Highly Efficient Biocompatible Neuroprotectants with Dual Activity as

Article pubs.acs.org/jmc

Highly Efficient Biocompatible Neuroprotectants with Dual Activity as Antioxidants and P2Y Receptor Agonists Sagit Azran,† Daniel Förster,‡ Ortal Danino,† Yael Nadel,† Georg Reiser,‡ and Bilha Fischer*,† †

Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel Otto von Guericke University, Leipziger Strasse 44, 39120 Magdeburg, Germany

‡

S Supporting Information *

ABSTRACT: Currently, there is a need for novel, biocompatible, and effective neuroprotectants for the treatment of neurodegenerative diseases and brain injury associated with oxidative damage. Here, we developed nucleotide-based neuroprotectants acting dually as antioxidants and P2Y-R agonists. To improve the potency, selectivity, and metabolic stability of ATP/ADP, we substituted adenine C2-position by Cl and Pα/Pβ position by borano group, 6−9. Nucleotides 6−9 inhibited oxidation in cell-free systems (Fe(II)-H2O2), as detected by ESR (IC50 up to 175 μM), and ABTS assay (IC50 up to 40 μM). They also inhibited FeSO4-induced oxidative stress in PC12 cells (IC50 of 80−200 nM). 2-Cl-ADP(α-BH3), 7a, was found to be the most potent P2Y1-R agonist currently known (EC50 7 nM) and protected primary cortical neurons from FeSO4 insult (EC50 170 nM). In addition, it proved to be metabolically stable in human blood serum (t1/2 7 vs 1.5 h for ADP). Hence, we propose 7a as a highly promising neuroprotectant.

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the main receptor involved in this process.10 Recently, the involvement of P2Y1-R in neuroprotection has been investigated.9,11 The exact mechanism of the neuroprotection is still unknown. It was suggested that the activation of P2Y1 receptor leads to IL-6 release, which is supplied to neurons and reduce the oxidative stress.9 Highly reactive OH radicals, among reactive oxygen species (ROS), are known to play a key role in oxidative stress in various health disorders including neurodegenerative diseases, brain ischemia, stroke, cancer, or diabetes.12−18 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. 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 diseases19 and ischemia.20 Conditions such as hypoxia-ischemia further aggravate the potential for iron release from transferrin or ferritin due to a decrease in pH and the creation of an electrolytic imbalance within the brain extracellular fluid.21 Therefore, there is a clear need for Fenton reaction inhibitors, whether radical scavengers or metal-ion chelators, to prevent the production of hydroxyl radical and its subsequent damage. Known antioxidants include substances such as clioquinol,22 verapamil,23

INTRODUCTION Neurodegenerative diseases and brain injury are associated with oxidative damage.1 Development of antioxidant agents is one of the promising approaches in the search for treatment of these health disorders. Another approach suggests the activation of P2Y-receptors (P2Y-Rs), widely expressed in the nervous system and proposed to be involved in neuroprotection, for the treatment of oxidative damage.2 The members of the P2 receptor (P2-Rs) family, consisting of ligand-gated ion channels (P2X-Rs) and G protein-coupled receptors (P2Y-Rs), are activated by endogenous extracellular nucleotides.3 Eight different P2Y receptors, P2Y1,2,4,6,11,12,13,14, have been characterized by molecular means.4 P2 receptors are distributed in a large variety of tissues and are widely expressed in the nervous system. Neurons and astrocytes express both classes of P2-Rs, while P2Y1-R is the predominant receptor in neurons.5 While ionotropic P2X receptors are mainly involved in fast synaptic neurotransmission, P2Y receptors mediate slow neuromodulatory effects.5 In the brain, P2Y-R mediated signaling is involved in nervous tissue remodeling following trauma, stroke, ischemia, or neurodegenerative disorders.2,6 Release of ATP and, in turn, ADP and adenosine production, in the brain occurs under normal and pathological conditions7 and is important for neuron-to-astrocyte intercellular communications. During brain injury, i.e., following ischemia, necrosis, or trauma, in which massive release of ATP occurs, some of the responses to this release are neuroprotective.8,9 Some reports attributed this neuroprotective effect of ATP to its interaction with P2X-Rs and P2Y-Rs by which the P2Y1-R appears to be © XXXX American Chemical Society

Received: February 7, 2013

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dx.doi.org/10.1021/jm400197m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

desferroxamine,24 and tridentate triazolyl,25 or naturally occurring antioxidants, e.g., vitamin C and E26 and caffeic acid.27 However, many of these chelators are poorly water-soluble, and some of them demonstrate pro-oxidant activities at high doses.28 In contrast, nucleotide analogues are water-soluble metal-ion chelators.29 For instance, ATP forms stable complexes with various metal ions (e.g., Mg(II), Zn(II), and Cu(II)).30 Recently, we explored the application of natural and synthetic nucleotides and inorganic phosphates as biocompatible, nontoxic, and water-soluble inhibitors of the Fe(II)-induced Fenton reaction.31 Using ESR, we studied the modulation of Fe(II)induced •OH production, from the decomposition of H2O2, by nucleotides and phosphates. We found that the phosphate moiety, not the nucleoside, determines the pro/antioxidant properties of a nucleotide, suggesting a chelation-based modulation. For instance, ATP-γ-S proved 100 and 20 times more active antioxidant at Fe(II)/H2O2 system than ATP and the potent antioxidant Trolox,31 respectively. Yet, a major limitation of this nucleotide antioxidant is its enzymatic instability. A boranophosphate moiety replacing a phosphate group is expected to improve the metabolic stability of a nucleotide derivative.32 In addition, although borane derivatives are not natural constituents of the organism, they are not toxic.33 Here, we report on the design and evaluation of novel biocompatible neuroprotective agents capable of dual activity as both antioxidants and P2Y1-R agonists. Specifically, we report the synthesis of analogues nucleotide and dinucleotide 6−11 (Figures 1, 2), their evaluation as P2Y1,11-R agonists, Fenton

