Neuroprotectants

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Identification of Highly Promising Anti-Oxidants / Neuroprotectants Based on Nucleoside 5’-Phosphorothioate Scaffold. Synthesis, Activity, and Mechanisms of Action Sagit Azran, Ortal Danino, Daniel Förster, Sarah Kenigsberg, Georg Reiser , Mudit Dixit, Vijay Singh, Dan T. Major, and Bilha Fischer J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00575 • Publication Date (Web): 08 Oct 2015 Downloaded from http://pubs.acs.org on October 9, 2015

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Journal of Medicinal Chemistry

Identification of Highly Promising Anti-Oxidants / Neuroprotectants Based on Nucleoside 5’-Phosphorothioate Scaffold. Synthesis, Activity, and Mechanisms of Action

Sagit Azran,a Ortal Danino,a Daniel Förster,b Sarah Kenigsberg,a Georg Reiser,b Mudit Dixit,a Vijay Singh,a Dan T. Major,a and Bilha Fischer*a

a

Department of Chemistry and the Lise Meitner-Minerva Center of Computational Quantum Chemistry, Bar-Ilan University, Ramat-Gan 52900 Israel b

Otto von Guericke University, Leipziger Str. 44, 39120, Magdeburg, Germany

ACS Paragon Plus Environment


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Abstract With a view to identify novel and biocompatible neuroprotectants, we designed nucleoside-5’-thiophosphate analogues, 6-11. We identified 2-SMe-ADP(α-S), 7A, as a most promising neuroprotectant. 7A reduced ROS production in PC12 cells under oxidizing conditions, IC50 0.08 vs. 21 µM for ADP. Furthermore, 7A rescued primary neurons subjected to oxidation, EC50 0.04 vs. 19 µM for ADP. 7A is a most potent P2Y1R agonist EC50 0.0026 µM. Activity of 7A in cells involved P2Y1/12-R as indicated by blocking P2Y12-R or P2Y1-R. Compound 7A inhibited Fenton reaction better than EDTA, IC50 37 vs. 54 µM, due to radical scavenging, IC50 12.5 vs. 30 µM for ADP, and Fe(II)-chelation, IC50 80 vs. > 200 µM for ADP (ferrozine assay). In addition, 7A was stable in human blood serum, t1/2 15 vs. 1.5 h for ADP, and resisted hydrolysis by NPP1/3, 2-fold vs. ADP. Hence, we propose 7A as a highly promising neuroprotectant.


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Introduction Neurodegenerative diseases and brain injury are associated with oxidative damage.1 Development of antioxidant agents is one of the approaches in the search for treatment of these health disorders. Another approach suggests the activation of P2Y-receptors (P2YRs), widely expressed in the nervous system and proposed to be involved in neuroprotection, for the treatment of oxidative damage.2, 3 The members of the P2 receptor (P2R) superfamily, consisting of ligand-gated ion channels (P2X-Rs) and G protein-coupled receptors (P2Y-Rs), are activated by endogenous extracellular nucleotides.4 Eight human P2Y-Rs subtypes are known so far (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11−P2Y14).5 The P2Y2,4,6-Rs are activated by uracil nucleotides, while the P2Y1,2,11 receptors are activated by adenine nucleotides (ATP, (1), or ADP, (2)) (Fig. 1). Neurons and astrocytes express both P2Y and P2X receptors. While ionotropic P2X receptors are mainly involved in fast synaptic neurotransmission, P2Y receptors mediate slow neuro-modulatory effects.6 In the brain, P2Y-R mediated signaling is involved in nervous tissue remodeling following trauma, stroke, ischemia or neurodegenerative disorders.3,

7

P2Y1-R and P2Y11-R mRNA are present in the human brain in large

quantities as compared to mRNAs in other tissues. Neurons and astrocytes express both sub-types of P2-Rs, while P2Y1-R is the predominant receptor in neurons.6 Neuronal activity-dependent release of 1 is one of the principal mechanisms underlying neuron- to -astrocyte intercellular communications.8 In addition to the rapid neurotransmitter-like action of 1 in normal brain function, it has become evident that some of the responses to 1 released during brain injury are neuroprotective.9 Some reports attributed this neuroprotective effect of 1 to its interaction with P2X-Rs and P2YRs by which the P2Y1-R appears to be the main receptor involved in this process.10 For



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instance, neuroprotection from methylmercury insult was reported to be induced by extracellular 1 through P2Y1-R.11 P2Y1-R stimulation by 2-SMe-ADP, (4), reduced ischemic neuronal lesions in mouse.12 In addition to 4, ATP-γ-S, (3), and ADP-β-S, (5), have also shown to be activators of P2Y1-R,13 which enhance neuroprotection8. Iron plays a vital role in various physiological functions and normal brain function. However, high level of iron can lead to significant oxidative damage via radical production within the brain, as in neurodegenerative diseases14 and ischemia.15 Specifically, OH radicals are generated from the less damaging reactive oxygen species, superoxide radical anion and hydrogen peroxide, in Fenton or Haber-Weiss reactions catalyzed by Fe(II)/Cu(I), or Fe(III)/Cu(II), respectively. Therefore there is a clear need for therapeutic agents, acting as Fenton reaction inhibitors, and targeting the elimination of toxic OH radicals, either by radical scavenging or by metal ion chelation mechanisms. In a quest for biocompatible and water soluble Fe (II)/Cu(I) chelators we considered nucleotide analogues. Specifically, purine nucleotide analogues are natural metal-ion chelators binding metal-ions by the purine ring N7 nitrogen atom, and / or the 5'-phosphate chain.16-18 Recently, we have studied natural nucleotides and the corresponding phosphorothioate analogues, as well as inorganic phosphates as inhibitors of Fenton reaction. We found that certain natural and synthetic adenine nucleotides at sub-millimolar concentrations prevented OH radical production from H2O2 in the presence of Cu(I)/Fe(II) ions better than standard antioxidants.16,

17

specifically, 3 proved a 100- and 20-times more active antioxidant at Fe(II)/H2O2 system than 1 and the potent antioxidant Trolox,16 respectively. Furthermore, we identified 3 as a highly promising antioxidant and neuroprotective agent in PC12 cells and primary neurons exposed to oxidative stress,19 which was significantly more potent than 5 and GDP-β-S. Yet, a major limitation of 3 is its enzymatic instability.


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Owing to the need for biocompatible neuroprotectants we designed and evaluated here adenosine 5’-phosphorothioate analogues capable of dual activity as both antioxidants and P2Y1-R agonists. Specifically, we report the synthesis of nucleotide analogues 6-11, their evaluation as P2Y1,11-R agonists, Fenton reaction inhibitors, radical scavengers, and inhibitors of ROS formation in PC12 cells and primary neurons under oxidative stress conditions. In addition, we report on the metabolic stability of the most promising nucleotide analogue. Results and discussion Selection of nucleotides with potential antioxidant- and P2Y1-R agonist- activity Previously we found that 3, was a potent antioxidant inhibiting OH radical production in the Fe(II)-H2O2 system.16 In addition, we found that 3 is a highly promising neuroprotectant rescuing primary neurons from insults such as FeSO4 and Aβ42.19 These findings have encouraged us to further explore the potential of adenosine 5'phosphorothioate analogues as antioxidants and neuroprotectants. Hence, we synthesized a series of adenosine 5'-phosphorothioate analogues, 6-11, and studied their antioxidant and neuroprotective activity as well as metabolic stability. We explored the structure-activity relationship of analogues 6-11. Specifically, we studied the following modifications: (1) the position of the phosphorothioate moiety (i.e. ADP-αS, 6, vs. 5); (2) presence of electron donating group on the adenine C2-position vs. electron withdrawing group (i.e. 2-SMe-ADP(α-S), 7, vs. 2-Cl-ADP(α-S), 8); (3) presence of di- vs. tri-phosphate group (8 vs. 2-Cl-ATP(α-S), 9); and (4) the effect of βphosphorothioate analogue bearing different C2-substituents (2-SMe-ADP(β-S), 10, vs. 2-Cl-ADP(β-S), 11). We selected SMe and Cl substitutions at the adenine C2 position of analogues 7, 10 and 8, 9, 11, respectively, since they were found to improve selectivity and potency of the ligand at the P2Y1 receptor.20 Recently, we reported on adenosine 5’-



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boranophosphate analogues which proved to be highly promising and selective P2Y1-R agonists (EC50 of 0.007-0.15 µM).21 Here, we targeted the synthesis and investigation of the related thiophosphate analogues 6-11 expecting them to be not only active agonists at P2Y1-R, but also better Fe(II) chelators (i.e. better antioxidants) than the corresponding boranophosphate analogues. Synthesis of nucleoside-5'-phosphorothioate analogues Compounds 6, 7, 8, and 9, were synthesized in a 4-step one-pot reaction from C2modified methoxymethylidene protected adenosine analogues, 15, 16 and 17 upon addition of 2-Cl-1,2,3-benzdioxaphosphorin-4-one in dry dioxane and dry pyridine to give phosphites, 18 (Scheme 1). The latter were treated with pyrophosphate tributylammonium salt in dry DMF to generate the cyclic intermediates, 19. Treatment of 19

with

S8

led

to

cyclic

thiotriphosphate

intermediates

20.

Subsequently,

ethylenediamine was added to generate 21 analogues upon elimination of cyclic phosphorodiamidate. Removal of the methoxymethylidene group involved a hydrolysis step at pH 2.3 and then at pH 9. Products 6-8, were obtained at 42-51% yield, respectively. During the synthesis of 8, analogue 9 was obtained as a byproduct at an 8% yield. Compounds 10, and 11 were obtained from 2-SMe-AMP, 22, and 2-Cl-AMP, 23, tributylammonium, trioctylammonium salts, respectively, in a 2-step one-pot reaction (Scheme 2). The latter AMP analogues were activated with carbonyldiimidazole (CDI) to give 22a and 23a, respectively, and then reacted with thiophosphate salt (bistributylammonium) in the presence of ZnCl2 for preventing conjugation via the sulfur atom. This synthetic method proved highly efficient as compared to a two-step reaction described for the preparation of related compounds.22 This one pot synthesis resulted in products 10 and 11 in 75% and 50% yields, respectively.


