CholesteroNitrones for Stroke - Journal of Medicinal Chemistry (ACS

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CholesteroNitrones for Stroke Maria I. Ayuso, Mourad Chioua, Emma Martínez-Alonso, Elena Soriano, Joan Montaner, Jaime Masjuán, Dimitra J. Hadjipavlou-Litina, José Marco-Contelles, and Alberto Alcázar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00755 • Publication Date (Web): 04 Aug 2015 Downloaded from http://pubs.acs.org on August 11, 2015

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Journal of Medicinal Chemistry

CholesteroNitrones for Stroke Maria I. Ayuso,1 Mourad Chioua,2,* Emma Martínez-Alonso,1 Elena Soriano,3 Joan Montaner,4 Jaime Masjuán,5 Dimitra J. Hadjipavlou-Litina,6 José Marco-Contelles,2,* and Alberto Alcázar 1,* 1

Department of Investigation, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain Laboratory of Medicinal Chemistry, Institute of Organic Chemistry (CSIC), Madrid, Spain 3 SEPCO, Institute of Organic Chemistry General (CSIC), Madrid, Spain 4 Institut de Recerca Vall d’Hebron, U. Autònoma de Barcelona and Hospital Vall d’Hebron, Barcelona, Spain 5 Department of Neurology, Hospital Ramón y Cajal, IRYCIS, Madrid, Spain 6 Department of Pharmaceutical Chemistry, Faculty Health Sciences, Aristotle U. Thessaloniki, Thessaloniki 54124, Greece 2

KEYWORDS: Nitrones; Steroids; Brain ischemia; Neuroprotection; Ischemic stroke; Reactive oxygen species. ABSTRACT: This study describes CholesteroNitrone 2 as an antioxidant and neuroprotective agent against ischemic injury. Neuroprotection was assessed using in vitro and in vivo experimental ischemia models. The compound significantly increased cell viability, induced neuroprotection following ischemic reperfusion, and decreased neurological deficit scores in treated animals, supporting the next pre-clinical studies as a potential agent for the treatment of stroke.

To date, reperfusion therapies (intravenous thrombolysis and endovascular treatment) are accepted as effective for the treatment of acute ischemic stroke. However, only a limited number of patients with stroke are receiving it due to the short therapeutic window on top of other clinical criteria. Therefore, there is an urgent need to develop additional effective therapies for ischemic stroke to extend the benefit of reperfusion therapies or to try to protect brain cells if administered soon after stroke, according to the neuroprotection therapeutic strategy.1 Nitrones are well known organic compounds that have extensively been used as spin-traps and powerful free radical scavengers for their ability to form adducts with reactive oxygen species (ROS).2 Consequently, due to their unique chemical properties and biological activity,3 nitrones have been proposed as therapeutic agents against several ROSrelated disorders such as brain ischemia.4-6 Phenyl tert-butyl nitrone (PBN, Chart 1) was the first nitrone that was shown to possess a neuroprotective action in several animal models of brain ischemia,4 and confer protection to endothelial cells against oxidative stress-induced injury, but the mechanisms for this protection remains unclear, and may be more complex than just through free radical scavenging.5 Disodium 4-[(Z)(tert-butyl-oxidoazaniumylidene)methyl]benzene-1,3-disulfonate NXY-059 (1) (Chart 1) also showed potential as therapeutic agents against stroke; however, agent 1 has not been successful in clinical studies due to various deficiencies in the design and analysis of preclinical and clinical studies.6-8 This background has supported our current efforts devoted to the search of new nitrones as neuroprotective agents for the development of new drugs for stroke.9 In this context, and being aware that steroids have been also proposed as neuroprotective

agents, able to prevent inflammation in the central nervous system,10 we have considered the synthesis and biological evaluation of new steroid-nitrone hybrids (CholesteroNitrones, ChNs) as permeable suitable drugs for the treatment of stroke. Very surprisingly, although steroidal nitrones are readily available molecules, their biological activities have been scarcely documented, and particularly, in the field of stroke we are not aware of any previous reported research. Thus, in this study, we report the synthesis, theoretical calculations in silico, antioxidant capacity, the in vitro and in vivo neuroprotective properties of the (E)- and (Z)- isomers of N((8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methyl heptan-2-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-1H-cyclo penta[a]phenanthren-3(2H,6H,10H)-ylidene)methanamine oxides,11 (2) and (3), respectively. The antioxidant and neuroprotective effect of these ChNs has been evaluated here for the first time by the usual protocols and cell viability assay on neuronal cultures subjected to experimental ischemia. We have found that ChN 2 is an antioxidant and neuroprotective agent when given after ischemia in neuronal cultures, and significantly decreases neuronal death after global cerebral ischemia in rats. Chart 1. Nitrones PBN and 1-3 N O PBN N O 1