(EC50 4.5 nM) than ATP, 1.32 Because ADP, 2, is the endogenous agonist of P2Y1-R, we synthesized here 2-Cl-ADP(α-B), 7, as a potentially more active agonist. Another analogue based on 8 was compound 9, where the β,γ-bridging oxygen atom was replaced by a CCl2 group. This compound was designed to overcome the instability of 8 to hydrolysis by enzymes such as nucleoside triphosphate diphosphohydrolase (NTPDase) and alkaline phosphatase (AP).32 The CCl2 modification was selected based on our previous findings on the enzymatic stability of ATP analogues bearing a β,γ-dihalomethylene group. These analogues were highly resistant to hydrolysis by AP, NTPDases1,2,3,8 and NPP1,3 (less than 5% hydrolysis, as compared to ATP).34 Another approach to improve the stability of nucleotides is by applying a dinucleotide scaffold. Dinucleoside polyphosphates activate both P2X and P2Y receptors.35 Previously, we reported dinucleotide Ap5(γ-B)A, 10, as a most potent P2Y1-R agonist (1.5-fold more active than 2-MeS-ADP).36 Yet, the resistance of 10 to hydrolysis by ecto-nucleotide pyrophosphatase (e-NPP1) was insufficient. Boranoisostere at Pα was expected to stabilize this dinucleotide because NPP1 cleaves dinucleotides between Pα and Pβ positions. Indeed, lately, we have shown that Npn(α -B)N′ proved to be ∼22-fold more resistant to hydrolysis by e-NPP1, as compared to the corresponding NpnN′ analogues.37 Therefore, here we have designed analogue 11 in which the nonbridging oxygen atom at one of Pα positions is substituted by a borano group. Synthesis of Potential P2Y1-R Ligands. Synthesis of PαBorano-Analogues 7, 9, and 11. Methoxymethylidene protected 2-Cl-adenosine, 13, was used for the synthesis of 2Cl-adenosine-5′-O-(α-boranodiphosphate), 7, by a one-pot phosphorylation reaction (Scheme 1).32,38 First, 2-Cl-1,3,2benzdioxaphosphorin-4-one and dry pyridine in dry dioxane was added to 13 in DMF to give phosphite, 14, which was treated with pyrophosphate tributylammonium salt in dry DMF to generate the cyclic intermediate, 15. Treatment of 15 with a 2 M solution of BH3·SMe2 complex in THF led to cyclic boronated triphosphate intermediate 16. Subsequently, ethylenediamine was added to generate 17 upon elimination of cyclic phosphorodiamidate.38 Removal of the methoxymethylidene group involved a hydrolysis step at pH 2.3 and then at pH 9. Analogue 2-Cl-ADP(α-B), 7, was obtained from nucleoside 13 at a 38% yield. For the preparation of 9, methoxymethylidene protected 2-chloroadenosine, 13, was treated with PCl3, followed by reaction with 0.5 M dichloromethylene diphosphonate bis(tributylammonium) salt to form intermediate 19 (Scheme 2).39 The latter was treated with a Lewis acid, BH3·SMe2, at 0 °C, and the reaction mixture was stirred at RT for 30 min. Finally, hydrolysis of the cyclic intermediate 20 in 0.5 M TEAB and deprotection of the methoxymethylidene group generated 2-ClATP-(α-B, β,γ-CCl2), 9, as a pair of diastereoisomers at an overall 30% yield after LC separation. For the synthesis of dinucleotide 11, analogue 21 was activated with CDI in dry DMF to generate the P-donor40 phosphoroimidazolide, 22. MgCl2 was added to the reaction mixture to enhance the reactivity of phosphoroimidazolide (Scheme 3).36 ATP(α-B), 24, was added as a P-acceptor to produce Ap5(α-B)A, 11, as a pair of diastereoisomers at a 67% yield after LC separation. ̀ The identity and purity of the products were established by 1 H and 31P NMR, HR mass spectrometry, and HPLC in two solvent systems. 31P NMR spectra of products 7, 9, and 11 showed a typical Pα signal as a doublet at about 83 ppm.

Figure 1. ADP and ATP analogues studied here.

Figure 2. Dinucleotides analogues studied here.

reaction inhibitors, radical scavengers, and inhibitors of ROS formation in PC12 cells and primary neurons under oxidative stress conditions. In addition, we report the metabolic stability of the most promising nucleotide analogue.

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RESULTS Design of Adenosine Nucleotides As Potential P2Y1-R Agonists. Previously, we identified 2-Cl-ATP(α-B) (a isomer), 8a, as a promising P2Y1-R ligand which was 100-fold more potent B



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Scheme 1a

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-1,3,2benzdioxaphosphorin-4-one, dry DMF, dry dioxane, RT, 10 min; (c) 1 M P2O7H22−(Bu3N+H)2 in dry DMF, Bu3N, RT, 5 min; (d) 2 M BH3·SMe2 in THF, RT, 15 min; (e) ethylenediamine, RT, 10 min; (f) (1) 10% HCl, pH 2.3, RT, 3 h, and (2) 24% NH4OH, pH 9, RT, 45 min. 7a/b was obtained from 13 in 38% yield after LC separation.

Scheme 2a

Reaction conditions: (a) PCl3, trimethylphosphate, Proton Sponge, 0 °C, 30 min; (b) 0.5 M dichloromethylene diphosphonate bis(tributylammonium) in dry DMF, Bu3N, 0 °C, 1 h; (c) 2 M BH3·SMe2 in THF, 0 °C, 5 min, and then RT, 60 min; (d) 0.5 M TEAB, pH 7, RT, 1 h; (e) (1) 10% HCl, pH 2.3, RT, 3 h, (2) 24% NH4OH, pH 9, RT, 45 min. 9a/b was obtained in 30% yield after LC separation. a

C



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Scheme 3a

a

Reaction conditions: (a) (1) carbonyldiimidazole, dry DMF, RT, 2.5 h, (2) dry MeOH, RT, 5 min; (b) ATP (α-B), 24, in dry DMF, MgCl2, RT, overnight; (c) (1) 10% HCl, pH 2.3, RT, 3 h, and (2) 24% NH4OH, pH 9, RT, 45 min. 11a/b was obtained in an overall 67% yield after LC separation.

Because of the chiral center at Pα, analogues 7, 9, and 11 are each obtained as a pair of diastereoisomers in a 1:1 ratio. In both 1H and 31P NMR spectra, there was a slight difference between the chemical shifts for the two diastereoisomers of 7, 9, and 11. For instance, for 7 diastereoisomers, two sets of signals were observed for H8, at 8.53 and 8.51 ppm. These isomers were well separated by reverse-phase HPLC with about 6−10 min difference in their retention times. The first eluting isomer was designated the a isomer, and the other was designated the b isomer. Assignment of the Absolute Configuration of Chiral Analogues 7, 9, and 11. A previous study elucidated the absolute configuration of 8 a and b isomers as Rp and Sp, respectively.41 Here, we employed 1H NMR spectroscopy to elucidate the absolute configuration around Pα for both diastereoisomers of analogues 7, 9, and 11. A difference in the chemical shift of H8 was observed between the two diastereoisomers of these analogues. The signal of H8 of the b isomer in the 1H NMR spectrum was more shielded than that of H8 of the a isomer, e.g., 8.53 (a isomer) vs 8.51 (b isomer) ppm for analogue 7. Shielding of H8 of the b isomer is probably due to the proximal negatively charged BH3 group (Figure 3). Pα lies much further away from H8 in the a than b isomer, thus, the Rp configuration can be attributed to a isomer, and Sp to the b isomer in nucleotide analogues 7, 9, and 11.41 Evaluation of Adenosine Nucleotide Analogues 7, 9, and 11 as P2Y1/11-Rs Agonists. The activity of nucleotides 7, 9, and 11 at the phylogenetically similar P2Y1/11-Rs42 was studied by measuring [Ca2+]i mobilization induced by these analogues and comparing it to that of the endogenous agonist of P2Y1-R and P2Y11-R, ADP, ATP, respectively. These studies were performed in 1321N1 astrocytoma cells stably expressing the human P2Y1/11 receptors. Concentration−response curves

Figure 3. Rp and Sp configuration is attributed to diastereoisomers 7a and 7b, respectively.

were derived for a range of nucleotide concentrations. The results are shown in Figure 4 and Figure S1 (Supporting Information) and summarized in Table 1. Previously, we have reported Ap5(γ-B)A, 10, as a highly potent rat P2Y1-R agonist (EC50 60 nM vs 100 nM for 2-MeSADP).36 However, at human P2Y1-R, 10 demonstrated an EC50 value of only 1.2 μM. Compound 11, expected to be more stable than 10, showed at P2Y1-R a 6-fold higher activity (EC50 0.2 μM) for the a isomer and poor activity for the b isomer (EC50 3 μM), as compared to 10. Isomer 8a32 was found to be 34-fold more potent than the corresponding b isomer (EC50 74 vs 2500 nM), yet less potent than ADP (EC50 24 nM). To enhance the metabolic stability of 8a, we substituted the bridging oxygen with β,γ-dichloromethylene group to give analogue 9. We found that 9a isomer was less potent than the corresponding 8a at P2Y1-R(EC50 150 nM), and 9b isomer was more potent than the corresponding 8b isomer (EC50 950 nM). The most potent hP2Y1-R agonist of the series studied here was 7a (EC50 7 nM), being 2-fold more potent than 2-MeSADP, 3, the most potent P2Y1R agonist currently known. Isomer 7b was 85-fold less active than the a isomer. Compounds 7a and 7b were P2Y1-R selective, showing up to 530-fold lower D



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Figure 4. Concentration−response curve for agonists 1 and 2 (A), 3 and 4 (B), 6a and 6b (C), and 7a and 7b (D). Data were obtained by determining the agonist-induced change in [Ca2+]i of 1321N1 cells stably expressing the human P2Y1GFP receptor. Cells were preincubated with 2 μM fura-2 AM for 30 min, and change in fluorescence (ΔF340 nm/F380 nm) was detected.