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Products 6-9, were obtained as pairs of diastereoisomers, due to a chiral center at Pα. These diastereoisomers were separated on HPLC showing 3-5 min difference of their retention times. The first and second eluting diastereisomers were denoted as A- and Bdiastereoisomers, respectively. Previously we have determined the absolute configuration of closely related nucleotide analogues.23 Based on that analysis we conclude that the first eluting isomer of 6-9, H8 of which is less shielded, is Rp isomer, and the second eluting isomer is Sp isomer. Evaluation of nucleotide analogues 6-11 as P2Y1/11-Rs agonists The activity of nucleotides 6-11 at the phyllo-genetically related P2Y1/11-Rs,24 was studied by measuring [Ca2+]i mobilization induced by these analogues and comparing it to that of the endogenous agonists of P2Y1-R and P2Y11-R, 2 and 1, respectively. These studies were performed in 1321N1 astrocytoma cells stably expressing the human P2Y1/11 receptors. Concentration-response curves were derived for a range of nucleotide concentrations (Figs. 2 and 3). The resulting EC50 values for the compounds evaluated, which are summarized in Table 1, allow comparison of the respective receptor affinities for P2Y1-R and P2Y11-R. At the P2Y1-R, isomers 6 A/B, (EC50 0.08 and 0.13 µM, respectively) were less active than 2, (EC50 0.024 µM) (Table 1 and Fig. 2), indicating that the replacement of the Pα non-bridging oxygen atom by sulfur atom did not improve the potency of the compound at the P2Y1-R. However, modification at the C2 position by Cl/SMe group, i.e., compounds 7 A/B, and 8 A/B, improved the agonist activity of 6. Introduction of SMe group at the C2 position of analogue 6 resulted in a highly active agonist, 7A, (EC50 0.0026 µM) being 5-fold more potent than 4, (EC50 0.013 µM), thus making 7A the most potent P2Y1-R agonist currently known. Isomer 7B was 6.5 fold less active than A isomer. Compounds 8A (EC50 0.03 µM) and 8B (EC50 1.5 µM) were 1.2 and 62-fold less



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active than 2, respectively. At the triphosphate analogues, 9 A/B, sulfur and Cl modifications did not improved activity at P2Y1-R as compared to 1 and their EC50 values were close to that of 1 (EC50 0.85 µM). For all Pα-S modified nucleotide analogues A isomer was more active than B isomer at P2Y1-R. Replacement of the terminal non-bridging oxygen atom of 2, (EC50 0.024 µM) by a sulfur atom 5, reduced the potency of 2 (EC50 1.27 µM). Modification at the C2 position of 5, by Cl/SMe to give 10, and 11 (EC50 0.037 and 0.09 µM, respectively) resulted in 34- and 14-fold, respectively, improved agonist activity at the P2Y1-R vs. 5 (EC50 1.27 µM). At P2Y11-R, compounds 6 A/B (EC50 4.5 and 8.7 µM, respectively) were less active than 2 (1.7 µM) indicating that the sulfur atom at the Pα-position is not tolerated by P2Y11-R (Table 1 and Fig. 3). Compound 7 containing SMe modification showed a similar activity to that of 2 for B isomer (EC50 1 µM), and reduced agonist activity for A isomer (EC50 3.2 µM). Compound 8 B isomer (EC50 0.5 µM) substituted by 2-Cl was 2-fold more active than 2, while A isomer was equipotent to 2 (EC50 1.4 µM). The triphosphate analogues 9 A/B showed reduced potency for A isomer (EC50 9.1 µM), as compared to 2 (EC50 1.7 µM) and 1 (EC50 6.7 µM), and improved potency for B isomer (EC50 1.1 µM) as compared to 2 and 1. A clear preference was observed for B isomer over A isomer at the Pα-S modified nucleotides tested at P2Y11-R. Compound 5 showed extremely poor activity with EC50 of 32 µM, while C2-Cl/SMe modification at 10 and 11 resulted in EC50 of 0.9 and 1 µM, respectively. These results suggest that the enhancement of P2Y11-R agonist activity is mainly attributed to the Cl/SMe modification at the C2 position of the adenosine ring. Compounds 7B, 8A, 9B and 10 were about 2-fold more active than 2 at P2Y11-R but they were not P2Y11-R selective since they also have agonist activity at P2Y1-R. In addition,


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compounds 8B and 11 were 3.4- and 2-fold respectively, more active than 2 at P2Y11-R, while their agonist activity at the P2Y1-R was low. However, the most active agonist at P2Y1-R, 7A, was 2-fold less active than 2 at the P2Y11-R. Evaluation of nucleotide analogues 6-11 as Fenton reaction inhibitors We used ESR to monitor the modulation of ·OH formation from H2O2 in the Fe(II)induced Fenton reaction by analogues 6-11. As the hydroxyl radical formed in the reaction is extremely short-lived, we used 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap.25 DMPO-OH adduct was then detected by ESR. The addition of nucleotides to Fe(II)-H2O2 mixture lowered DMPO-OH signal due to metal-ion chelation and/ or radical scavenging.16 We found that nucleoside-5'-thiophosphate analogues are good Fenton reaction inhibitors being 2.5-8-fold more active than 2 and EDTA, respectively, and far more active than 1 (Table 2). The differences between the activity of compounds 10-11 containing a sulfur atom at the Pβ position (IC50 19-20 µM), and compounds 6-8 containing a sulfur atom at the Pα position (IC50 31-39 µM) may be related to the fact that under Fenton reaction conditions oxidation of ADP-α-S moiety to the corresponding disulfide dimer provides two terminal distant phosphate groups for Fe(II)-coordination, while oxidation of the ADP-β-S moieties provides four neighboring phosphate groups for Fe(II) coordination.16 Specifically,

previously

we

found

that

under

Fenton

reaction

conditions,

phosphorothioate compounds underwent rapid oxidation to form the corresponding disulfide dimers.16 We found that, 3, disulfide-dimer has six phosphate coordination sites and may form an octahedral complex with Fe(II). Occupation of all Fe(II) coordination sites by this chelator precludes the binding of H2O2 to Fe(II)-chelates and the subsequent Fenton reaction. ATP-α-S, however, was a 30-fold weaker inhibitor as compared to 3.16



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This may be due to the fact that ATP-α-S dimer contains only four phosphate coordination sites. In addition, we found that there was no difference in the activity between both isomers of the Pα-S compounds, e.g. IC50 values of 7 isomers A/B are 37 and 38 µM, respectively (Fig. 4). This result supports our hypothesis that the disulfide dimer is the actual Fenton reaction inhibitor. Compound 2 binds Fe(II) preferentially in a "closed" structure in which the coordination to the metal-ion is both through the phosphate chain and the adenine N7-nitrogen atom.26 Therefore substitution of an electron withdrawing- or donating-group on the nucleobase will affect electron density on N7-nitrogen atom and hence, affinity to metal-ion. However, comparison of C2-modified nucleotides to the corresponding parent compounds, 6, showed that substitution of C2 by an electron withdrawing group, as Cl, or an electron donating group, as SMe, had no influence on the antioxidant activity. Namely, the modified adenine moiety is probably not directly involved (although an outer sphere coordination is possible) in Fe(II)-chelation, and it is probably the disulfide dimer that is the actual chelator via the two terminal phosphate groups. Evaluation of the radical scavenging activity of analogues 6-11 using ABTS decolorization assay Fenton reaction can be inhibited by both metal-ion chelators and radical scavengers. Hence, we evaluated nucleotide analogues 6-11 also as radical scavengers by the 2,2'azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

(ABTS) decolorization assay.27

ABTS•+ is formed by the oxidation of ABTS with potassium persulfate, and absorbs at 0.645, 0.734 and 0.815 µM. The scavenging of ABTS•+ radical is determined as a function of the antioxidant concentration, and is calculated relative to the reactivity of Trolox as a standard.


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We found thiophosphate analogues 6-11 to be good radical scavengers with IC50 values around 12 µM as compared to IC50 of 18 and 30 µM for Trolox and 2, respectively (Table 3). This improved antioxidant activity of analogues 6-11 is probably due to the thiophosphate group which can donate an electron to ABTS•+ and subsequently form a disulfide dimer. Evaluation of the relative binding of Fe(II) ions to nucleotide 7A and ADP In order to evaluate the ability of 7A to chelate Fe(II)-ions we performed competitive studies of the relative binding of Fe(II) by 7A vs. ferrozine (Fig. 5). Specifically, we measured the absorbance of the formed colored Fe(II)-ferrozine complex in the presence or absence of 7A and ADP. Compound 7A inhibited 50% of the formation of Fe(II)ferrozine complex at 80 µM, while, ADP at maximal concentration of 200 µM inhibited only 40% of Fe(II)-ferrozine complex formation. These findings are consistent with our previous studies on nucleotides as inhibitors of formation of Fe(II)-ferrozine complex vs. Fe(II)-chelators such as DTPA, Trolox, and citrate.28 In that study we found that nucleoside-thiophosphate analogues were better Fe(II)-chelators vs. the corresponding natural nucleotides, e.g.

