Improving the Efficacy of Quinolylnitrones for Ischemic Stroke Therapy, QN4 and QN15 as New Neuroprotective Agents after Oxygen–Glucose Deprivation/Reoxygenation-Induced Neuronal Injury

In our search for new neuroprotective agents for stroke therapy to improve the pharmacological profile of the compound quinolylnitrone QN23, we have prepared and studied sixteen new, related and easily available quinolylnitrones. As a result, we have identified compounds QN4 and QN15 as promising candidates showing high neuroprotection power in a cellular experimental model of ischemia. Even though they were found to be less active than our current lead compound QN23, QN4 and QN15 provide an improved potency and, particularly for QN4, an expanded range of tolerability and improved solubility compared to the parent compound. A computational DFT-based analysis has been carried out to understand the antioxidant power of quinolylnitrones QN23, QN4 and QN15. Altogether, these results show that subtle, simple modifications of the quinolylnitrone scaffold are tolerated, providing high neuroprotective activity and optimization of the pharmacological potency required for an improved design and future drug developments in the field.


Introduction
Stroke is a pathology caused by the interruption of the blood supply to the brain, frequently of ischemic nature (87% of cases), mainly caused by a thrombus, but also due to the rupture of a blood vessel (hemorrhagic stroke) [1]. By cutting off the supply of oxygen and nutrients, stroke causes damage to the brain tissue [2]. A very severe stroke can cause sudden death, but it more likely constitutes a major cause of neurological disability, and the second cause of dementia after Alzheimer's disease [3]. Restoration of the normal circulatory conditions may reverse the damage, but in case it is not allowed, compromised tissue may also turn into infarcted tissue [4]. Thus, a proper reperfusion should be the first goal for any ischemic stroke therapy, but other new therapies are sought to extend the benefit of reperfusion therapies, ameliorate the restoration of cellular homeostasis and improve neuroprotection against the damage caused by reperfusion itself, i.e., oxidative stress. This is the case of neuroprotection based on antioxidants such as nitrones, wellknown organic molecules showing strong radical scavenging properties [5].
In this context, and based on a collaborative project with Dr. Alcázar's (Hospital Ramón y Cajal, Madrid, Spain) and Prof. Oset-Gasque's (UCM, Madrid, Spain) teams, In this context, and based on a collaborative project with Dr. Alcázar's (Hospital Ramón y Cajal, Madrid, Spain) and Prof. Oset-Gasque's (UCM, Madrid, Spain) teams, in the last decade we have dedicated a great effort to develop an ambitious project targeted to the identification of novel small nitrones for the therapy of cerebral ischemia (CI) [6]. So far, we have reported new α-phenyl-N-tert-butyl nitrone analogues [7][8][9][10][11], N-substituted, C-dialkoxyphosphorylated nitrones [12], cholesteronitrones [13,14] and quinolylnitrones (QNs) [15][16][17][18]. Particularly, in the development of QNs, we have successfully demonstrated that (Z)-N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)methanimine oxide (QN23) [15] ( Figure 1) is a new and potent antioxidant agent, showing strong power to scavenge diverse radical oxygenated species (ROS) and efficient in vitro and in vivo neuroprotective profile [15,19]. QN23 (Figure 1) was synthesized following the general method shown in Scheme 1 from 2-chloro-6-methoxyquinoline-3-carbaldehyde and N-tert-butylhydroxylamine hydrochloride, in 82% yield, and isolated as a single and pure Z-isomer [15].  In a previous study, we reported the neuroprotection capacity of QN23, compared with the NXY-059 as reference nitrone, in primary neuronal cultures subjected to 4 h of oxygen and glucose deprivation (OGD) conditions, followed by treatment with the nitrones added at the beginning of a 24 h recovery period [15]. Neuronal cultures were treated with different concentrations of selected nitrones and their effect was evaluated after 24 h of recovery after OGD by a cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In these experiments, the cell viability without OGD was considered as 100% control, while 4 h OGD resulted in a significant reduction in cell viability (67.6%) compared with 100% control (p <0.0001, one-sample t-test). The OGD-induced cell injury was partially reversed after 24 h of recovery, but it did not reach the control value (77.1%; p < 0.001 compared with 4 h OGD by Student's t-test). The reference neuroprotective nitrone NXY-059, assayed ranging from 1 μM to 1 mM, only produced effective neuroprotection at 250 μM (88.9%; p < 0.05 compared with 24 h of recovery by Dunnett's post-test after ANOVA), while QN23 significantly increased cell viability at the whole 1−250 μM concentration range, reaching cell viabilities near to control values at 100 μM (94.9%; p < 0.01 compared with 24 h of recovery by Dunnett's post-test after ANOVA) [15]. In Table 1, we have gathered and quantitatively compared the neuroprotective effect of QN23 (100−250 μM) and NXY-059, showing and confirming the remarkable neuroprotection provided by QN23 [15]. These results prompted us to start the in vivo CI model studies with this compound. The already-reported positive results have allowed us to propose ligand QN23 for advanced pre-clinical studies, as a promising candidate for the treatment of ischemic stroke [15]. Taking QN23 as a reference, we have kept focused on the development of new and improved quinolylnitrones derivatives in terms of neuroprotection activity. In this work, we shall describe the synthesis and the neuroprotective properties of sixteen novel QN23 analogues, in our attempt to keep researching on this privileged scaffold for the potential treatment of ischemic stroke.