(IC50 170 μM). We found that 8b was the most active compound in this series, with IC50 of 36 μM, and was ca. 2-fold more active than 8a (IC50 66 μM). Methylenedichloride modification in analogues 8, analogues 9, reduced IC50 (131 vs 66 μM) for the a isomer and 60 vs 36 μM for the b isomer. Compound 7a exhibited an IC50 (175 μM) similar to that of ADP, while the b isomer has an IC50 of 120 μM. In the dinucleotide series, we found that positioning the boranophosphate isostere at Pα, rather than Pγ, improves antioxidant activity by 2-to 3-fold. Thus, Ap5-αB-A 11a/b exhibited IC50 values of 47/59 μM, respectively, as compared to Ap5-γ-B-A, 10, (IC50 161 μM). Yet, the parent compound, Ap5A, 4, was also relatively active (IC50 75 μM). Evaluation of the Antioxidant Activity of Analogues 6−11 Using the ABTS Decolorization Assay. Next, we evaluated the antioxidant activity of analogues 6−11 that is due to radical scavenging. For this purpose, we used the improved ABTS decolorization assay.44 ABTS•+ is formed by the oxidation of ABTS with potassium persulfate and has absorbance at 645, 734, and 815 nm. 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 (Table 3). We found that analogues 6−9 inhibited ABTS•+, with IC50 values around 24 μM as compared to IC50 of 18 and

activity at the P2Y11-R (EC50 values of 3700 and 1000 nM, respectively, Figure 5). Analogues 6a and 6b lacking the 2-Cl substitution were 43- and 22-fold less active than 7a/b at the P2Y1-R with EC50 of 300 and 13000 nM, respectively. Compounds 9a and 9b exhibited activity at the P2Y11R compounds with EC50 of 1400 and 130 nM, respectively. Compound 9b was 52-fold more potent than ATP. It is the most potent P2Y11R currently known. Furthermore, it is P2Y11R selective because its activity at P2Y1R is 73-fold lower than that of 2-MeS-ADP (EC50 13 nM). Evaluation of Analogues 6−11 as Fenton Reaction Inhibitors. To study the antioxidant effect of our compounds we used ESR to monitor the modulation of •OH formation from H2O2 by the Fe(II)-induced Fenton reaction. 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.43 DMPO−OH adduct was then detected by ESR. The addition of chelators to Fe(II)-H2O2 mixture lowers the DMPO−OH signal due to metal-ion chelation and/or radical scavenging.31 The inhibition of radical production by compounds 6−11 (expressed in IC50 values, Table 2) was compared to the inhibitory effect of the metal-ion chelator EDTA (IC50 54 μM) and natural nucleotides such as ATP (IC50 > 500 μM) and ADP E



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Table 1. Potencies of Nucleotides 1−11 at hP2Y1/11-Ra

(NTPDases) and nucleotide pyrophosphatase/phosphodiesterase (NPPs) are the principal enzymes that metabolize extracellular nucleotides. As 7a is the most potent compound in this series, we tested the resistance of 7a/b to hydrolysis by NPP1,3 and NTPdase1,2,3,8. We compared 7a/b to ADP-α-B, 6a/b, and ADP to evaluate the effect of Cl at the C2 position, and the borano group, on the stability of 7a/b. We found that analogues 6a and 7a were hardly hydrolyzed by NPP1 (2.5−5.6%, Table 5), and analogue 6b was not metabolized at all by NPP1,3. Compounds 6−7 were found to be stable to NTPDase1,8 hydrolysis after 1 h incubation. Surprisingly, compound 7b was hydrolyzed at ∼46% by NTPDase2 and compound 7a was hydrolyzed at ∼15% and ∼22% by NTPDase2 and NTPDase3, respectively. The higher hydrolysis rate of 2-Cl-ADP-α-B, 7, as compared to ADP-α-B, 6, may be due to H-bonding of the chlorine atom at the adenine C2 with one of the amino acids inside the NTPDase2,3 catalytic-site and consequently the phosphate chain of 7 may be appropriately located near the catalytic amino acid residues. Resistance of Analogue 7a 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. Previous studies have used human blood serum46 to demonstrate the metabolic stability of phosphonate modified nucleotide analogues.39,47 Here, we examine the stability of the most promising analogue identified here, 7a, in human blood serum as compared to ADP and ADP-α-B, 6a. These analogues were incubated in human blood serum and RPMI-1640 at 37 °C 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 (Figure 6). After 6 h, 53% of 7a was detected by HPLC, while 56% of 6a and only 32% of ADP remained, indicating that the major contribution to stability enhancement is the boranophosphate substitution. Antioxidant Activity of 7a Involves also P2Y12-R Activation. Like P2Y1-R, P2Y12 purinergic receptor is activated by endogenous ADP and a synthetic agonist, 2-MeS-ADP. The activity of these nucleotides at P2Y12-R is blocked by 2-MeSAMP.48 Here, we investigated the possible involvement of P2Y12-R in the antioxidant activity of the most promising compound, 7a. For this purpose, we studied the effect of 7a on PC12 cells expressing P2Y12-R49 under oxidizing conditions, with or without antagonist. We selected PC12 cells for this study, rather than primary neurons, because the former express P2Y12-R but not P2Y1-R.50 Specifically, PC12 cells were treated with 50 μM 2-MeSAMP for 20 min. DCFH-DA was added to cells for 20 min. After DCFH-DA was removed and oxidation initiated by addition of FeSO4 (0.16 μM) to the wells, 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 0.04−100 μM, 43% up to 70% decrease in the antioxidant activity of 7a in the presence of 2-MeS-AMP was measured (Figure 7). These results suggest that the antioxidant activity of 7a also involves the activation of P2Y12-R in PC12 cells. Evaluation of the Neuroprotective Effect of Nucleotides 7a/b on Primary Neurons Subjected to Oxidative Stress Caused by FeSO4. Next, we evaluated the neuroprotective effect of 7a, which was the most promising analogue identified here. Primary cortical neurons were treated with 3 μM FeSO4 and 0.01−5 μM 7a/b for 24 h. Cell viability was measured

EC50 values (μM) agonist

P2Y1-R

SEM

P2Y11-R

1 2 3 4 5 6a 6b 7a 7b 8a 8b 9a 9b 10 11a 11b

0.85 0.024 0.013 0.76 n.m 0.3 13.0 0.007 0.6 0.074 2.5 0.15 0.95 1.2 0.2 3.0

0.047 0.02 0.0045 0.215

6.7 1.7 nm nm 1.5 ndr 2.9 3.68 1 nm nm 1.4 0.13 nm ndr ndr

0.07 1.55 0.0007 0.018 0.03 0.017 0.033 0.002 0.051 0.045 0.075

SEM 0.87 0.45

0.005 0.001 0.37 0.084

0.0125 0.021

a EC50 values for [Ca2+]i elevation were obtained from concentration− response curves based on fura 2/AM F340 nm/F380 nm ratio measurements. ndr, no detectable response; nm, not measured; SEM, standard error of the mean. EC50 values were obtained from concentration−response curves (Figure 4). Each data point represents mean values of at least 40 cells measured. SEM of the EC50 values was determined from n = 3 concentration−response curves.