IC50 values of ADP-β-S and ADP were 85 and 250 µM,

respectively vs. DTPA, Trolox, and citrate, 90, 150 and 300 µM, respectively. Our findings here and our previous results support the assumption that a sulfur atom replacing oxygen in the phosphate moiety improves the antioxidant activity of the molecule due to improved affinity to Fe(II)17,

29

and improved chemical and metabolic stability of the

compounds.30-32

Inhibition of ROS production in PC12 cells under oxidizing conditions by thiophosphate analogues 6-11



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Previously we studied the modulation of Fe(II)-induced oxidative stress in PC12 cells by nucleotides and dinucleotides, using DCFH-DA,19 a radical sensitive indicator.33 When DCFH-DA

is

oxidized

by

ROS,

it

is

converted

to

2',

7'-

dichloro-

fluorescein (DCF), and emits green fluorescence. The fluorescence intensity, a function of the ROS concentration in the cells, is measured quantitatively by a fluorimeter. Here, we measured analogues 6-11 as potential inhibitor of ROS formation in PC12 cell treated with FeSO4. Table 4 summarizes IC50 values obtained for phosphorothioate analogues 6-11 vs. the corresponding parent nucleotides (1, 2 and 5). A reduction of 50% of total ROS concentration, was observed at 21 µM 2, while replacement of Pα of 2 by thiophosphate in 6, resulted in slightly less active analogues, IC50 31 and 36 μM for isomers A/B, respectively. However, substitution of the adenine C2-position by SMe in addition to thiophosphate at Pα, 7A, improved ADP’s antioxidant activity 250-fold (IC50 0.08 µM). Analogue 7A was 7-fold more potent than 7B (IC50 0.6 µM). In addition, substitution of Cl at the C2 position in 8 improved antioxidant activity vs. 2 100- and 200- times for A and B isomers, respectively. However, replacement of the Pβ-nonbridging oxygen atom by sulfur atom, 5, has not changed the antioxidant activity of 2 (IC50 20 µM). Interestingly, substitution of 2-Cl/SMe in addition to thiophosphate at Pβ resulted in improved antioxidant activity. Specifically, 11 and 10, inhibited ROS production with IC50 values of 5 and 12 µM vs. 20 µM for 5. The least active compound in this series was 9, with IC50 of 100 µM for both isomers. Analysis of the SAR of the tested compounds at PC12 cells under oxidizing conditions revealed that: (A) A sulfur atom at either Pα or Pβ does not improve antioxidant activity of 2. The introduction of a sulfur atom at Pα or Pβ position, in 6 A/B or 5, resulted in 2-fold less active and equiactive compounds, respectively, to 2. (B) Substitution of Cl/SMe at the C2 position of the adenine ring improves antioxidant activity. 2-SMe substitution at 6A, improves


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antioxidant activity of 7A 470-fold. 2-Cl substitution in 8A resulted in a 350-fold more active compound. These results support our hypothesis that the mechanism of the antioxidant activity also involves P2Y-R activation (see above). (C) Triphosphate analogue 9 is a poor antioxidant. Compound 1 did not reach 50% inhibition of ROS production up to 50 µM. Neither introduction of sulfur atom at Pα position, nor 2-Cl modification at the nucleobase, improved the antioxidant activity of 9 A/B. This finding also supports that anti-oxidant activity of 2 analogues involves activation of ADPbinding- P2Y-Rs (e.g. P2Y1-R and P2Y12-R). Protectant activity of 2-SMe-ADP(α α-S), 7A, involves also P2Y1-R and P2Y12-R activation P2Y1-R and P2Y12 are purinergic receptors activated by endogenous ADP and a synthetic agonist, 2-MeS-ADP. It is well known that both receptors enhance neuroprotective activity.11, 12 The activity of P2Y1- and P2Y12-Rs can be blocked by MRS217934 and 2MeS-AMP, respectively.35 Here, we investigated the possible involvement of P2Y1-R and P2Y12-R in protection of cells against oxidative stress by the most promising compound identified here, 2-SMeADP(α-S), 7A (Fig. 7). First, we studied the effect of 7A on P2Y12-R, using PC12 cells, under oxidizing conditions, with or without the antagonist 2-MeS-AMP (Fig. 7A). We selected PC12 cells for this study, rather than primary neurons, since the former express P2Y12-R, but not P2Y1-R.36,37 Specifically, PC12 cells were treated with 2-MeS-AMP and then with DCFH-DA, finally, oxidation was initiated by addition of FeSO4. The effect of the antagonist on percentage of inhibition of ROS formation (vs. control not containing 2-MeS-AMP), was found to be negligible (ca. 2%). When this experiment was repeated with analogue 7A, added at a final concentration of 0.01-25 µM, up to 40% decrease in the antioxidant activity of 7A in the presence of 2-MeS-AMP was measured.



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Next, we evaluated the involvement of P2Y1-R in the antioxidant activity of 7A, using Ntera-2 cells, under oxidizing conditions, with or without the antagonist, MRS2179 (Fig. 7B). We used Ntera-2 cells, since they endogenously express a functional P2Y1 receptor, while other P2Y subtypes, except perhaps P2Y4, are not functionally expressed.38 Therefore, Ntera-2 cells provide a useful neuronal-like cellular model for studying the precise signaling pathways and physiological responses mediated by a native P2Y1 receptor. At Ntera-2 cells, we found that 7A inhibited 50% of ROS formation at 0.2 µM. When we incubated Ntera-2 cells with the antagonist MRS2179, prior to treatment with 7A, the antioxidant activity was reduced by 35%. MRS2179 alone (no 7A was added) did not affect the ROS reduction. These results suggest that the antioxidant activity of 7A also involves the activation of P2Y1-R and P2Y12-R. Resistance of 7 A/B to hydrolysis by ecto-nucleotidases The therapeutic merit of nucleotide analogues is dependent on their metabolic stability, specifically on their resistance to hydrolysis. Ecto-nucleotide pyrophosphatase / phosphodiesterase (eNPP) family is one of the principal enzyme families that metabolize extracellular nucleotides. Since 7A was found here to be the most potent antioxidant and P2Y1-R agonist, we tested the resistance of 7 A/B to hydrolysis by eNPP1, 3. We compared the hydrolytic stability of 7 A/B to that of its parent nucelotide 2, and the primary endogenous substrate of eNPP1/3, 1. We found that although 7 A/B did undergo hydrolysis by eNPP1 and 3 (23.7 – 42.0%, Table 5), both analogues 7 A and B were considerably more stable than 2 (74.5 and 106% hydrolysis of 2 by eNPP1 and eNPP3, respectively). Interestingly it was the B analogue which was found to be more resistant to enzymatic hydrolysis proving to be four times more stable than 1 and 3- and 4- times more stable than 2 at NPP1 and eNPP3, respectively. As eNPP1/3 hydrolyze the phosphate chain mostly between Pα-Pβ,39 it


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seems that the replacement of the Pα non bridging oxygen with a sulfur atom has a role to play in inhibiting 7A/B hydrolysis as well as possibly to enable stronger binding to the zinc containing catalytic site.40’41 Analogue 7A is relatively resistant to hydrolysis in human blood serum The usage of nucleotides as therapeutic agents is limited due to their rapid dephosphorylation by enzymes in physiological systems. Blood serum contains such enzymes and, therefore, provides a good model system for estimation of the in vivo stability of nucleotide analogues.42,

43

Here, we evaluated the stability of the most

promising analogue identified here, 7A, in human blood serum as compared to 2, and 6A. These analogues were incubated in human blood serum and RPMI-1640 at 37 ºC for 0.5– 24 h. The hydrolysis rate of the nucleotide analogues was determined by measuring the change in the integration of the HPLC peaks for each analogue over time. Compound 2 was hydrolyzed to AMP, followed by further degradation to adenosine, with a half-life of 1.5 h (Fig. 6). Yet, analogue 7A was hydrolyzed with a half-life of 15 h, and analogue 6A displayed a half-life of 14.5 h, indicating that the major contribution to stability enhancement of 2 is the thiophosphate substitution. Analogue 7A is a highly potent neuroprotectant Encouraged by the beneficial properties of 7A, we evaluated its neuroprotective effect. Primary cortical neurons were treated with 3 µM FeSO4 and 0.01-25 µM 7 A/B for 24 h, and cell viability was measured by MTT assay (Fig. 8). We found that 2, protects primary neurons in the presence of FeSO4, with EC50 19 µM, while 7A was found to be a 475-time more potent neuroprotectant (EC50 0.04 µM). In addition, 7A was 15-fold more active than B isomer. These results are in good correlation with the higher potency of 7A at P2Y1-R, thus implying the involvement of this receptor in protection of primary



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neurons. In addition, P2Y12-R may possibly be also involved in the neuroprotective activity of 7A. Analogue 7A helps maintain normal neuron morphology In order to establish the effects of 7A, on cell morphology, especially on cytoskeleton elements, we co-applied 7A and FeSO4 to primary cortical neurons, and viewed the cells by light microscopy (Fig. 9). Viewing cells after treatment with 3 µM FeSO4 for 24 h (Fig. 9B) as compared to control (Fig. 9A), clearly showed a change in the morphology of the primary cortical neuron cells. After treatment with FeSO4, the amount of cells decreased dramatically. In addition, the cytoskeleton elements were damaged and the cells lost their extensions as compared to control. When cortical neurons were treated with increasing amounts of 7A (0.2, 5 and 100 µM) (Fig. 9C, 9D and 9E), the number of vital neuronal cells increased correspondingly. Furthermore, the morphology of the cells at 5 and 100 µM 7A was almost similar to that of the native neurons (Figs. 9D and 9E).

EC50 values of P2Y1-R agonists 1-11 are correlated with the docking score and desolvation energies To rationalize the difference of binding interactions of P2Y1R agonists 1-11, we performed docking studies of these nucleotides into crystal structures of P2Y1R. Two crystal structures of the human P2Y1R (pdb codes: 4XNV and 4XNW) are available.44, 45 The crystal structure 4XNV is a complex of P2Y1R with a non-nucleotide antagonist,

1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-(4-(trifluoromethoxy)phenyl)

urea (BPTU). In this structure, BPTU binds outside the protein, at the protein-lipid interface in the transmembrane domain (Fig. 10A). The other crystal structure, 4XNW, is a complex with a nucleotide-based antagonist (1′R,2′S,4′S,5′S)-4-(2-Iodo-6methylaminopurin-9-yl)-1-[(phosphato)methyl]-2(phosphato)bicycle[3.1.0]-hexane


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(MRS2500). In this complex, MRS2500 binds at the top of extracellular side of the protein within the loops and alpha helices (Fig. 10B). Unfortunately, no crystal structure of human P2Y1R with agonists has yet been reported. We note that the human P2Y12R complex with an agonist, 4, is available (pdb code: 4PXZ).45 In this complex, the agonist binds deep inside the receptor transmembrane region, with the purine base interacting with hydrophobic and polar residues, while the phosphate moiety interacts with several positively charged residues. It is tempting to attempt modeling of P2Y11R using the homology modeling based on the P2Y1R or P2Y12R structures. Unfortunately, the sequence identities and similarities (Table 6) between these receptors are not high enough to obtain meaningful homology models. We considered both the 4XNV and 4XNW receptor structures for docking ligands 1-11. In the 4XNW receptor, all the ligands show binding to the receptor, with high docking scores (Table 7). Surprisingly, all the ligands bind at the top of the extracellular side of the receptor within the loop region (Fig. 10D). These binding poses are similar to that in the crystal structure with the antagonist MRS2500. We subsequently docked the ligands into the 4XNV structure. In the 4XNV receptor, all ligands bind inside the bundle of alpha helices. In this receptor, the adenine moiety of the ligands makes contact with hydrophobic residues, whereas the phosphate chain interacts with positively charged residues (Fig. 10C). We note that in the 4XNV receptor, all the ligands bind in similar binding poses deeper into the receptor pocket than MRS2500 (in 4XNW). Interestingly, these binding pose are rather similar to the experimental binding pose of agonist 4 in P2Y12R, although not quite as deep inside the transmembrane helical bundle.



