe, D.J., Lansbury, P.T., Fink, A.L., Teplow, D.B., Amyloid, 2000, 7, 166-178 Francis, P.T., Palmer, A.M., Snape, M., Wilcook, G.K., /. Neurol., Neurosurg.

Psychiatry, 1999, 66, 137-147

Fujita, T., Tozaki-Saitoh, H., Inoue, K., Glia, 2009, 57(3), 244-257

Furia, T.E., CRC Handbook of Food Additives, 2 ^nd edn, 1980, vol. 2, pp. 271-294

Garzon-Rodriguez, W., Yatsimirsky, A.K., Glabe, C.G., Bioorg. Med. Chem. Lett.,

1999, 9, 2243-2248

Goody, R.S., Eckstein, F., /. Am. Chem. Soc, 1971, 93, 6252-6257

Green, D.E., Bowen, M.L. Scott, L.E., Storr, T., Merkel, M., Bohmerle, K.,

Thompson, K.H., Patrick, B.O., Schugarb, H.J., Orvig, C, Dalton Trans., 2010, 39, 1604-

1615

Huang, X., Atwood, C.S., Moir, R.D., Hartshorn, M.A., Vonsattel, J.P., Tanzi, R.E., Bush, A.L, /. Biol. Chem., 1997, 272, 26464-26470

Iqbal, K., Del, A., Alonso, C, Chen, S., Chohan, M.O., El-Akkad, E., Gong, C.X., Khatoon, S., Li, B., Liu, F., Rahman, A., Tanimukai, H., Grundke-Iqbal, I., Biochim. Biophys. Acta, Mol. Basis Dis., 2005, 1739, 198-210 Jomova, K., Vondrakova, D., Lawson, M., Valko, M., Mol. Cell. Biochem., 2010, 345, 91-104

Karr, J.W., Akintoye, H., Kaupp, L.J., Szalai, V.A., Biochemistry, 2005, 44, 5478-

5487

Kowalska, J., Lewdorowicz, M., Darzynkiewicz, E., Jemielity, J., Tetrahedron Lett, 2007, 48, 5475-5479

Kukulski, F., Levesque, S.A., Lavoie, E.G., Lecka, J., Bigonnesse, F., Knowles, A.F., Robson, S.C., Kirley, T.L., Sevigny, J., Purinergic Signalling, 2005, 1, 193-204

Lakatos, A., Zsigo, E., Hollender, D., Nagy, N.V., Fueloep, L., Simon, D., Bozso, Z., Kiss, T., Dalton Trans., 2010, 39, 1302-1315

Lau, L.F., Brodney, M.A., Curr. Top. Med. Chem., 2008, 2, 1-24

LeVine, H., Dingb, Q., Walkerc, J. A., Vossc, R.S., Augelli-Szafran, C.E., Neurosci. Lett., 2009, 465, 99-103

Lim, G.P., Yang, F., Chu, T., Chen, P., Beech, W., Teter, B., Tran, T., Ubeda, O., Ashe, K.H., Frautschy, S.A., Cole, G.M., /. Neurosci., 2000, 20, 5709-5714

Lomakin, A., Teplow, D.B., Kirschner, D.A., Benedek, G.B., Proc. Natl. Acad. Set U. S. A., 1997, 94, 7942-7947

Lovell, M.A., Robertson, J.D., Teesdale, W.J., Campbell, J.L., Markesbery, W.R., /. Neurol. Set, 1998, 158, 47-52

Miura, T., Suzuki, K., Kohata, N., Takeuchi, H., Biochemistry, 2000, 39, 7024-

7031

Ohtsuki, S., Terasaki, T., Pharmaceutical research, 2007, 24(9), 1745-1758 Panza, F., Frisardi, V., Imbimbo, B.P., Capurso, C, Logroscino, G., Sancarlo, D.,

Seripa, D., Vendemiale, G., Pilotto, A., Solfrizzi, V., CNS Neurosci. Ther., 2010, 16, 272-