30 μM for Trolox and ADP, respectively. However, dinucleotides 10−11 inhibited ABTS•+, with IC50 above 60 μM. Evaluation of Cell Toxicity by Analogues 7, 9, and 11. Nucleotide derivatives 7, 9, and 11 at various concentrations up to 200 μM were tested for cell toxicity by the MTT assay using PC12 cells and found to have no effect on cell viability after 48 h (data not shown). Inhibition of ROS Production in PC12 Cells under Oxidizing Conditions by Analogues 6−11. The modulation of Fe(II)-induced oxidative stress in PC12 cells by nucleotides and dinucleotides, was investigated using DCFH-DA,45 an indicator sensitive to radicals. When DCFH-DA is oxidized by ROS, it is converted to 2′,7′-dichlorofluorescein (DCF) and emits green fluorescence. The fluorescence intensity, a function of the ROS concentration in the cells, was measured quantitatively by a spectrofluorometer (Table 4). A reduction of 50% of the total ROS concentration was observed at 21 μM ADP, 2, while replacement of Pα by boranophosphate in ADP-α-B, 6, resulted in a 21-fold more potent analogues (IC50 1 μM for both isomers). Furthermore, substitution of the adenine C2position by Cl in addition to boranophosphate at Pα, 7a, triggered a 262-fold increase of antioxidant activity (IC50 0.08 μM) vs ADP. Analogue 7a was 2.5-fold more potent than 7b (IC50 0.2 μM), whereas ATP inhibited ROS production by only 45% at 100 μM, analogues 8a/b inhibited ROS formation with IC50 of 1 and 2.6 μM, respectively. Substitution of the ATP Pβ-Pγbridging oxygen atom by CCl2 group, 9a/b, (IC50 0.18 and 0.19 μM, respectively) improved antioxidant activity about 5-fold for a isomer as compared to 8a. Replacing Pγ in Ap5A by a boranophosphate, analogue, 10, brought about a 6-fold enhancement of antioxidant activity, while positioning the boranophosphate moiety at Pα, analogue, 11a, resulted in 70-fold improvement (IC50 5 and 0.4 μM, respectively). Resistance of 6a/b and 7a/b to Hydrolysis by Ectonucleotidases. Nucleotide triphosphate diphosphohydrolase F



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Figure 5. Concentration−response curve for agonists 1 and 2 (A), 5 (B), 6b (C), 7a and 7b (D), and 9a and 9b (E). Data were obtained by determining the agonist-induced change in [Ca2+]i of 1321N1 cells stably expressing the human P2Y11GFP receptor. Cells were preincubated with 2 μM fura-2 AM for 30 min, and change in fluorescence (ΔF340 nm/F380 nm) was detected.

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DISCUSSION Activity of Nucleotides 6−11 at hP2Y1/11-R. Activity data of nucleotides 6−11 at hP2Y1/11-Rs led us to several SAR observations.

by the MTT assay. Figure 8 shows enhancement of primary neuron cells viability due to their treatment with 7a and 7b. Compound 7a showed a most effective protection with EC50 value of 0.17 μM, while the b isomer was less active with EC50 value of 0.4 μM. G



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Table 2. Inhibition of OH Radical Production by Adenine Nucleotides and Dinucleotides 6−11a

Table 4. Inhibition of Fe(II)-Induced ROS Production in PC12 Cells by Nucleotides 6−11 vs 1, 2, and 4a

compd

IC50 (μM)

compd

IC50 (μM)

EDTA 1 2 4 6a 6b 7a 7b 8a 8b 9a 9b 10 11a 11b

54 ± 5 na 170 ± 6.36 75 ± 4 47 ± 1.86 41 ± 2.02 175 ± 11.96 120 ± 1.56 66 ± 4.3 36 ± 2.5 131 ± 8 60 ± 2 161 ± 15 59 ± 3.53 47 ± 3.5

Trolox 1 2 4 6a 6b 7a 7b 8a 8b 9a 9b 10 11a 11b

20 ± 2.4 na 21 ± 1.5 29 ± 1.6 1 ± 0.1 1 ± 0.2 0.08 ± 0.002 0.2 ± 0.005 1 ± 0.28 2.6 ± 0.4 0.18 ± 0.03 0.19 ± 0.4 5 ± 0.47 0.4 ± 0.02 1.2 ± 0.06

Values represent mean ± SD of three experiments (P < 0.05). na: the nucleotide did not inhibit 50% of ROS production.

a

a

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

Table 5. Hydrolysis of Analogues 6 and 7 by Ectonucleotidasesa

Table 3. Scavenging of ABTS•+ by Nucleotides 6−11 vs Trolox compd

IC50 (μM)

Trolox 1 2 4 6a 6b 7a 7b 8a 8b 9a 9b 10 11a 11b

18 ± 1.41 60 ± 0.49 30 ± 0.54 >60 26 ± 0.5 24 ± 1.62 40 ± 1.72 39 ± 0.53 23 ± 0.6 24 ± 0.7 24 ± 2.12 27 ± 3 61 ± 1.83 >60 >60

relative hydrolysis (% ± SD of ADP hydrolysis) human ectonucleotidase

6a

6b

7a

7b

NPP1 NPP3 NTPDase1 NTPDase2 NTPDase3 NTPDase8

5.4 ± 1.1 3.6 ± 0.7 ND ND ND ND

ND ND ND ND 6 ± 0.2 6 ± 0.2

5.6 ± 0.5 2.5 ± 0.6 ND 15 ± 1 22 ± 3 5 ± 0.1

2.5 ± 0.6 2.9 ± 0.8 ND 46 ± 5 ND ND

a

ND = not detected.

C. Pα Borano Substituted Adenine-Nucleotide Diastereoisomers Are Recognized with Opposite Diastereoselectivity by Either P2Y1-R or P2Y11-R. Here, we found the a isomer of analogues 6, 7, 8, 9, and 11 to be more active than the b isomer at the P2Y1-R, while the opposite diastereoselectivity at the P2Y11-R was observed with 9a/b. This is consistent with our previous reports on the selectivities of P2Y1/11-Rs. P2Y1-R displayed preference for the Rp isomer of borano-phosphate adenine nucleotides, whereas the P2Y11-R preferred the Sp isomers.41,51 D. CCl2 Replacement of the Pβ-Pγ-Bridging Oxygen Atom Increases P2Y11 vs P2Y1R Selectivity and Renders 9b the Most Potent P2Y11R Currently Known. Resistance of nucleotides to hydrolysis by NTPDase and alkaline phosphatase may be achieved by replacing the bridging oxygen atom between Pβ-Pγ with a dihalomethylene group.39 Therefore, we synthesized compounds containing dichloromethylene group at Pβ-Pγ position, analogues 9a/b. We found that the addition of the dichloromethylene group reduced agonist activity at P2Y1R. Analogue 9b proved to be a most potent P2Y11-R agonist, being 51.5-fold more potent than ATP. In addition, we found that 9b isomer is 10-fold more potent than 9a isomer. These results are consistent with our previous reports on the opposite diastereoselectivity of P2Y11 vs P2Y1 receptor.51 E. Pα-B, Pγ-B Dinucleotides Are Not Active at hP2Y1-R. Previously, we reported 10 to be a most potent rP2Y1-R agonist, yet this compound suffered from metabolic instability.36 In this study, we found compound 10 to be less active

A. The Length of the Nucleotide Phosphate Chain Determines Activity at P2Y1-R. 2-Cl-ADP-α-B, analogue 7a, was found to be the most potent compound studied here, being 10-fold more active than the corresponding triphosphate analogue 8a, in agreement with the preference of P2Y1-R for ADP.5 Compounds 7a/b were found to be P2Y1-R selective showing no activity at P2Y11-R. B. 2-Cl Modification Renders ADP-α-B the Most Potent P2Y1R Agonist Currently Known. Comparison of 6a (EC50 0.3 μM) to ADP (EC50 0.024 μM) indicates that the borane substitution had no significant effect on the affinity of the ligand toward P2Y1-R. Previously we reported that modification at C2 either by electron-donating or -withdrawing groups increased the affinity of the ligand to P2Y1-R.32 Indeed, here, we found that 2-Cl substitution rendered 7a 3.5-fold and 43-fold more active than ADP and 6a, respectively. Furthermore, 7a was found to be 1.8-fold more active than 2-MeS-ADP, 3, (EC50 13 nM), thus making 7a the most potent P2Y1-R agonist currently known. H