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We propose that an important difference in the 4XNW and 4XNV structures is the position of Asp204. In the former crystal structure, this residue protrudes deeper into the transmembrane region than in the latter, possibly to minimize repulsion with the antagonist phosphate chain. As a result, a binding pocket deeper inside the transmembrane region is partially blocked in 4XNW, hence not allowing agonist binding. In the 4XNV structure, this alternative binding pocket is available for agonist binding, and indeed, is occupied in our docking simulations. Furthermore, this suggests that the position of Asp204 is variable, depending on the ligand. Nonetheless, the calculated interaction energies for some of the ligands were found to be unfavorable in the 4XNV structure. We ascribe this to repulsion between Asp204 and the ligand phosphate moieties (Table 7). Therefore, we rotated Asp204 by 90 degrees and redocked the ligands. Interestingly, in the modified receptor (4XNVMOD) we found high docking scores (favorable binding) for all the ligands. The CDOCKER interaction energies of all ligands with 4XNV, 4XNW and 4XNVMOD are shown in Table 7. To understand the experimental trends of the EC50 values, we predicted the EC50 values using regression analysis with solvation energy (PSol), with CDOCKER interaction energies (PCDOCK) and with both solvation and CDOCKER interaction energies (PCDOCK+Sol). Based on the desolvation energies alone, we can rationalize the difference between several ligands. For instance, we obtained the following desolvation trend ATP, 1 > 2-Cl-ATP(α-S), 9 A/B ~ ADP, 2 ~ 2-SMe-ADP, 4 > ADP(α-S), 6 A/B ~ 2SMe-ADP(α-S), 7 A/B (1 has highest desolvation energy). This suggests that 1 pays a greater free energy penalty on binding to P2Y1R than 2. On the other hand, the P2Y1-R binding site has evolved to bind 2, and hence is likely unable to fully compensate for ATP’s loss of solvent interactions via binding site interactions. Hence, 1 is a poorer agonist than 2. Similarly, 5 and 6 A/B is easier to desolvate than 2, due to weaker


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interaction of the thiophosphate moiety with water than phosphate. Attempts to correlate PCDOCK values only with EC50 suggest that the optimal fit is obtained using the 4XNVMOD structure for docking (Fig. 11B and S1B and S2B). Nonetheless, the PSol values and PCDOCK values alone do not reproduce the experimental trends of EC50 (Fig. 11, Fig. S1 and Fig. S2). Interestingly, PCDOCK+Sol values for the 4XNVMOD receptor show similar EC50 trends as that obtained experimentally. We note that such agreement with the experimental EC50 values was not obtained using the 4XNW and 4XNV structures (Fig. S1 and S2). Figure 12 shows the binding pocket of 4XNVMOD receptor with ADP and 2-SMeADP(α-S). The positive residues Arg128 and Arg310 are found to interact with all the ligands as shown for ADP and 2-SMe-ADP.46 Interestingly, mutagenesis of P2Y1-R also suggests these to be active in ligand binding. Gln307 and Asp204 are also suggested to participate in ligand recognition based on mutagenesis data,46 and these residues are also found to interact with our docked ligands in the 4XNVMOD receptor. Various ligand-receptor interactions observed in the current docking simulations are in agreement with our early predictions by modeling studies done in absence of crystal structures23,

47

For instance, repulsive interaction between the phosphate moiety and

Asp204, π−π stacking of the adenine ring with Phe131 and binding of the phosphate moiety with Arg128 and Arg310 were all predicted in our earlier model. We concluded that the EC50 values of P2Y1R agonists 1-11 cannot be explained solely on the basis of either ligand interaction energy or desolvation energy, but rather on their combination. The binding of agonists inside the modified protein pocket is clearly different from that of antagonists in the crystal structures. The adenine moiety of the agonists interacts with hydrophobic residues via π−π interaction (Phe131) and the phosphate-chain



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interacts with positively charged residues (Arg128 and Arg310). This is in agreement with mutagenesis data

46

and our early studies.23, 47 The predicted EC50 values clearly

distinguish between the ATP-based, ADP-based and sulfur containing ADP and ATP analogues. We correlate this with desolvation and docking energies. Yet, the docking simulations are not sufficiently accurate to distinguish between diastereoisomers, e.g. 7A/B. Conclusions In this study we targeted the identification of a potent neuroprotectant based on an adenosine 5'-thiophoshate scaffold. Out of the series of analogues 6-11, we have found 7A, as a most promising protectant rescuing PC12 cells and primary neurons from oxidative stress initiated by FeSO4 with EC50 value of 0.04 µM. Furthermore, compound 7A helped maintain the normal morphology of the neurons undergoing oxidative insult. Based on our findings here, we propose that the neuroprotective action of 7A is due to its activity as an antioxidant, both as a Fe(II)-chelator and OH radical scavenger in Fenton reaction. In addition, 7A is a highly potent P2Y1-R agonist which also activates P2Y11-R and P2Y12-R. In particular, the activation of P2Y1-R is known to result in neuroprotection. EC50 values of P2Y1-R agonists 1-11 correlate well with a combination of desolvation and docking energies. Finally, 7A was found to be relatively metabolically stable both to isolated ectonucleotidases and in human blood serum. In the light of the above data, we propose 7A as a promising neuroprotectant. The results of in-vivo studies using 7A will be published in due course. Experimental Chemistry. General. All air and moisture sensitive reactions were carried out in flamedried, argon-flushed, two-neck flasks sealed with rubber septa, and the reagents were


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introduced by syringe. Progress of reactions was monitored by TLC on pre-coated Merck silica gel plates (60F-254). Visualization was accomplished by UV light. Flash chromatography was carried out on silica gel (Davisil Art. 1000101501). Separations were carried out using also an HPFC automated flash purification system (Biotage SP1 separation system (RP)). Compounds were characterized by NMR using Bruker AC200, DPX-300 or DMX-600 spectrometers. 1H NMR spectra were recorded at 200, 300, 600 or 700 MHz. Chemical shifts are expressed in ppm downfield from Me4Si (TMS), used as an internal standard. Nucleotides were characterized also by 31P NMR in D2O, using 85% H3PO4 as an external reference on Bruker AC-200 and DMX- 600 spectrometers. High resolution mass spectra were recorded on an AutoSpec Premier (Waters UK) spectrometer by chemical ionization. Nucleotides were analyzed under ESI (electron spray ionization) conditions on a Q-TOF micro-instrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a Sephadex DEAE-A25 column, swollen in 1M NaHCO3 at 4◦C for 1 day. The resin was washed with deionized water before use. The LC separation was monitored by UV detection at 0.28 µM. A buffer gradient of NH4HCO3 was applied as detailed below. Final purification of the nucleotides was achieved on an HPLC (Merck-Hitachi) system, using a semi-preparative reverse-phase column (Gemini 5u C-18 110A, 250×10.00 mm, 5 micron, Phenomenex, Torrance, USA). The purity of the nucleotides was evaluated with an analytical reverse-phase column system (Gemini 5u C-18 110A, 150 mm ×4.60 mm; 5 µm; Phenomenex, Torrance, CA) using two solvent systems: solvent system I, (A) 100 mM triethylammonium acetate (TEAA), pH 7:(B) CH3CN; solvent system II, (A) 0.01 M KH2PO4, pH = 4.5:(B) CH3CN. The details of the solvent system gradients used for the separation of each product are given below. The purity of the nucleotides was generally ≥90%. All commercial reagents were used without further purification,



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unless otherwise noted. All reactants in moisture sensitive reactions were dried overnight in a vacuum oven. All phosphorylation reactions were carried out in flamedried, argon-flushed, two-neck flasks sealed with rubber septa. Nucleosides were dried under vacuum overnight. Phosphorus oxychloride was distilled and kept under nitrogen. The

tri-n-butylammonium

pyrophosphate

and

tri-n-butylammonium phosphate

solutions were prepared as previously described.48 Typical procedure for preparation of adenosine nucleoside 5'-diphosphate–α α-S derivatives.

2-Chloro-2',3'-O-methoxymethylidene adenosine, 16, (140 mg, 0.41

mmol, 1 eq) was dissolved in DMF (3.9 mL), and a freshly prepared solution of 2chloro-1,3,2-benzodioxaphosphorin-4-one (0.452 mmol, 1.1 eq) in dry dioxane and dry pyridine added. After stirring for 10 min, a freshly prepared 0.5 M solution of bis(tri-nbutylammonium) pyrophosphate (0.61 mmol, 1.5 eq) in DMF and tri-n-butylamine (1.64 mmol, 4 eq) were simultaneously added. Precipitation occurred immediately after the addition of the reagents but disappeared with further stirring. Sulfur (4.1 mmol, 10 eq) was added, and the mixture was stirred for 15 min. Ethylenediamine (2.05 mmol, 5 eq) was then added. A brown precipitate was immediately formed. After stirring for 60 min, deionized water (0.13 mL) was added and the brown precipitate gradually dissolved. The reaction mixture was filtered through Buchner funnel and then diluted with deionized water and washed twice with di-ethyl ether. The aqueous layer was then freeze-dried. Methoxymethylidene protecting group was removed by acidic hydrolysis (10% HCl solution was added until pH 2.3 was obtained). After 3 h, at RT, the pH was rapidly raised to 9 by addition of 24% NH4OH solution (pH 11), and the solution was stirred at RT for 45 min and then freeze-dried. The residue was subjected to ionexchange chromatography (on DEAE A25 Sephadex, swollen overnight in 1M NaHCO3 at 4ºC). The column was eluted with an ammonium bicarbonate gradient of 0-


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0.2 M (300 mL each), and then 0.2-0.4 M (150 mL each). Finally, relevant fractions containing the products were separated on HPLC, as described below, and finally were passed through a Dowex 50WX8-200 ion-exchange resin Na+-form column and eluted with deionized water to obtain the corresponding sodium salts after freeze-drying.