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Synthesis of ChNs 2 and 3. The reaction of commercially available 4-cholesten-3-one (or 5-cholesten-3-one) with Nmethylhydroxylamine gave a mixture of E- and Z-isomers of ChNs 2 and 3 11 (Figure 1S, Supporting Information), respectively, easily separated by column chromatography. The structure and assignment of the configuration of the double bond in each ChN has been achieved by their analytical and spectroscopic data, including by selective nOe experiments in the 1H NMR spectra (Figures 2S, 3S, Supporting Information). Theoretical calculations. First of all, several theoretical parameters were studied in order to characterize the physicochemical properties of the ChNs (Supporting Information). Thus, the computed frontier orbitals for both E- and Z-isomers revealed subtle differences. ChN 2 showed a lower LUMO (lowest occupied molecular orbital) and HOMO (highest occupied molecular orbital) energies than the 3 isomer. Although both isomers showed a similar distribution over the unsaturated system, a slightly higher concentration of the orbitals lobes on C1 was found for the E-isomer than for the Z-isomer, as the orbital coefficients C1 revealed by the structural and electronic data (Table 1S, Supporting Information). These results suggest a higher reactivity for ChN 2. We have computed other parameters related to reactivity indices such as chemical resistance (η), electronic chemical potential (µ), and electrophilicity index (ω). η Is resistant to deformation or change in donating or withdrawing electrons; µ is used to measure the escaping tendency of electrons from regions with higher µ to areas of lower µ; and ω serves as a measure of the stabilization energy when atoms or molecules drawing maximal electrons. Because of this, a comparison between the two ChNs (Table 1S, Supporting Information) indicates that 2 would be the structure that requires the lowest energy for mechanisms involved in the radical scavenging activity, thus possessing higher antioxidant capacity. Thus, ChN 3 should be more stable and less reactive than the E-isomer. The calculated average polarizability for 2 is 497.31au and that for 3 is 489.24 au, confirming that both molecules have the capacity to polarize other atoms or molecules and are moderately soluble in polar solvents. In addition, our computed results suggest that these ChNs should present a good brain penetration profile, with a logBB value of 0.92 (Table 2S, Supporting Information). Finally, we have estimated the neuroprotective effect. As the neuroprotective profile is strongly dependent on the orbital energies, our results suggest higher neuroprotection for the 2 than 3 (Table 2S, Supporting Information). The formation of ROS is an unavoidable event for aerobic organisms,12 and their involvement in central nervous system ischemia is under intensive study.13 Due to the extreme reactivity and tendency of ROS to initiate and participate in chain reactions, the role of antioxidants as a defence system is highly recognised.14 Consequently, ChNs 2 and 3 have been tested for their antioxidant abilities (Supporting Information). Neuroprotection evaluation of ChNs against experimental ischemia at short-term recovery. Exposure of neuronal cultures to oxygen−glucose deprivation (OGD) for 4 h (OGD 4 h) induced a significant decrease in cell viability (67.3%, p