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Figure 6. Stability of nucleotide 7a, compared to 6a and ADP, in human blood serum. The assay mixture containing 0.1 mg of each analogue in deionized water (4.5 μL), human blood serum (180 μL), and RPMI-1640 medium (540 μL) was incubated at 37 °C for 0−24 h. The hydrolysis rate of the nucleotide analogues was determined by measuring the change in the integration of the respective HPLC peaks with time. Values represent mean ± SD of two experiments (P < 0.05).

at the hP2Y1-R (EC50 1.2 μM) as compared to Ap5A (EC50 0.76 μM). Recently, we reported that dinucleotide analogues bearing borane substitution at Pα are more resistant to hydrolysis by NPP1 than analogues bearing borane at Pβ.37 Here, we found that Ap5A analogue in which the Pα nonbridging oxygen was replaced by a borane group, 11a, was also more active than Ap5A at hP2Y1-R (EC50 0.2 vs 0.76 μM). F. 2-Cl-ADP-α-B Also Activates P2Y12R. Like P2Y1-R, P2Y12-R is activated by ADP and 2-MeS-ADP. Hence, we evaluated the possible involvement of the most promising neuroprotectant identified here, 7a, in P2Y12-R activation at PC12 cells (expressing P2Y12-R but not P2Y1R) under oxidizing conditions with or without P2Y12-R antagonist, 2-MeS-AMP. Decrease up to 70% in the antioxidant activity of 7a in the presence of 2-MeS-AMP implies the activation of P2Y12-R by 7a. Nucleotide Analogues 6−11 are Fenton Reaction Inhibitors. Here we found that the Pα-borano substitution improved significantly the inhibition of the Fenton reaction by the ADP analogues. Specifically, 6a/b exhibited IC50 47 and 41 μM, respectively, vs 170 μM for ADP. However, introduction of Cl at the adenosine C2 position in analogues 6a/b to give 7a/b reduced the activity of these analogues. This may be due to the fact that ADP binds Fe(II) in a “closed” structure in which the coordination to the metal-ion is both with the phosphate chain and the N7-nitrogen of the base residue.52 2-Cl substitution reduces electron density at the nucleobase, and therefore N7-coordination with the metal-ion is reduced. Surprisingly, 2-Cl-ATP(α-B) 8b was found to be the most active compound in this series (IC50 36 μM) and 2-fold more active than 8a (IC50 66 μM). Here, Cl substitution probably did not affect the compound activity and can be explained by the fact that ATP-Fe(II) complex exists mostly in the “open” structure52 in which the coordination to the metal-ion occurs only with the phosphate chain. Replacing the bridging oxygen atom in 8a/b with the CCl2 group in 9a/b decreased the antioxidant activity. We assume that the reason for that may be the reduction of the electron density on the nonbridging oxygen by the CCl2 group and hence reduction of chelation of Fe(II).

Figure 7. Reduction of antioxidant activity of 7a in PC12 cells under oxidizing conditions upon blocking P2Y12-R with 50 μM 2-SMe-AMP.

Figure 8. Neuroprotective activity of nucleotides 7a/b. Cortical neurons were treated with increasing concentrations (0.01−5 μM) of the nucleotides, FeSO4 (3 μM) for 24 h. Cell viability was measured by the MTT assay. The results shown are the mean ± SD of three independent experiments in triplicate (P < 0.05). I



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the neuroprotective effect of nucleotide 7, which we found to be a highly potent hP2Y1-R agonist. We explored the neuroprotective effect of 7a/b at primary neurons subjected to Fe(II) oxidation. Upon FeSO4 treatment, only 20% of the neuronal cells remained vital, while treatment with 7a/b improved the vitality of the neuronal cells up to 60% at 1 μM 7a (Figure 6). We found that the a isomer is a better neuroprotectant than the b isomer. These results are in good correlation with a higher potency of 7a at P2Y1-R, thus implying the involvement of this receptor in protection of primary neurons. Furthermore, the possible involvement of P2Y12-R was established by inhibition of antioxidant activity of 7a in PC12 cells expressing P2Y12-R but not P2Y1-R. The application of 7a as a drug candidate depends on its BBB permeation. If our future in vivo studies will show insufficient BBB permeability of 7a, we will apply known methodologies, such as the use of magnetic nanoparticles,53 inclusion into liposomes,54 or nucleotide prodrugs55 to allow transport of 7a across the BBB and, hence, neuroprotection.

Comparison of the dinucleotides 10 and 11a/b to Ap5A (IC50 75 μM) revealed that the location of the borane group influences the antioxidant activity of this compounds. A Pγ borane group in dinucleotide 10 decreased the antioxidant activity (IC50 161 μM). However, a Pα−borane group improved antioxidant activity. In most cases the b isomer was more active than the a isomer. In general it seems that the borane group improves the antioxidant activity. However, the exact mechanism is still unknown and requires further investigation. Nucleotide Analogues 6−11 Function Also As Radical Scavengers. As the Fenton reaction can be inhibited by both metal-ion chelators and radical scavengers, we also evaluated analogues 6−11 as radical scavengers by the ABTS decolorization assay. We found that dinucleotides 10−11 are mild radical scavengers, and it seems that they act mainly as Fe(II) chelators. Furthermore, nucleotides 6−9 reached IC50 values close to that of ADP and Trolox. Although none of the tested compounds showed high activity as a radical scavenger, this mechanism of antioxidant activity of the tested nucleotides cannot be ignored. 2-Cl-ADP(α-B), 7a, is a Highly Potent Antioxidant in PC12 Cells under Oxidative Stress. The antioxidant potential of nucleotides 6−11 in cell-free systems encouraged us to study their potential to protect cells from oxidative damage. For this purpose, we used PC12 cells as a model for neuronal cells. Analysis of the SAR of the tested compounds revealed that: A. The Borane Moiety Improves the Antioxidant Activity of the Nucleotides. The introduction of BH3-substitution in ADPα-B, 6, at Pα improves the antioxidant activity 21-fold as compared to ADP at PC12 cells under oxidative stress. The beneficial effect of the BH3-group was observed also for the dinucleotide scaffold, where 11a was 60-fold more active than Ap5A. Yet, when the borane group was located at Pγ, in compound 10, it resulted in only 6-fold activity enhancement vs the parent compound. B. Substitution of Cl at the C2 Position of the Adenine Ring Improves Antioxidant Activity. 2-Cl substitution at ADPα-B, 7a, improves antioxidant activity of 6a 12-fold. Indeed, previously we found that 2-Cl substitution improves agonist activity of an adenine nucleotide at P2Y-R.32 Because PC12 cells express P2Y2,4,6,12-Rs,49 we assume that the mechanism of the antioxidant activity also involves P2Y-R activation. C. Replacement of the Bridging Oxygen Atom with CCl2 Group Improves Antioxidant Activity. Substitution of CCl2 at Pβ-Pγ position of 2-Cl-ATP-α-B improved antioxidant activity 5-fold. Previously, we found that 2-SMe-ATP(α-B, β,γ-CCl2) is highly resistant to hydrolysis by NTPDase and NPP1,3.47 Therefore, we assume that the stability of 9 vs 8 may be the reason for its higher antioxidant activity. D. A Borane Group at Pα Improves Antioxidant Activity Better than at Pγ. Replacement of the nonbridging oxygen atom by borane group either at Pγ (10) or Pα (11) improved activity of the parent compound, Ap5A, 4. Apparently, borane group placed at the Pα-position 11a resulted in a 12-fold more active compound than that with a borane group at Pγ (10), while isomer 11b was 4-fold more active. This observation may be due to the lower resistance of Ap5-γ-B-A to NPP1 hydrolysis of the phosphate chain between Pα-Pβ.36 These findings are also consistent with our previous study showing that the a isomer of Pα-borane modified Np3/4N is more stable than the b isomer.37 Nucleotide 7a is a Potent Neuroprotectant. The neuroprotective effect of ATP was attributed to its binding to P2X-Rs and P2Y-Rs.7 These findings encouraged us to explore