2-Methylthioadenosine-5'-O-(α α-thio)-diphosphate (7A/B) The separation of diastereoisomers, 7A and 7B, was accomplished using a semipreparative reverse-phase Gemini 5u column and isocratic elution with 87:13 (A) 100 mM TEAA, pH 7:(B) CH3CN at a flow rate of 4 mL/min. Fractions containing purified isomers [Rt: 9.2 min (7A); 10.8 min (7B isomer)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles, with the solid residue dissolved each time in deionized water. Diastereoisomers 7A and 7B were obtained at a 23% overall yield (36 mg) after LC separation. Compound 7A, 1H NMR (D2O, 200 MHz): δ 8.51 (s, 1H, H-8), 6.15 (d, J=6.2 Hz, 1H, H-1'), 4.7 (m, H-2' signal is hidden by the water signal), 4.61 (m, 1H, H-3'), 4.4 (m, 1H, H-4'), 4.28 (m, 2H, H-5', H-5''), 2.61 (m, 3H, CH3) ppm.

31

P NMR (D2O, 200 MHz)

δ: 43.46 (d, J=31.1 Hz, Pα-S), -10.76 (d, J=31.1 Hz, Pβ) ppm. HR MALDI (negative): calcd for C11H16N5O9P2S2 458.9859, found 487.9870. Purity data obtained on an analytical column- Rt: 4.62 min (98% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Rt: 7.53 min (93% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Compound 7B, 1H NMR (D2O, 200 MHz): δ 8.48 (s, 1H, H-8), 6.15 (d, J=4.2 Hz, 1H, H-1'), 4.69 (m, H-2', H-3'), 4.33 (m, 1H, H-4'), 4.25 (m, 2H, H-5', H-5''), 2.61 (m, 3H, CH3) ppm. 31P NMR (D2O, 200 MHz) δ: 41.33 (d, J=31.3 Hz, Pα-S), -5.916 (d, J=31.1 Hz, Pβ) ppm. HR MALDI (negative): calcd for C11H16N5O9P2S2 487.9859, found



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487.9852. Purity data obtained on an analytical column- Rt: 7.86 min (96% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Rt: 10.28 min (92% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min).

2-Chloroadenosine-5'-O-(α α-thio)-diphosphate (8A/B) The separation of diastereoisomers, 8A and 8B, was accomplished using a semipreparative reverse-phase Gemini 5u column and isocratic elution with 90.5:9.5 (A) 100 mM TEAA, pH 7:(B) MeOH at a flow rate of 4.5 mL/min. Fractions containing purified isomers [Rt: 8.49 min (8A); 11.69 min (8B)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles, with the solid residue dissolved each time in deionized water. Diastereoisomers 8A and 8B were obtained at a 42% overall yield (84 mg) after LC separation. Compound 8A, 1H NMR (D2O, 200 MHz): δ 8.58 (s, 1H, H-8), 6.03 (d, J=5.8 Hz, 1H, H-1'), 4.6 (m, 1H, H-2'), 4.54 (m, 1H, H-3'), 4.39 (m, 1H, H-4'), 4.24 (m, 2H, H-5', H5'') ppm.

31

P NMR (D2O, 200 MHz) δ: 44.51 (d, J=30.4 Hz, Pα-S), -3.425 (d, J=30.2

Hz, Pβ) ppm. HR MALDI (negative): calcd for C10H13Cl1N5O9P2S1 475.9592, found 475.964. Purity data obtained on an analytical column- Rt: 3.92 min (90% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Rt: 5.16 min (93% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Compound 8B, 1H NMR (D2O, 200 MHz): δ 8.55 (s, 1H, H-8), 6.01 (d, J=4.8 Hz, 1H, H-1'), 4.7 (m, 1H, H-2' signal is hidden by the water signal), 4.63 (m, 1H, H-3'), 4.39 (m, 1H, H-4'), 4.26 (m, 2H, H-5', H-5'') ppm. 31P NMR (D2O, 200 MHz) δ: 41.44 (d,


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J=30.78 Hz, Pα-S), -5.91 (d, J=31.2 Hz, Pβ) ppm. HR MALDI (negative): calcd for C10H13Cl1N5O9P2S1 475.9592, found 475.9630. Purity data obtained on an analytical column- Rt: 8.96 min (91% purity) using solvent system I (isocratic elution of 92:8 A:B over 20 min at a flow rate of 1 mL/min). Rt: 10.12 min (90% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). 2-Chloroadenosine-5'-O-(α α-thio)-triphosphate (9A/B) This analogue was obtained as a by-product from the above described synthesis of 8. After LC separation, the relevant fractions were pooled and freeze-dried three times to yield a white solid. Final separation of the diastereomers and purification of the relevant fractions was carried out on an HPLC system, using a semi-preparative reverse-phase column, under the conditions described below. The purity of the nucleotides was evaluated on an analytical reverse-phase column system, in two solvent systems as described below. Finally, aqueous solutions of the products were passed through a Dowex 50WX8-200 ion-exchange resin Na+-form column and the products were eluted with deionized water to obtain the corresponding sodium salts after freeze-drying. The separation of diastereoisomers, 9A and 9B, was accomplished using a semipreparative reverse-phase Gemini 5u column and isocratic elution with 90.5:9.5 (A) 100 mM TEAA, pH 7:(B) MeOH at a flow rate of 4.5 mL/min. Fractions containing purified isomers [Rt: 10.75 min (9A); 15.49 min (9B)] were collected and freeze-dried. Excess buffer was removed by repeated freeze-drying cycles, and the solid residue was dissolved each time in deionized water. Diastereoisomers 9A and 9B were obtained at 8% overall yield (20 mg) after LC separation.



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Compound 9A, 1H NMR (D2O, 200 MHz): δ 8.66 (s, 1H, H-8), 6.05 (d, J=6 Hz, 1H, H1'), 4.3 (m, 2H, H-2', H-3'), 3.97 (m, 1H, H-4'), 3.7 (m, 2H, H-5', H-5'') ppm. 31P NMR (D2O, 200 MHz) δ: 44.4 (d, J=30 Hz, Pα-S), -5.23 (d, J=18.9 Hz, Pγ), -21.753 (m, Pβ) ppm. HR MALDI (negative): calcd for C10H13Cl1N5O9P2S1 555.9256, found 555.9261. Purity data obtained on an analytical column- Rt: 6.67 min (90% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Rt: 5.69 min (92% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Compound 9B, 1H NMR (D2O, 200 MHz): δ 8.64 (s, 1H, H-8), 6.03 (d, J=5.4 Hz, 1H, H-1'), 4.68 (m, 2H, H-2', H-3'), 4.56 (m, 1H, H-4'), 4.33 (m, 2H, H-5', H-5'') ppm. 31P NMR (D2O, 200 MHz) δ: 43.55 (d, J=30 Hz, Pα-S), -5.21 (d, J=20.3 Hz, Pγ), -21.86 (dd, J=20.4, 26 Hz, Pβ) ppm. HR MALDI (negative): calcd for C10H13Cl1N5O9P2S1 555.9256, found 555.9243. Purity data obtained on an analytical column- Rt: 9.71 min (91% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Rt: 9.29 min (90% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Typical procedure for preparation of adenosine 5'-monophosphate derivatives. A solution of 2-Cl-adenosine, (13), (200 mg, 0.365 mmol) in dry trimethyl phosphate (2 mL) was cooled to -15°C using an ethylene glycol-dry ice bath; then Proton Sponge® (427 mg, 1.99 mmol, 3 eq) was added. After 20 min, distilled phosphorus oxychloride (91 µL, 0.96 mmol, 2 eq) was added drop-wise. Stirring continued for 3h at -15 °C. TLC on a silica gel plate (isopropanol: 25% NH4OH: H2O 11:2:7), indicated the disappearance of the starting material and the formation of a more polar product. 1M TEAB solution (8 mL, pH 8) was then added until neutralization, and the clear solution was stirred at room temperature for 45 min. The solution was freeze-dried overnight.