95%. General procedure for cholesteronitrone synthesis. A solution of the ketone (1 mmol), dry Na2SO4 (3 mmol), and triethylamine (2 mmol) were suspended in EtOH. Then, the hydroxylamine hydrochloride (1.5 mmol) was added. The mixture was stirred for 30 sec and then exposed to MWI (250 W) at 90 °C during the time indicated for each compound. When the reaction was over (TLC analysis), the solvent was removed under reduced pressure and diluted with water, extracted with AcOEt, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The resultant solid was purified by column chromatography to give pure compounds E-(2), and Z-(3).11 Method A. 4-Cholesten3-one (385 mg, 1 mmol), Na2SO4 (426 mg, 3 mmol), Et3N (0.30 mL, 2 mmol), and methylhydroxylamine hydrochloride (126 mg, 1.5 mmol) in ethanol (10 mL), after 3 h, and column chromatography (CH2Cl2/MeOH, from 1% to 2%), gave a mixture of 2 and 3 (396 mg, 96%, in 1:3 ratio). Method B. Following the general procedure, reaction of commercial 5-cholesten-3-one (385 mg, 1 mmol), Na2SO4 (426 mg, 3 mmol), Et3N (0.30 mL, 2 mmol), and methylhydroxylamine hydrochloride (126 mg, 1.5 mmol) in Ethanol (10 mL), after 2 h, and column chromatography (CH2Cl2/MeOH, from 1% to 2%), gave a mixture of 2 and 3 (407 mg, 98%, in a 3:1 ratio, respectively). Repeated and careful chromatography allowed us to obtain pure samples of 2 and 3 for biological analysis. (E)-N-((8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-((R)-6methyl heptan-2-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-1Hcyclopenta[a]phenanthren-3(2H,6H,10H)-ylidene) methanamine oxide (2). White solid; Rf (0.21, CH2Cl2/MeOH, 5%); mp 139-141 °C; IR (KBr) νmax 2939, 2868, 2849, 1466, 1215 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.97 (d, J= 2.0 Hz, 1H, 4 CH), 3.72 (s, 3H, NCH3), 3.23 (d, J= 18.4 Hz, 1H, 2CH), 2.34 (m, 2H, 6CH2), 2.21 (m, 1H, 2CH), 1.99 (m, 2H, CH2), 1.80 (m, 2H, CH2), 1.60 (s, 3H, CH3), 1.36 (m, 10H, 5CH2), 1.12 (m, 6H, 6CH2), 1.04 (s, 3H, 19CH3), 0.99 (m, 2H, CH2), 0.91 (d, J = 6.4 Hz, 3H, 21CH3), 0.88 (d, J = 1.3 Hz, 3H, 26CH3), 0.86 (m, 3H, 27 CH3), 0.70 (s, 3H, 18CH3); 13C NMR (101 MHz, CDCl3) δ 156.8 3 ( C), 146.4 (5C), 112.9 (4CH), 56.1 (17CH), 55.9 (14CH), 53.5 (9CH), 46.0 (13C), 42.3 (NCH3), 39.6 (C), 39.4 (C), 37.9 (10C), 36.1 (C), 35.77 (C), 35.73 (C), 34.4 (C), 33.4 (C), 32.2 (25CH2), 28.1 (16CH2), 27.9 (2CH2), 24.2 (15CH2), 23.8 (24CH2), 22.7 (26CH3), 22.5 (27CH3), 21.4 (CH2), 21.3 (11CH), 18.6 (19CH3), 17.8 (21CH3), 11.9 (18CH3). MS (EI) m/z: 413 (M, 37%)+, 398 (M-CH3, 27%), 397(M-O, 70), 137(C8H11NO, 100%); MS (ESI) m/z: 414.2 (M + H)+, 436.2 (M + Na)+, 827.8 (2M)+, 849.7 (2M + Na)+.