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CONCLUSIONS Oxidative stress, implicated in various health disorders, is a multifunctional condition. Hence, treatment of neurons under oxidative stress requires the application of a multifunctional drug. Here, we have identified a highly promising neuroprotectant, analogue 7a, which rescued primary neurons subjected to oxidative stress, with EC50 value of 170 nM. On the basis of our studies, we suggest that the mechanism of action of 7a involves P2Y1-R activation (EC50 7 nM, 2-fold more active than the most P2Y1-R agonist known so far), Fe(II)- chelation (IC50 175 vs 54 μM for EDTA), and radical scavenging (IC50 40 vs 18 μM for Trolox). Our findings imply that P2Y12-R may also be activated by 7a. In addition, this compound proved to efficiently inhibit ROS formation in PC12 cells under oxidative stress (IC50 80 nM) and was nontoxic up to a concentration of at least 200 μM. Moreover, this compound proved to be metabolically stable in human blood serum (t1/2 7 h), and enzymatically stable in the presence of NTPDase1,8 and NPP1,3 (0−5% hydrolysis). Hence, we propose nucleotide 7a as a highly promising neuroprotectant. In vivo studies of BBB permeable prodrugs of 7a will be reported in due course.

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EXPERIMENTAL SECTION

Chemistry. General. All air and moisture sensitive reactions were carried out in flame-dried, argon flushed, two-neck flasks sealed with rubber septa, and the reagents were introduced by syringe. Progress of reactions was monitored by TLC on precoated Merck silica gel plates (60F-254). Visualization was accomplished by UV light. Flash chromatography was carried out on silica gel (Davisil Art. 1000101501). The separation on the automatic column was carried out using an HPFC automated flash purification system (Biotage SP1 separation system (RP)). Compounds were characterized by NMR using Bruker AC-200, 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 microinstrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a Sephadex DEAE-A25 column, swollen in 1 M NaHCO3 at room temperature for 1 day. The resin was washed with deionized water before use. The LC separation was monitored by UV detection at 280 nm. A buffer gradient J



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of NH4HCO3 was applied as detailed below. Final purification of the nucleotides was achieved on an HPLC (Merck-Hitachi) system, using a semipreparative reverse-phase column (Gemini 5u C-18 110A, 250 mm × 10.00 mm, 5 μm, 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 ≥95%. All commercial reagents were used without further purification unless otherwise noted. All reactants in moisture sensitive reactions were dried overnight in a vacuum oven. All phosphorylation reactions were carried out in flame-dried, argon-flushed, two-neck flasks sealed with rubber septa. Nucleosides were dried in vacuo overnight. Phosphorus oxychloride was distilled and kept under nitrogen. The tri-n-butylammonium pyrophosphate and tri-n-butylammonium phosphate solutions were prepared as described previously.56 Synthesis of Analogues 6, 8, 10, 12, and 24. ADP(α-B), 6, 2-Cl-ATP(α-B), 8, 2-Cl-adenosine, 12, Ap5(γ-B)A, 10, and ATP(α-B), 24, were synthesized according to literature procedures.32,57,36 2-Cl-Adenosine 5′-O-α-boranodiphosphate (7). 2-Chloro2′,3′-O-methoxymethylideneadenosine, 13, (0.57 mmol) was dissolved in DMF (1.5 mL)/ pyridine (2.8 mmol, 5 equiv), and a freshly prepared solution of 2-chloro-1,3,2-benzodioxaphosphorin-4-one (0.627 mmol, 1.1 equiv) in freshly distilled dioxane was added. After stirring for 10 min, a freshly prepared 1 M solution of bis(tri-n-butylammonium) pyrophosphate (0.85 mmol, 1.5 equiv) in DMF and tri-n-butylamine (2 mmol, 4 equiv) were simultaneously added. Precipitation occurred immediately after the addition of the reagents but disappeared with further stirring. A 2 M solution of BH3:Me2S in THF (4.56 mmol, 8 equiv) was added, and the mixture was stirred for 15 min. Ethylenediamine (2.8 mmol, 5 equiv) was then added. A white precipitate was immediately formed. After stirring for 60 min, deionized water (1 mL) was added and the white precipitate gradually dissolved. After 10 min, the reaction solution mixture was evaporated and then diluted by deionized water and washed twice with ethyl ether. The aqueous layer was then freeze-dried. The 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 ion-exchange chromatography (on DEAE Sephadex, swollen overnight in 1 M NaHCO3 at 4 °C). The column was eluted with anammonium bicarbonate gradient of 0−0.2 M (200 mL each) and then 0.2−0.4 M (200 mL each). Finally, aqueous solutions of the products separated on HPLC, as described below, 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. Separation of 7a and 7b. The separation of diastereoisomers, 7a and 7b was accomplished using a semipreparative reverse-phase Gemini 5μ column and isocratic elution with 83:17 (A) 100 mM TEAA, pH 7: (B) MeOH at a flow rate of 4 mL/min. Fractions containing purified isomers (Rt: 11 min (7a); 15 min (7b)) 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 38% overall yield (115 mg) after LC separation. 2-Cl-Adenosine 5′-O-α-boranodiphosphate (7a). 1H NMR (D2O, 600 MHz): δ 8.53 (s, 1H, H-8), 6.03 (d, J = 5.6 Hz, 1H,H-1′), 4.71(dd, J = 5.6, 4.4 Hz, 1H, H-2′), 4.57 (dd, J = 4.4, 4 Hz, 1H, H-3′), 4.37 (m, 1H, H-4′), 4.2 (m, 1H, H-5′), 4.10 (m, 1H, H-5″), 0.45 (m, 3H, BH3) ppm. 31P NMR (D2O, 200 MHz) δ: 82.2 (m, Pα-BH3), −9.4 (d, Pβ). HR MALDI (negative): calcd for C10H16B1Cl1N5O9P2 458.0199, found 458.022. Purity data obtained on an analytical column: retention time 10.09 min (95% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min).