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The semisolid obtained after freeze-drying was chromatographed on an activated Sephadex DEAE-A25 column. The resin was washed with deionized water and loaded with the crude reaction residue dissolved in a minimal volume of water. The separation was monitored by UV detection at 280 nm. A buffer of 0-0.2 M NH4HCO3 was used. The relevant fractions were collected and freeze-dried three times until a constant weight was obtained, to yield the product as a white solid (220 mg, 79%). [PSO3H]2-(Bu3NH+)2

salt

[PSO3H]2-(Bu3NH+)2

salt

was

prepared

from

the

corresponding sodium salt (tribasic hydrate). The thiophosphate sodium salt was passed through a column of activated Dowex 50WX-8 200 mesh, H+ form. The column elutant was collected in an ice-cooled flask containing tributylamine (1 eq) and EtOH. The resulting solution was freeze-dried to yield the salt as colorless oil. The oil was dried by repeated evaporation with absolute EtOH (3 times), followed by co-evaporation with anhydrous DMF for three times to obtain colorless oil. [PSO3H]2-(Bu3NH+)2 salt was stored in a desiccator at -20°C. Typical procedure for preparation of adenosine 5'-O-(β-thio)-diphosphate derivatives. 2-Cl-AMP (Bu4N+) salt (0.26 mmol, 1 eq) and anhydrous DMF (2.5 mL) were stirred in a two-necked flask to form a colorless solution. CDI (214 mg, 1.32 mmol, 5 eq) was added to the solution. The reaction was stirred at room temperature for 3 h. Next, dry MeOH (0.053 mL, 1.325 mmol, 5 eq) was added to the reaction flask and the solution was stirred for 8 min. Subsequently [PSO3H]2-(Bu3NH+)2 salt (1.59 mmol, 6 eq) dissolved in anhydrous DMF (5 mL) and anhydrous ZnCl2 (283 mg, 2.12 mmol, 8 eq) were added to form a colorless solution. After 2 h, the reaction was quenched by addition of EDTA solution (0.93 g, 2.01 mmol in 30 mL of deionized water) and 1M TEAB was added until pH 8 was attained. The solution was then freezedried. The resulting white solid residue was separated on a Sephadex DEAE-A25



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Page 28 of 63

column applying a linear gradient of water (200 mL) to 0.2 M TEAB (200 mL) and then 0.2 M-0.4 M TEAB (total volume of 800 mL). The solution was freeze-dried at least 4 times to yield a yellowish solid in 50% yield (65 mg). Final purification was carried out on an HPLC system, using a semi-preparative reverse-phase column. The purity of the nucleotides was evaluated on an analytical reverse-phase column system, in two solvent systems as described below. The products, obtained as triethylammonium salts, were generally ≥ 90% pure. Finally, aqueous solutions of the products were passed through a sodium form Dowex 50WX8-200 ion-exchange resin column and the products were eluted with deionized water to obtain the corresponding sodium salts after freeze-drying. 2-Methylthioadenosine-5'-O-(β-thio)-diphosphate (10) Analogue 10 was obtained in a 75% overall yield (142 mg) after LC separation. Compound 10, 1H NMR (D2O, 200 MHz): δ 8.38 (s, 1H, H-8), 6.1 (d, J=4.6 Hz, 1H, H-1'), 4.68 (m, 2H, H-2', H-3'), 4.64 (m, 1H, H-4'), 4.25 (m, 2H, H-5', 5'') ppm.

31

P

NMR (D2O, 200 MHz) δ: 33.88 (d, J=31.3 Hz, Pβ-S), -11.11 (d, J=31.3 Hz, Pα) ppm. HR MALDI (negative): calcd for C11H16N5O9P2S2 487.9850, found 487.9863. Purity data obtained on an analytical column- Rt: 8.47 min (90% purity) using solvent system I (isocratic elution of 92:8 A:B over 20 min at a flow rate of 1 mL/min). Rt: 6.59 min (95% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). 2-Chloroadenosine-5'-O-(β-thio)-diphosphate (11) Purification of analogue 11 was accomplished using a semipreparative reverse-phase Gemini 5u column and isocratic elution with 92:8 (A) 100 mM TEAA, pH 7:(B) CH3CN at a flow rate of 4.5 mL/min. The fraction containing the purified analogue (Rt


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15.89 min) was freeze-dried. Excess buffer was removed by repeated freeze-drying cycles, and the solid residue was dissolved each time in deionized water. Analogue 11 was obtained in a 50% overall yield (65 mg) after LC separation. Compound 11, 1H NMR (D2O, 200 MHz): δ 8.49 (s, 1H, H-8), 6.01 (d, J=5.8 Hz, 1H, H-1'), 4.60 (m, 2H, H-2', H-3'), 4.36 (m, 1H, H-4'), 4.20 (m, 2H, H-5', 5'') ppm.

31

P

NMR (D2O, 200 MHz) δ: 36.97 (d, J=30 Hz, Pβ-S), -11.47 (d, J=30 Hz, Pα) ppm. HR MALDI (negative): calcd for C10H13Cl1N5O9P2S1 475.9592, found 475.9600. Purity data obtained on an analytical column- Rt: 4.8 min (89% purity) using solvent system I (isocratic elution of 92:8 A:B over 20 min at a flow rate of 1 mL/min). Rt: 3.8 min (96% purity) using solvent system II (isocratic elution of 96:4 A:B over 20 min at a flow rate of 1 mL/min). 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 µM fura 2/AM and 0.02% pluronic acid in Na-HBS buffer (Hepes buffered saline: 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 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 [Ca2+]i was monitored by detecting the respective emission intensity of fura 2/AM at 510 nm with 340 nm and 380 nm excitations.49 The average maximal amplitude of the responses and the respective standard errors were calculated from ratio of the fura 2/AM fluorescence intensities with excitations at 340 nm and 380 nm. We only analysed GFP-labelled cells. Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and SigmaPlot (SPSS Inc., Chicago, IL, USA) were used to derive the concentration-response curves and EC50 values from the average response amplitudes obtained in at least three independent



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experiments.50,

51

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 GFP-tagged P2Y receptors are suitable for pharmacological and physiological studies, as previously reported.52-54 ABTS radical cation decolorization assay27 ABTS radical cation (ABTS•+) was produced by reacting 7 mM aqueous ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. The radical was stable under these conditions for more than two days. ABTS•+ solution was diluted with water, pH 7.4, to an absorbance of 0.80 at 734 nm. After addition of 180 µL of diluted ABTS•+solution to 2.5-48 µL of antioxidant compounds or Trolox standard (final concentration 0–60 µM) in water, the absorbance reading at 734 nm was taken exactly 7 min after initial mixing. Appropriate solvent blanks were run in each assay. All determinations were carried out at least three times, in triplicate. The percentage of inhibition is calculated and plotted as a function of concentration of antioxidants and of Trolox for the standard reference data. Determination of the relative binding of ferrous ions to analogue 7A vs. 2 The relative binding of ferrous ions to nucleotides was determined by a competitive assay as described in the literature.55 Briefly, FeSO4 (75 µM, 38 µL, final concentration: 10 µM) was mixed with ferrozine (140 µΜ, 70 µL, final concentration: 20 µM) and with the nucleotide analogue in DDW at final concentrations of 25-200 µM, and agitated for 10 min at room temperature. The absorbance of the formed colored Fe(II)ferrozine complex in the presence or absence of nucleotides was measured at 562 nm.


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ESR OH radical assay 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 2x105 in experiments with Fe(II). 1 mM Ammonium iron(II) sulfate (10 µL) was added to 5-500 µM tested compound (110 µL) solutions. Afterwards, 1 mM Tris buffer, pH 7.4, (60-70 µL) was added to the mixture. After mixing for 30 sec, 100 mM DMPO (10 µL) were quickly added followed by the addition of 100 mM H2O2 (10 µL). Final sample pH values for the Fe(II) systems ranges between 7.2 and 7.4. Each ESR measurement was performed 150 s after the addition of H2O2. All experiments were performed at room temperature, at a final volume of 100 µL. Determination of ROS production in cultured PC12 cells33 PC12 cells were grown in Dulbecco's modified Eagle's medium and seeded into medium in 96-well tissue culture plates for 24 h. A stock solution of 2',7'dichlorofluorescein-diacetate (DCFH-DA) was prepared by dissolving 2 mg/mL of the material in ethanol, and kept in the dark at -20oC. For the experiments, the DCFH-DA stock solution was diluted 100 times in PBS, and 25 µL of the diluted solution was added to each well in the microplate, incubated for 20 min, and removed from the cells. After DCFH-DA was removed, the nucleotides were added to the cells at a final concentration of 0.2-200 µM. Oxidation was initiated by the addition of FeSO4 (2 µM, 12 µL, final concentration: 0.16 µM) to the wells. The plates were incubated for 1 h at 37 oC, during which time absorbance was read by a Tecan fluorimeter at 485/530 nm. Evaluation of the resistance of 7 A/B to hydrolysis by eNPP1,3



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The percentage of hydrolysis of analogue 7A/B by human eNPP1,3 was evaluated as follows: human eNPP1 or eNPP3 extract 19 µg or 32 µg, respectively, was added to 0.473 mL of the incubation mixture (1 mM CaCl2, 200 mM NaCl, 10 mM KCl, and 100 mM Tris, pH 8.5) and pre-incubated at 37 °C for 3 min. Reaction was initiated by the addition of 4 mM 7A/B (0.012 mL). The reaction was stopped after 2 h or 3 h for NPP1 or NPP3, respectively, by adding ice-cold 1 M perchloric acid (0.350 mL). These samples were centrifuged for 1 min at 10000 g. Supernatants were neutralized with 2 M KOH (140 mL) in 4 °C and centrifuged for 1 min at 10000 g. The reaction mixture was filtered and freeze-dried. Each sample was dissolved in HPLC-grade water (200 µL), and 20 µL was injected to an analytical HPLC column (Gemini analytical column (5µ C-18 557 110A; 150 mm × 4.60 mm)). The samples were separated on an analytical reverse-phase HPLC using isocratic elution applying 90% 100 mM TEAA (pH 7) and 10% CH3CN, flow rate 1 mL/min. The hydrolysis rates of analogues 7A/B by eNPP1 or eNPP3 were determined by measuring the change in the integration of the HPLC peaks for each analogue over time versus 1 as control. To determine the percentage of degradation due to enzymatic hydrolysis, each of the samples was compared to a control to which acid, but 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 formed due to acidic hydrolysis. Determination of the involvement of P2Y1/12-R in the antioxidant activity of compound 7A PC12 or Ntera-2 cells were grown in Dulbecco's modified Eagle's medium and seeded into medium in 96-well tissue culture plates for 24 h. 50 µM 2-MeS-AMP (3.75 µL) or 40 µM MRS2179, was added to each well in the microplate and incubated for 20 min.