284

Pratico, D., Trends Pharmacol. Set, 2008, 29, 609-615

Richter, Y., Fischer, B., JBIC, J. Biol. Inorg. Chem., 2006, 11, 1063-1074

Ritchie, C.W., Bush, A.I., Mackinnon, A., Macfarlane, S., Mastwyk, M.,

MacGregor, L., Kiers, L., Cherny, R., Li, Q.X., Tammer, A., Carrington, D., Mavros, C,

Volitakis, I., Xilinas, M., Ames, D., Davis, S., Beyreuther, K., Tanzi, R.E., Masters, C.L.,

Arch. Neurol., 2003, 60, 1685-1691

Ritche, C.W., Bush, A. I., Masters, C.I., Expert Opin. Invest. Drugs, 2004, 13,

1585-1592 Salvador, G.A., Uranga, R.M., Giusto, N.M., Int. J. Alzheimers Dis., 2010, 2011 Scott, L.E., Orvig, C, Chem. Rev., 2009, 109, 4885-4910

Selkoe, D.J., Neuron, 2001, 32, 177-180

Shearer, J., Szalai, V.A., /. Am. Chem. Soc, 2008, 130, 17826-17835

Shinozaki, Y., Koizumi, S., Ishida, S., Sawada, J., Ohno, Y., Inoue, K., Glia, 2005, 49(2), 288-300

Sigel, H., Griesser, R., Chem. Soc. Rev., 2005, 34, 875-900

Simons, M., Schwarzler, F., Liitjohann, D., Bergmann, K.V., Beyreuther, K., Dichgans, J., Wormstall, H., Hartmann, T., Schulz, LB., Ann. Neurol., 2002, 52, 346-350

Singh, A.N., Newborn, J.S., Raushel, F.M., Bioorganic Chemistry, 1988, 16(2), 206-214

Soscia, S.J., Kirby, J.E., Washicosky, K.J., Stephanie, T., Ingelsson, M.M., Hyman, B., Burton, M.A., Goldstein, L.E., Duong, S., Tanzi, R.E., Moir, R.D., PLoS One, 2010, 5, e9505

Stern, N., Major, D.T., Gottlieb, H.E., Weizman, D., Fischer, B., Org. Biomol. Chem., 2010, 8, 4637

Storr, T., Scott, L.E., Bowen, M.L., Green, D.E., Thompson, K.H., Schugar, H.J., Orvig, C, Dalton Trans., 2009, 3034-3043

Toyota, K., Ishikawa, Y., Shirabe, K., Yoshifuji, M., Okada, K., Hirotsu, K., Heteroat. Chem., 1993, 4, 279-285

Tulapurkar, M.E., Kaubinger, W., Nahum, V., Fischer, B., Reiser, G., British Journal of Pharmacology, 2004, 142(5), 869-878

Tulapurkar, M.E., Zundorf, G., Reiser, G., Journal of Neurochemistry, 2006, 96(3), 624-634

Ubl, J.J., Vohringer, C, Reiser, G., Neuroscience (Oxford), 1998, 86(2), 597-609 Weaver, J., Pollack, S., Biochem. J, 1989, 261, 787-792

Weaver, J., Zhan, H., Pollack, S., Br. J. Haematol., 1993, 83, 138-144

White, A.R., Barnham, K.J., Huang, X., Voltakis, L, Beyreuther, K., Masters, C.L., Cherny, R.A., Bush, A.L, Cappai, R., JBIC, J. Biol. Inorg. Chem., 2004, 9, 269-280

Yatsunyk, L.A., Rosenzweig, A.C., /. Biol. Chem., 2007, 282, 8622-8631

Zlokovic, B.V., /. Neurochem., 2004, 89, 807-811

Zylberg, J., Ecke, D., Fischer, B., Reiser, G., Biochemical Journal, 2007, 405(2),