Retention time: 9.16 min (96% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). 2-Cl-Adenosine 5′-O-α-boranodiphosphate (7b). 1H NMR (D2O, 600 MHz): δ 8.51 (s, 1H, H-8), 6.03 (d, J = 5.6 Hz, 1H, H-1′), 4.71(dd, J = 5.6, 5 Hz, H-2′, 1H), 4.57 (dd, J = 5, 4 Hz, 1H, H-3′), 4.37 (m, 1H, H-4′), 4.23 (m, 1H, H-5′), 4.14 (m, 1H, H-5″), 0.30 (m, 3H, BH3) ppm. 31P NMR (D2O, 200 MHz) δ: 82.1 (m, Pα-BH3), −9.0 (d, Pβ). HR MALDI (negative): calcd for C10H16B1Cl1N5O9P2 458.0199, found 458.022. Purity data obtained on an analytical column: retention time 14.29 min (95% purity) using solvent system I (isocratic elution of 93:7 A:B over 20 min at a flow rate of 1 mL/min). Retention time: 10.8 min (97% purity) using solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Preparation of Bis(tributylammonium) Dichloromethylene Diphosphonate Salt. A H+-Dowex 50WX-8 200 mesh activated by washing with 10% NaOH (150 mL) until the pH of the effluent was basic. Then the column was washed with distilled water until the pH of the effluent reached neutral. Finally, the column was washed with 10% HCl (300 mL), followed by distilled water until the effluent reached acidic and neutral pH, respectively. A flask containing 2 equiv of Bu3N in EtOH was placed in an ice bath under the column and stirred. Disodium dichloromethylene diphosphonate salt in distilled water was poured onto the column, and the column was washed with distilled water until the pH of the effluent was neutral. The effluent dropped into the Bu3N/EtOH solution. The final solution of bis(tributylammonium)dichloromethylene diphosphonate salt was then freeze-dried and then dried by coevaporation with dry DMF. 2-Cl-Adenosine 5′-O-α-borano-β−γ-dichloro-methylenetriphosphate (9). 2′,3′-O-Methoxymethylidene 2-Cl-adenosine, 13, (260 mg; 0.75 mmol) was dissolved in trimethylphosphate (5.7 mL) in a flame-dried two-neck flask under N2. 1,8-Bis(dimethylamino) naphthalene (324 mg; 1.5 mmol; 2 equiv) was added at 0 °C, and the reaction was stirred for 20 min until a clear solution was attained. PCl3 (138 μL; 1.5 mmol; 2 equiv) was added at 0 °C and a white solid precipitated. The suspension was stirred at 0 °C for 30 min. Then, a 0.5 M solution of bis(tributylammonium) dichloromethylene diphosphonate salt (657 mg; 12.2 mmol; 3 equiv) in dry DMF (3.2 mL) and tributylamine (0.72 mL; 3.03 mmol; 4 equiv) were added at 0 °C, and the reaction mixture was stirred for 60 min. A 2 M solution of BH3·SMe2 complex in THF (8.6 mL; 15.1 mmol; 20 equiv) was added at 0 °C, and the reaction mixture became clear. The solution was stirred for 5 min at 0 °C and then for 1.5 h at RT. Finally, a 0.5 M TEAB solution (10 mL) was added at RT, and the mixture was stirred for 60 min and then freeze-dried. The 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 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 residue dissolved in a minimal volume of water. The separation was monitored by UV detection at 280 nm. A buffer gradient of 0−0.5 M NH4HCO3 (1 L of each solution) were applied. The different fractions were pooled and freeze-dried three times to yield a yellow solid. Finally, aqueous solutions of the products separated on HPLC, as described below, 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. Separation of 9a and 9b. The separation of diastereoisomers 9a and 9b was accomplished using a semipreparative reverse-phase Gemini 5μ column and isocratic elution with 93:7 (A) 100 mM TEAA, pH 7:(B) CH3CN at a flow rate of 5 mL/min. Fractions containing purified isomers (Rt: 8.12 min (9a); 9.73 min (9b)) 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 9a and 9b were obtained at a 30% overall yield (101 mg) after LC separation. 2-Cl-Adenosine 5′-O-α-borano-β,γ-dichloromethylene-triphosphate (9a). 1H NMR (D2O, 600 MHz): δ 8.49 (s, 1H, H-8), 5.98 K



Article

(d, J = 6 Hz, 1H, H-1′), 4.8 (1H, H-2′, signal is hidden by the water signal), 4.56 (m, 1H, H-3′), 4.3 (m, 2H, H-4′, H-5′), 4.1 (m, 1H, H5″), 0.80 (m, 3H, BH3) ppm. 31P NMR (D2O, 200 MHz): δ 85.58 (m, 1P, Pα-BH3), 8.10 (d, J = 19 Hz, 1P,Pγ), −0.63 (d,d, J = 19, 19.1 Hz, 1P, Pβ) ppm. HR MALDI (negative): calcd for C11H17B1Cl3N5O11P3 603.9291, found 603.932. Purity data obtained on an analytical column: retention time, 12.3 min (96% purity) using solvent system I (isocratic elution of 93:7 A:B over 25 min at a flow rate of 1 mL/min). Retention time: 8.96 min (94% purity) solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). 2-Cl-Adenosine 5′-O-α-borano-β,γ-dichloro-methylene-triphosphate (9b). 1H NMR (D2O, 600 MHz): δ 8.48 (s, 1H, H-8), 5.97 (d, J = 5.6 Hz, 1H, H-1′), 4.6 (1H, H-2′, signal is hidden by the water signal) 4.47 (m, 1H, H-3′), 4.33 (m, 1H, H-4′), 4.23 (m, 1H, H-5′), 4.19 (m, 1H, H-5″), 0.83 (m, 3H, BH3) ppm. 31P NMR (D2O, 200 MHz): δ 84.8 (m, 1P, Pα-BH3), 7.3 (d, J = 19.8 Hz, 1P, Pγ), 1.6 (dd, J = 19.8, 20 Hz, 1P, Pβ) ppm. HR MALDI (negative) calcd for C11H17B1Cl3N5O11P3 603.9291, found 603.932. Purity data obtained on an analytical column: retention time, 17.1 min (95% purity) using solvent system I (isocratic elution of 93:7 A:B over 25 min at a flow rate of 1 mL/min). Retention time: 11.66 min (97% purity) solvent system II (isocratic elution of 97:3 A:B over 20 min at a flow rate of 1 mL/min). Diadenosine 5′,5″-P1,P5,α-Boranopentaphosphate (11). Trin-butylammonium-trin-octylammoniumadenosine 5′-diphosphate salt (101 mg, 0.194 mmol) was dissolved in dry DMF (1.5 mL) and added to a flame-dried, nitrogen-flushed two-neck round-bottom flask containing CDI (157 mg, 0.97 mmol, 5 equiv). The reaction was stirred at RT. After 2 h, TLC (NH4OH:H2O:2-propanol 2:7:11) showed the presence of a less polar product and the complete disappearance of the starting material. MeOH (0.19 mL, 0.97 mmol, 5 equiv) was added to consume remaining CDI, and after 10 min, a solution of 24 (0.193 mmol, 1 equiv) in dry DMF (1.5 mL) and MgCl2 (73 mg, 0.766 mmol, 4 equiv) was added. The solution was stirred at RT, and TLC monitoring after 24 h showed the presence of a more polar product. Water was added, and the solution was freezedried. The 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 semisolid obtained after freezedrying was chromatographed on an activated Sephadex DEAE-A25 column. The resin was washed with deionized water and loaded with the crude residue dissolved in a minimal volume of water. Separation was monitored by UV detection at 280 nm. A buffer gradient of 0.2−0.7 M NH4HCO3 (800 mL of each solution) was applied. The different fractions were pooled and freeze-dried three times to yield a white solid. Finally, aqueous solutions of the products separated on HPLC, as described below, 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. Separation of Diadenosine 5′,5″-P1,P5,α-Boranopentaphosphate (11a and 11b). The separation of diastereoisomers, 11a and 11b, was accomplished using a semipreparative reverse-phase Gemini 5μ column and isocratic elution with 92.5:7.5 (A) 100 mM TEAA, pH 7:(B) CH3CN at a flow rate of 5 mL/min. Fractions containing purified isomers (Rt: 7.09 min (11a); 9.48 min (11b)) 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 11a and 11b were obtained at a 67% overall yield (129 mg) after LC separation. Diadenosine 5′,5″-P1,P5,α-Boranopentaphosphate (11a). 1H NMR (D2O; 600 MHz): δ 8.46 (s, 1H, H-8A), 8.40 (s, 1H, H-8B), 8.12 (s, 1H, H-2A), 8.11 (s, 1H, H-2B), 6.05 (d, J = 6.4 Hz, 2H, H-1′), 4.75 (2H, H-2′A, H-2′B signal is hidden by the water signal), 4.61 (m, 2H, H-3′A), 4.58 (m, 2H, H-3′B), 4.38 (m, 2H, H-4′A, H-4′B), 4.29 (m, 2H, H-5′A, H-5′B), 4.23 (m, 1H, H-5″A), 4.17 (m, 1H, H-5″B), 0.42 (m, 3H, BH3) ppm. 31P NMR (240 MHz, D2O) δ: 84.73 (m, 1P, Pα-BH3), −10.83 (d, 1P, Pβ), −22.61 (m, 3P, Pα,β,γ) ppm. HR MALDI (negative):