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Anal. Calcd for C28H47NO: C, 81.29; H, 11.45; N, 3.39. Found: C, 80.98; H, 11.29; N, 3.44. (Z)-N-((8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-((R)-6methyl heptan-2-yl)-7,8,9,11,12,13,14,15,16,17-decahydro-1Hcyclopenta[a]phenanthren-3(2H,6H,10H)-ylidene) methanamine oxide (3). White solid; Rf (0.20, CH2Cl2/MeOH, 5%); mp 153-155 °C; IR (KBr) νmax 2936, 2868, 1629, 1214 cm−1; 1 H NMR (400 MHz, CDCl3) δ 6.78 (s, 1H, 4CH), 3.66 (s, 3H, NCH3 ), 2.44 (m, 4H, 2CH2), 1.88 (m, 4H, 2CH2 ), 1.37 (m, 14H, 7CH2), 1.04 (s, 3H, 19CH3), 0.98 (m, 2H, CH2), 0.91 (d, J= 6.4 Hz, 3H, 21CH3), 0.88 (d, J = 1.4 Hz, 3H, 26CH3), 0.85 (d, J = 1.4 Hz, 3H, 27CH3), 0.70 (s, 3H, 18CH3); 13C NMR (101 MHz, CDCl3) δ 123.7 (3C), 120.3 (5C), 113.7 (4CH), 56.0 (17CH), 55.9 (14CH), 53.5 (9CH), 46.4 (13C), 42.3 (NCH3), 39.6 (C), 39.4 (C), 37.9 (10C), 36.0 (C), 35.72 (C), 35.71 (C), 35.4 (C), 32.9 (C), 32.2 (C), 28.1 (C), 27.9 (C), 24.1 (16CH2), 23.7 (15CH2), 23.6 (24CH2), 22.7 (26CH3), 22.5 (27CH3), 21.3 (11CH2), 18.6 (19CH3), 17.8 (21CH3), 11.9 (18CH3). MS (EI) m/z: 413 (M, 37%)+, 398 (M-CH3, 27%), 397(M-O, 70), 137 (C8H11NO, 100%); MS (ESI) m/z: 414.2 (M + H)+, 827.8 (2M)+, 849.7 (2M + Na)+. Anal. Calcd for C28H47NO: C, 81.29; H, 11.45; N, 3.39. Found: C, 81.03; H, 11.33; N, 3.30. ACKNOWLEDGMENTS AA wishes to thank M. Gómez-Calcerrada and Valerio Frezza for their technical assistance. This work was supported by ISCIII and FEDER grants to AA (PI1/00334 and PI14/00705). ABBREVIATIONS ChN, CholesteroNitrones; DPPH, 2,2-diphenyl-1-picrylhydrazyl; NDS, neurological deficit score; OGD, oxygen−glucose deprivation; PBN, phenyl tert-butyl nitrone; R5d, 5 d of reperfusion/recovery after ischemia; ROS, reactive oxygen species. ASSOCIATED CONTENT Supporting Information. Detailed theoretical calculations, biochemical experimental procedures and additional results. AUTHOR INFORMATION Corresponding authors: Dr. Alcázar. Phone: +34913369016. Email: [email protected]. Dr. Marco-Contelles: Phone: +34915622900. E-mail: [email protected]; Dr. Chioua: Phone: +34915622900. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. REFERENCES (1) Barone, F. C. Post-stroke pharmacological intervention: promoting brain recovery from injury in the future. Neuropharmacology 2010, 59, 650-653. (2) Villamena, F. A.; Zweier, J. L. Detection of reactive oxygen and nitrogen species by EPR spin trapping. Antioxid. Redox Signaling 2004, 6, 619-629. (3) Villamena, F. A.; Das, A.; Nash, K. M. Potential implication of the chemical properties and bioactivity of nitrone spin traps for therapeutics. Future Med. Chem. 2012, 4,1171-1207. (4) Molnar, M.; Lennmyr, F. Neuroprotection by S-PBN in hyperglycemic ischemic brain injury in rats. Upsala J. Med. Sci. 2010, 115, 163-168. (5) Kim, S.; de, A. V. G. V.; Bouajila, J.; Dias, A. G.; Cyrino, F. Z.; Bouskela, E.; Costa, P. R.; Nepveu, F. Alpha-phenyl-N-tertbutyl nitrone (PBN) derivatives: synthesis and protective action