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Determination of ROS production in cultured cells, was performed with DCFH-DA with or without the addition of 7A at a range of concentrations (0.01 – 25 µM), and initiation of oxidation by addition of FeSO4 (2 µM, 12 µL, final concentration: 0.16 µM) to the wells. ROS production was evaluated by the DCFH-DA assay as detailed above. Determination of cell viability by MTT assay Primary cortical neurons were seeded into grown in Dulbecco's modified Eagle's: F12 medium in 96-well tissue culture plates for 5 d. After 5 d, the tested compounds were added to the well. After 24 h, the culture medium was removed. Cell viability was assessed using an MTT assay which is a marker of mitochondrial activity.56 MTT (1 mg/mL) in PBS, pH 7.4, was added to the wells, following incubation (2 h at 37˚C). MTT solution was removed, and the formazan product was dissolved in DMSO. Absorbance was read at 550 nm using a Tecan spectrophotometer, plate reader. Data are presented as a percentage relative to their vehicle controls. Preparation of primary neuron cell cultures Rat brain (1d old) were removed under sterile conditions, the cortex was dissected and separated from the remaining brain, roughly homogenised by repeating pippetation and then trypsin was added. The trypsine was removed from the dissociated cells by centrifugation at 4000 rpm, and dissociated cells were plated at a density of 4 ·105/mL into 96 multi well plates (Nunc, Naperville, IL, USA) that had previously been precoated with poly-ornithine (15 µg/mL). Cells were cultured in a serum-free medium composed of a mixture of Dulbecco’s modified Eagle’s medium and F12 nutrient (1 : 1 v/v) supplemented with 10% B-27 (Gibco, BRL), 5% glutamine, 1% penicillinstreptomycin-nystatin. After 24 h the medium was replaced with fresh medium. After



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72 h the cells were treated with Ara-C (Cytosine β-D-arabinofuranoside, inhibitor of DNA replication) at 50 mM for 48 h, which resulted in 90% neuronal cell culture. The medium was then replaced with fresh medium and cells were ready for experiments. Neuroprotection assay Primary cortical neurons were seeded in 96-well plates and treated with FeSO4 at final concentrations of 3 µM for 24 h. Prior to exposure to FeSO4 the cells were treated with nucleotides 7A/B at various concentrations (0.01-5 µM). After 24 h cells were tested for cell viability by the MTT assay. Computational methods The ligands were described using the CHARMm force field with an implicit water solvent model (generalized Born with molecular volume). Energy minimization of the ligands was performed using the smart minimizer (1000 steps of the steepest descent method followed by the conjugate gradient method). Prior to docking the ligands into the receptor, we generated a library of various ligand conformations (maximum 255) using the FAST conformation generation method with a maximum energy threshold of 20 kcal/mol. All the conformation were subjected to docking using a high throughput protocol designed within Pipeline Pilot.57, 58 The grid-based molecular docking program CDOCKER58 with the CHARMm force field59 was employed to dock all the generated conformations into the P2Y1R. The docked pose with the highest negative interaction energy (i.e. lowest energy) was considered for further analysis. The solvation energy of the ligands was calculated using the Delphi60 program. The solvation energy of each ligand is computed by averaging the solvation energy of the five lowest energy conformations obtained from the conformational search mentioned above. To facilitate comparison with experiment, we predict EC50 values using multi-regression analysis of the solvation energy, the CDOCKER interaction energy and of both the solvation


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energy and the CDOCKER interaction energy. The crystal structures of P2Y1R were obtained from protein data bank (pdb codes: 4XNV and 4XNW).44, 45 All calculations employed the Discovery Studio (DS 4.0) modeling platform (Biovia, Inc.)61

Associated Content Supporting Information Experimental and predicted EC50 values. Author information Corresponding Author * Fax: 972-3-6353907; tel.: 972-3-5318303 e-mail: [email protected] Author Contributions §

These authors contributed equally to this work.

Abbreviations: [Ca2+]i, intracellular Ca2+ concentration; CDI, carbonyldimidazole; eNPP,

ecto-nucleotide

pyrophosphatase/phosphodiesterase;

eNTPDase,

ecto-

nucleoside triphosphate diphosphohydrolase; ESI, electron spray ionization; HRMSMALDI, high-resolution mass spectrometry matrix-assisted laser desorption ionization; P2R, P2 receptor; RT, room temperature; SD, standard deviation; TEAA, triethylammonium acetate; TEAB, triethylammonium bicarbonate.

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Tulapurkar, M. E.; Zundorf, G.; Reiser, G. Internalization and desensitization

of a green fluorescent protein-tagged P2Y nucleotide receptor are differently controlled by inhibition of calmodulin-dependent protein kinase II. J. Neurochem. 2006, 96, 624-634. (54)

Zylberg, J.; Ecke, D.; Fischer, B.; Reiser, G. Structure and ligand-binding site

characteristics of the human P2Y11 nucleotide receptor deduced from computational modelling and mutational analysis. J. Biochem. 2007, 405, 277-286. (55)

Decker, E. A.; Welch, B. Role of ferritin as a lipid oxidation catalyst in

muscle food. J. Agric. Food Chem. 1990, 38, 674-677. (56)

Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B.

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936-942. (57)

Pilot, P., Version 9.2; Biovia. Inc.: San Diego, CA, 2014.


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(58)

Wu, G.; Robertson, D. H.; Brooks, C. L.; Vieth, M. Detailed analysis of grid‐

based molecular docking: A case study of CDOCKER—A CHARMm‐based MD docking algorithm. J. Comput. Chem. 2003, 24, 1549-1562. (59)

Brooks, B. R.; Brooks, C. L.; MacKerell, A. D.; Nilsson, L.; Petrella, R. J.;

Roux, B.; Won, Y.; Archontis, G.; Bartels, C.; Boresch, S. CHARMM: the biomolecular simulation program. J. Comput. Chem. 2009, 30, 1545-1614. (60)

Sitkoff, D.; Sharp, K. A.; Honig, B. Accurate calculation of hydration free

energies using macroscopic solvent models. J. Phys. Chem. 1994, 98, 1978-1988. (61)

Studio, D., Version 4.1.; Biovia: San Diego, CA, 2015.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1 Synthesis of C2-modified-adenosine 5'-O-α-thio-diphosphate 6-9 a

Reaction conditions: (a) (1) HC(OMe)3, p-TsOH, RT, overnight; and (2) Dowex

MWA-1 (weak base), RT, 3 h, 96% yield; (b) 2-Cl-1,3,2-benzdioxaphosphorin-4-one, dry pyridine, dry dioxane, RT, 10 min; (c) 0.5 M P2O7H22-(Bu3N+H)2 in dry DMF, Bu3N, RT, 5 min; (d) S8 , RT, 1 h; (e) ethylenediamine, RT, 10 min; (f) (1) 10% HCl, pH 2.3, RT, 3 h; and (2) 24% NH4OH, pH 9, RT, 45 min. Compounds 6A/B, 7A/B and 8A/B, were obtained in 51%, 47% and 42% yield, respectively, after LC separation. Byproduct 9A/B, was obtained in 8% yield.


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Scheme 2 Synthesis of C2-modified-adenosine 5'-O-β-thio-diphosphate 10 and 11 a

Reaction conditions: (a) (1) CDI, DMF, RT, 3 h; (2) MeOH, 8 min, RT, 3 h; (b)

(1)[PSO3H]2-(Bu3NH+)2, ZnCl2, DMF, RT, 2 h; (2) EDTA. 2-SMe-ADP(β-S), 10, and 2-Cl-ADP(β-S), 11, were obtained in 75% and 50% yield, respectively, after LC separation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 1 Potencies of nucleotide analogues 6-11 at hP2Y1/11-R. EC50 values for [Ca2+]i elevation were obtained from concentration-response curves based on fura 2/AM F340 nm/ F380 nm ratio measurements. EC50 values (µM) Agonist

P2Y1-R

s.e.m.

P2Y11-R

s.e.m.

ATP (1)

0.85

0.047

6.7

0.87

ADP (2)

0.024

0.02

1.7

0.45

2-MeS-ADP (4)

0.013

0.0045

n.m.

ADP-β-S (5)

1.27

0.34

32.7

1.85

ADP-α-S (6A)

0.080

0.0078

4.5

0.55

ADP-α-S (6B)

0.13

0.022

8.7

0.3

2-SMe-ADP(α-S) (7A)

0.0026

0.000025

3.2

0.5

2-SMe-ADP(α-S) (7B)

0.017

0.0037

1

0.03

2-Cl-ADP(α-S) (8A)

0.030

0.003

1.4

0.24

2-Cl-ADP(α-S) (8B)

1.5

0.0025

0.5

0.013

2-Cl-ATP(α-S) (9A)

0.73

0.018

9.1

1.43

2-Cl-ATP(α-S) (9B)

0.88

0.034

1.1

0.08

2-SMe-ADP(β-S) (10)

0.037

0.004

0.9

0.054

2-Cl-ADP(β-S) (11)

0.090

0.025

1

0.125

n.m. - not measured, s.e.m. -standard error of the mean. EC50 values were obtained from concentration-response curves (Fig. 2 and 3). Each data point represents mean values of at least 40 cells measured. s.e.m of the EC50 values was determined from n= 3 concentration-response curves.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2 Inhibition of OH radical production in Fenton reaction by adenine nucleotides 6-11 Compound

µM) IC50(µ

EDTA

54±5

ATP (1)

n.a

ADP (2)

170±6.36

ADP-β-S (5)

19±2.5

ADP-α-S (6A)

31±1.55

ADP-α-S (6B)

36±3


37±2.1


38±3

2-Cl-ADP(α-S) (8A)

36±2.8

2-Cl-ADP(α-S) (8B)

39±4

2-Cl-ATP(α-S) (9A)

37±0.7

2-Cl-ATP(α-S) (9B)

27±2

2-SMe-ADP(β-S) (10) 2-Cl-ADP(β-S) (11)

21±0.07 21±2.1

Antioxidant IC50 values represent the compound’s concentration that inhibits 50% of the OH radical amount produced in the control reaction. n.a. = Not available, the minimal amount of radical production exceeds 50%.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table 3 Scavenging of ABTS•+ by nucleotide analogues 6-11 vs. Trolox Compound

IC50(µ µM)

Trolox

18±1.4

ATP (1)

60±0.49

ADP (2)

30±0.54

ADP-β-S (5)

15±0.8

ADP-α-S (6A)

20±0.6

ADP-α-S (6B)

22±0.3


12.5±0.01


12±0.02

2-Cl-ADP(α-S) (8A)

11±0.7

2-Cl-ADP(α-S) (8B)

12±0.35

2-Cl-ATP(α-S) (9A)

25±0.6

2-Cl-ATP(α-S) (9B)

24±0.4


15±0.7

2-Cl-ADP(β-S) (11)

13±0.7


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4 Inhibition of Fe(II)-induced ROS production in PC12 cells by nucleoside 5’thiophosphate analogues 6-11. Compound

IC50(µ µM)

Trolox

20±2.4

ATP (1)

n.a

ADP (2)

21±1.5

ADP-β-S (5)

20±2.3

ADP-α-S (6A)

38±1.2

ADP-α-S (6B)

39±2.5


0.08±0.002


0.6±0.02

2-Cl-ADP(α-S) (8A)

0.2±0.04

2-Cl-ADP(α-S) (8B)

0.11±0.02

2-Cl-ATP(α-S) (9A)

106±3.8

2-Cl-ATP(α-S) (9B)

100±2


12±1.9 5±0.7

2-Cl-ADP(β-S) (11)

Values represent mean±S.D. of three experiments (P < 0.05). n.a - The nucleotide did not inhibit 50% of ROS production.