calcd for C20H31B1N10O21P5 913.0441, found 913.051. Purity data obtained on an analytical column: retention time, 7.37 min (94% purity) using solvent system I (isocratic elution of 93:7 A:B over 15 min at a flow rate of 1 mL/min). Retention time, 3.99 min (90% purity) solvent system II (isocratic elution of 97:3 A:B over 15 min at a flow rate of 1 mL/min). Diadenosine 5′,5″-P1,P5,α-Boranopentaphosphate (11b). 1H NMR (D2O; 600 MHz): δ 8.44 (s, 1H, H-8A), 8.38 (s, 1H, H-8B), 8.08 (s, 1H, H-2A), 8.07 (s, 1H, H-2B), 6.00 (m, 2H, H-1′), 4.70 (m, 2H, H2′A, H-2′B), 4.56 (m, 2H, H-3′A), 4.52 (m, 2H, H-3′B), 4.38 (m, 2H, H-4′A, H-4′B), 4.30 (m, 2H, H-5′A, H-5′B), 4.23 (m, 2H, H-5″A, H5″B), 0.48 (m, 3H, BH3) ppm. 31P NMR (240 MHz, D2O) δ: 83.96 (m, 1P, Pα-BH3), −10.83 (d, 1P, Pβ), −22.64 (m, 3P, Pα,β,γ) ppm. HR MALDI (negative): calcd for C20H31B1N10O21P5 913.0441, found 913.040. Purity data obtained on an analytical column: retention time, 10.53 min (94% purity) using solvent system I (isocratic elution of 93:7 A:B over 15 min at a flow rate of 1 mL/min). Retention time, 4.9 min (89% purity) solvent system II (isocratic elution of 97:3 A:B over 15 min at a flow rate of 1 mL/min). Evaluation of the Resistance of Analogues 6 and 7 to Hydrolysis by NPP1,3. The percentage of hydrolysis of analogues 6 and 7 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 of the incubation mixture (1 mM CaCl2, 200 mM NaCl, 10 mM KCl, and 100 mM Tris, pH 8.5) and preincubated at 37 °C for 3 min. Reaction was initiated by the addition of 0.015 mL of 4 mM analogue. The reaction was stopped after 30 min or 1 h for NPP1 or NPP3, respectively, by adding 0.350 mL of ice-cold 1 M perchloric acid. These samples were centrifuged for 1 min at 10000g. Supernatants were neutralized with 140 μL of 2 M KOH in 4 °C and centrifuged for 1 min at 10000g. The reaction mixture was filtered and freeze-dried. Each sample was dissolved in 200 μL of HPLC water, and only 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 with an analytical reverse-phase HPLC using linear elution 85−97% 100 mM TEAA (pH 7) and 15−3% CH3CN, flow rate 1 mL/min. 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 versus ADP as control. The percentage of compound degradation was calculated versus ADP to take into account 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 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. Evaluation of the Resistance of Analogues 6−7 to Hydrolysis by NTPDase1,2,3,8. The percentage of hydrolysis of the new analogues by human NTPDase1,2,3,8 was evaluated as follows: 2.8 μg or 4.3 μg of human NTPase1,2,3,8 extract, respectively, was added to 0.579 mL of the incubation mixture (10 mM CaCl2 and 160 mM Tris, pH 7.4) and preincubated at 37 °C for 3 min. Reaction was initiated by the addition of 0.012 mL of 4.24 mM analogue. The reaction was stopped after 1 h by adding 0.350 mL of ice-cold 1 M perchloric acid. These samples were centrifuged for 1 min at 10000g. Supernatants were neutralized with 140 μL of 2 M KOH in 4 °C and centrifuged for 1 min at 10000g. The reaction mixture was filtered and freeze-dried. Each sample was dissolved in 200 μL of HPLC water, and only 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 with an analytical reverse-phase HPLC using linear elution 78−97% 100 mM TEAA (pH 7) and 22−3% AcN, flow rate 1 mL/min. The hydrolysis rates of analogues 6 and 7 by NTPDase1,2,3,8 were determined by measuring the change in the integration of the HPLC peaks for each analogue over time versus ADP as control. The percentage of compound degradation was calculated versus ADP to account for the degradation of the compounds due to the addition of L



Article

Determination of Involvement of P2Y12-R in Antioxidant Activity of Compound 7a. PC12 cells were grown in Dulbecco’s Modified Eagle’s Medium and seeded into medium in 96-well tissue culture plates for 24 h. 2-MeS-AMP (50 μM, 3.75 μL) was added to each cell in the microplate and incubated for 20 min. Determination of ROS production in cultured PC12 cells was performed with DCFHDA with or without the addition of 7a at a range of concentrations (0.04−100 μM) and initiation of oxidation by addition of FeSO4 (2 μM, 12 μL, final concentration 0.16 μM) to the wells. See experimental details above. Determination of Cell Viability by MTT Assay. Primary cortical neurons were grown in Dulbecco’s Modified Eagle’s F12 Medium and seeded into medium in 96-well tissue culture plates for 5 d. Cell viability was assessed using an MTT assay, which is a marker of mitochondrial activity.64 After 5 d, the tested compounds were added to the well. After 24 h, the culture medium was removed before the addition of MTT (1 mg/mL) in PBS, pH 7.4. 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 and plate reader. Data were presented as a percentage relative to their vehicle controls. Preparation of Primary Neuron Cell Cultures. Rat brain (1 d old) were removed under sterile conditions, the cortex was dissected and separated from the remaining brain, roughly homogenized by repeating pippetation, and then trypsin was added. The trypsin 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-multiwell plates (Nunc, Naperville, IL, USA) that had previously been precoated with polyornithine (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% penicillin−streptomycin− nystatin. After 24 h, the medium was replaced with fresh medium. After 72 h, the cells were treated with Ara-C (cytosine β-Darabinofuranoside, inhibitor of DNA replication) at 50 mM for 48 h, which result 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.

acid to stop the enzymatic reaction. Therefore, 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 peak formed due to chemical acidic hydrolysis. 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 and 380 nm excitations.58 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 and 380 nm. We only analyzed GFP-labeled 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 experiments.59,60 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.61−63 ABTS Radical Cation Decolorization Assay.44 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. 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 2 × 105 in experiments with Fe(II). Ammonium iron(II) sulfate (1 mM, 10 μL) were added to 5−500 μM tested compound (1−10 μL) solutions. Afterward, 1 mM Tris buffer, pH 7.4, (60−70 μL) was added to the mixture. After mixing for 30 s, 100 mM DMPO (10 μL) were quickly added followed by the addition of 100 mM H2O2 (10 μL). Final sample pH values for the Fe2+ 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 in a final volume of 100 μL. Determination of ROS Production in Cultured PC12 Cells.45 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 −20 °C. 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 °C, during which time absorbance was read by a Tecan fluorometer at 485/530 nm.

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ASSOCIATED CONTENT

* Supporting Information S

Concentration−response curve for agonists 8−11 in 1321N1 cells stably expressing the human P2Y1GFP receptor. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 972-3-5318303. Fax: 972-3-6354907. E-mail: bilha. fi[email protected]. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ATP, adenosine triphosphate; ADP, adenosine diphosphate; P2Y-R, P2Y-receptor; NPP, nucleotide pyrophosphatase/ phosphodiesterase; NTPDase, nucleotide triphosphate diphosphohydrolase; SAR, structure−activity relationship; TEAA, triethylammonium acetate; MALDI, matrix-assisted laser desorption/ionization; [Ca2+]i, intracellular Ca2+ concentration; SD, standard deviation; HRMS-MALDI, high-resolution mass spectrometry matrix-assisted laser desorption ionization M



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Article

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