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against microvascular damages induced by ischemia/reperfusion. Bioorg. Med. Chem. 2007, 15, 3572-3578. (6) Fong, J. J.; Rhoney, D. H. NXY-059: review of neuroprotective potential for acute stroke. Ann. Pharmacother. 2006, 40, 461-471. (7) Floyd, R. A.; Kopke, R. D.; Choi, C. H.; Foster, S. B.; Doblas, S.; Towner, R. A. Nitrones as therapeutics. Free Radicals Biol. Med. 2008, 45, 1361-1374. (8) Savitz, S. I. A critical appraisal of the NXY-059 neuroprotection studies for acute stroke: a need for more rigorous testing of neuroprotective agents in animal models of stroke. Exp. Neurol. 2007, 205, 20-25. (9) Chioua, M.; Sucunza, D.; Soriano, E.; Hadjipavlou-Litina, D. J.; Alcázar, A.; Ayuso, I.; Oset-Gasque, M. J.; González, M. P.; Monjas, L.; Rodríguez-Franco, M. I.; Marco-Contelles, J.; Samadi, A. α-Aryl-N-alkyl nitrones, as potential agents for stroke treatment: synthesis, theoretical calculations, antioxidant, antiinflammatory, neuroprotective, and brain-blood barrier permeability properties. J. Med. Chem. 2012, 55, 153−168. (10) Stein, D. G. Is progesterone a worthy candidate as a novel therapy for traumatic brain injury? Dialogues Clin. Neurosci. 2011, 13, 352-359. (11) Barton, D. H. R.; Day, M. J.; Hesse, R. H.; Pechet, M. M. A new rearrangement of ketonic nitrones; a convenient alternative to the Beckmann rearrangement. J. Chem. Soc., Perkin Trans. 1 1975, 1764-1767. (12) Patel, R, P.; Cornwell, T.; Darley-Usmar, V. M. The biochemistry of nitric oxide and peroxynitrite: Implications for mitochondrial function. In Understanding the Process of Aging: the Roles of Mitochondria, Free Radicals, and Antioxidants; Cadenas E., Packer L., Eds.; Marcel Dekker: New York, 1999; pp 39–56. (13) Lewén, A.; Matz, P.; Chan, P. H. Free radical pathways in CNS injury, J. Neurotrauma 2000, 17, 871-890. (14) Ringel, F.; Schmid-Elsaesser, R.; Liang, A. C. Antioxidants for CNS ischaemia and trauma, Expert Opin. Ther. Pat. 2001, 11, 987-997. (15) Ayuso, M. I.; Martínez-Alonso, E.; Cid, C.; de Leciñana, M. A.; Alcázar, A. The translational repressor eIF4E-binding protein 2 (4E-BP2) correlates with selective delayed neuronal death after ischemia. J. Cereb. Blood Flow Metab. 2013, 33, 1173-1181. (16) Rami, A.; Bechmann, I.; Stehle, J. H. Exploiting endogenous anti-apoptotic proteins for novel therapeutic strategies in cerebral ischemia. Prog. Neurobiol. 2008, 85, 273-296. (17) Bjorkhem, I. Crossing the barrier: oxysterol as cholesterol transporters and metabolic modulators in the brain. J. Intern. Med. 2006, 260, 493-508. (18) Saito, A.; Maier, C. M.; Narasimhan, P.; Nishi, T.; Song, Y. S.; Yu, F.; Liu, J.; Lee, Y. S.; Nito, C.; Kamada, H.; Dodd, R. L.; Hsieh, L. B.; Hassid, B.; Kim, E. E.; González, M.; Chan, P. H. Oxidative stress and neuronal death/survival signaling in cerebral ischemia. Mol. Neurobiol. 2005, 31, 105-116. (19) Warner, D. S.; Sheng, H.; Batinic-Haberle, I. Oxidants, antioxidants and the ischemic brain. J. Exp. Biol. 2004, 207, 3221-3231. (20) Pulsinelli, W. A.; Brierley, J. B.; Plum, F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 1982, 11, 491-498. (21) Harukuni, I.; Bhardwaj, A. Mechanism of brain injury after global cerebral ischemia. Neurol. Clin. 2006, 24, 1-21. (22) Hossmann, K. A. Pathophysiology and therapy of experimental stroke. Cell. Mol. Neurobiol. 2006, 26, 1057-1083. (23) Bath, P. M.; Gray, L. J.; Bath, A. J.; Buchan, A.; Miyata, T.; Green, A. R. Effects of NXY-059 in experimental stroke: an individual animal meta-analysis. Br. J. Pharmacol. 2009, 157, 1157-1171.

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Cholesteronitrone (E)-N-((8S,9S,10R,13R,14S,17R)-10,13-Dimethyl17-(( R)-6-methylheptan-2-yl)-7,8,9,11,12,13,14,15, 16,17-decahydro-1 H-cyclopenta[ a]phenanthren3(2H,6H,10H)-ylidene)methanamine oxide ( 2) induces neuroprotection after transient brain ischemia

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