Table 5 Hydrolysis of ADP, 2, and 2-SMe-ADP(α-S), 7A/B, by humanectonucleotidases, eNPP1/3.

relative hydrolysis (% ± SD of ATP hydrolysis) human ectonucleotidase eNPP1 eNPP3

7A

7B

ADP

36.6% ± 2.4 42.0% ± 1.8

23.7% ± 4.4 25.6% ± 2.6

74.52% ± 2.6 106.3% ± 2.5

The percentages are relative to ATP, meaning ATP was calculated as being 100% hydrolyzed.



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Table 6 Sequence similarities and sequence identities between P2Y1R, P2Y11R and P2Y12R.

Receptor-pairs Sequence Identity Sequence similarity P2Y11-P2Y12

19.3%

30.9%

P2Y11-P2Y1

25.4%

40.4%

P2Y1-P2Y12

20.8%

36.6%

Table 7 CDOCKER interaction energies of ligands 1-12 with the P2Y1 receptor and absolute configuration of the phosphorous atoms of the docked ligands.

Ligand

CDOCKER Interaction energy

Absolute

(kcal/mol)

configuration

4XNV

4XNW

4XNVMOD

ATP (1)

-19.47

-81.22

-60.40

-

ADP (2)

-10.65

-101.02

-45.93

-

2-MeS-ADP (4)

-9.67

-105.77

-46.20

-

ADP-β-S (5)

0.72

-89.23

-40.51

-

ADP-α-S (6A)

0.17

-81.22

-34.88

RP

ADP-α-S (6B)

0.17

-71.89

-34.88

SP


1.89

-85.80

-34.38

RP


1.90

-85.92

-34.65

SP

2-Cl-ADP(α-S) (8A)

-1.60

-85.28

-36.09

RP

2-Cl-ADP(α-S) (8B)

-2.67

-79.92

-37.56

SP

2-Cl-ATP(α-S) (9A)

-18.48

-107.48

-50.72

RP

2-Cl-ATP(α-S) (9B)

-2.74

-106.65

-55.66

SP


-5.50

-90.7

-37.47

-

2-Cl-ADP(β-S) (11)

6.87

-86.3

-38.59

-


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 1 NH2 N O

O

N

O N

P O

P Y2

X3

P Y1

X2

N

O O

Z

X1

n

HO

OW

1: Z=H, n=1, X1=X2=X3=O, Y1=Y2=O, W=H 2: Z=H, n=0, X1=X3=O, Y1=O, W=H 3: Z=H, n=1, X1=X2=O X3=S, Y1=Y2=O, W=H 4: Z=SMe, n=0, X1=X3=O, Y1=O, W=H 5: Z=H n=0, X1=O, X3=S, Y1=O, W=H 6: A+B: Z=H, n=0, X1=S, X3=O, Y1=O, W=H 7: A+B: Z=SMe, n=0, X1=S, X3=O, Y1=O, W=H 8: A+B: Z=Cl, n=0, X1=S, X3=O, Y1=O, W=H 9:A+B: Z=Cl, n=1, X1=S, X3=X2=O, Y1=Y2=O, W=H 10: Z=SMe, n=0, X1=O, X3=S, Y1=O, W=H 11: Z=Cl, n=0, X1=O, X3=S, Y1=O, W=H



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 2

A

B

C


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 3

A

B

C



Fig. 4

Amount of DMPO-OH adduct (% 0f control)

120 2 7A 7B

100

80

60

40

20

0 0.0 0

.2 0.2

.4 0.4

0.6.6

Concentration (mM)

Fig. 5

% Inhibition of Fe-Ferozzine complex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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70 60 50 40 30 20

7A

10

ADP

0 25

50

75

100

150

200

Concentration (µM)


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Fig. 6

100 2 8A 7A

% Nucleotide

80

60

40

20

0 0

1 0.5

1

2 1.5

2

4

8

12

16

20

24

Time [h]

Fig. 7

A

B

60

80

*

7A

%Inhibiton of ROS production

70 %Inhibiton of ROS production

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7A+ 2-MeS-AMP

50 40 30 20 10

*

7A

70

7A+ MRS2179

60 50 40 30 20 10 0

0 2-MeSAMP

0.01

0.04

0.2

1

5

0.01

Concentration (µ µM)

0.04

0.2

1



5

25


Fig. 8

80

7A

70

7B

60

% of cell viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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50 40 30 20 10 0 FeSO4

0.01

0.04

0.2

1

5



25

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 9

A

B

C

D

Fig. 10


E


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 11


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure legend Fig. 1. Nucleotide analogues studied here. Fig. 2. Concentration-response curves for agonists at hP2Y1-R: ADP-α-S, 6A/B, and 2-SMe-ADP(α-S), 7A/B (A), 2-Cl-ADP(α-S), 8 A/B, ADP, 2, and 2-SMe-ADP, 4 (B) 2-SMe-ADP(β-S), 10, 2-Cl-ADP(β-S), 11, ADP, 2, and ADP-β-S, 5 (C). Data were obtained by determining the agonist-induced change in [Ca2+]i of 1321N1 cells stably expressing the human P2Y1GFP receptor. Cells were pre-incubated with 2 µM fura-2 AM for 30 min and change in fluorescence (∆F340 nm/F380 nm) was detected. Fig. 3. Concentration-response curves for agonists at hP2Y11-R: ADP-α-S, 6 A/B, and 2-SMe-ADP(α-S), 7A/B (A), 2-Cl-ADP(α-S), 8A/B, and ADP, 2 (B), 2-SMeADP(β-S), 10, 2-Cl-ADP(β-S), 11, ADP, 2, and ADP-β-S, 5 (C). Data were obtained by determining the agonist-induced change in [Ca2+]i of 1321N1 cells stably expressing the human P2Y11GFP receptor. Cells were pre-incubated with 2 µM fura-2 AM for 30 min and change in fluorescence (∆F340 nm/F380 nm) was detected. Fig. 4. Effects of 2-SMe-ADP(α-S), 7A/B, vs. ADP, 2, on the amount of DMPO-OH adduct formed under Fenton reaction conditions. The amount is given as the percentage of control, which contains only FeSO4, H2O2, and DMPO. Fig. 5. Chelation of ferrous ions by 2-SMe-ADP(α-S), 7A, and ADP. 10 µM of FeSO4 was incubated with 20 µM of ferrozine and with the tested compounds for 10 min at room temperature. The absorbance of the formed Fe(II)-ferrozine complex in the presence or absence of the tested compounds was measured at 562 nm. Values represent mean ± S.D of three experiments. Fig. 6. Hydrolysis of 2-SMe-ADP(α-S), 7A, ADP, 2 and ADP-α-S, 6A, in human blood serum and RPMI-1640 medium over 24 h at 37 ºC, as monitored by HPLC.


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Fig. 7. Reduction of the antioxidant activity of 2-SMe-ADP(α-S), 7A, in PC12 cells under oxidizing conditions upon blocking P2Y12-R with 2-SMe-AMP (A). Reduction of antioxidant activity of 7A, in Ntera-2 cells by blocking P2Y1-R with MRS2179 (B). The results shown are the mean ± SD of three independent experiments performed in triplicate (* P < 0.05, 7A vs. 7A with P2Y1/12-R inhibitors). Fig. 8. The neuroprotective activity of 2-SMe-ADP(α-S), 7A vs. 7B. Cortical neurons were treated with 7A/B 0.01-25 µM and 3 µM FeSO4 for 24 h. After 24 h, cell viability was measured by a MTT assay. The results shown are the mean ± S.D. of three independent experiments in triplicate (P < 0.05). Fig. 9. Effect of 2-SMe-ADP(α-S), 7A, and FeSO4 treatment on the morphology of primary cortical neurons. Neuronal cells were photographed before (A) and after treatment with 3 µM FeSO4 (B) for 24 h. In order to reduce the oxidative stress the cells were treated with 7A at 0.2 (C), 5 µM (D), and 100 µM (E) and photographed by light microscopy (100× magnification). The arrows mark the areas that maintain the normal morphology in the soma and the extensions. Fig. 10. Structures of P2Y1R: (A) Experimental crystal structure, 4XNV, with BPTU antagonist (B) Experimental crystal structure, 4XNW, with MRS-2500 antagonist (C) 2SMe-ADP(β-S),7A, docked in the 4XNVMOD receptor (D) 2-SMe-ADP(β-S),7A, docked in the 4XNW receptor. Green oval shapes indicate the binding pocket. Fig. 11. Experimental and predicted EC50 values. (A) Experimental EC50 values (B) Predicted EC50 values using ligand CDOCKER interaction energy with the 4XNVMOD receptor (C) Predicted EC50 values using solvation energy (D) Predicted EC50 values using both ligand CDOCKER interaction energy with the 4XNVMOD receptor and solvation energy.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 12. The binding cavity of 4XNVMOD receptor complex with (A) ADP(2) (B) 2SMe-ADP(α-S) (7A).


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