Design and Synthesis of New Quinolylnitrones
For the design and selection of the novel QNs, first of all we were curious about knowing the effect of the substitution of the O-methyl group at C6 by the O-propargyl In a previous study, we reported the neuroprotection capacity of QN23, compared with the NXY-059 as reference nitrone, in primary neuronal cultures subjected to 4 h of oxygen and glucose deprivation (OGD) conditions, followed by treatment with the nitrones added at the beginning of a 24 h recovery period [15]. Neuronal cultures were treated with different concentrations of selected nitrones and their effect was evaluated after 24 h of recovery after OGD by a cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In these experiments, the cell viability without OGD was considered as 100% control, while 4 h OGD resulted in a significant reduction in cell viability (67.6%) compared with 100% control (p <0.0001, one-sample t-test). The OGD-induced cell injury was partially reversed after 24 h of recovery, but it did not reach the control value (77.1%; p < 0.001 compared with 4 h OGD by Student's t-test). The reference neuroprotective nitrone NXY-059, assayed ranging from 1 µM to 1 mM, only produced effective neuroprotection at 250 µM (88.9%; p < 0.05 compared with 24 h of recovery by Dunnett's post-test after ANOVA), while QN23 significantly increased cell viability at the whole 1−250 µM concentration range, reaching cell viabilities near to control values at 100 µM (94.9%; p < 0.01 compared with 24 h of recovery by Dunnett's post-test after ANOVA) [15]. In Table 1, we have gathered and quantitatively compared the neuroprotective effect of QN23 (100−250 µM) and NXY-059, showing and confirming the remarkable neuroprotection provided by QN23 [15]. These results prompted us to start the in vivo CI model studies with this compound. The already-reported positive results have allowed us to propose ligand QN23 for advanced pre-clinical studies, as a promising candidate for the treatment of ischemic stroke [15]. Taking QN23 as a reference, we have kept focused on the development of new and improved quinolylnitrones derivatives in terms of neuroprotection activity. In this work, we shall describe the synthesis and the neuroprotective properties of sixteen novel QN23 analogues, in our attempt to keep researching on this privileged scaffold for the potential treatment of ischemic stroke.

Design and Synthesis of New Quinolylnitrones
For the design and selection of the novel QNs, first of all we were curious about knowing the effect of the substitution of the O-methyl group at C6 by the O-propargyl and O-benzyl groups, including in both cases the benzyl (Bn) and the tert-butyl (tert-Bu) as the N-substituent at the nitrone functional motif, a process that resulted in QNs 1-4 ( Figure 1). Next, we explored the replacement of the chlorine atom at C2 by: (a) a hydroxyl group, hence QN5; (b) a fluorine atom, resulting in QNs 6,7; (c) arylsulfones such as in QNs 8-11; The synthesis of QNs 1-16 has been carried out as shown in Scheme 2. We have applied the general methodology shown in Scheme 1, starting from commercial or readily available carbaldehydes and the appropriate N-alkylhydroxylamine hydrochlorides, as described in the Supplementary Material. and O-benzyl groups, including in both cases the benzyl (Bn) and the tert-butyl (tert-Bu) as the N-substituent at the nitrone functional motif, a process that resulted in QNs 1-4 ( Figure 1). Next, we explored the replacement of the chlorine atom at C2 by: (a) a hydroxyl group, hence QN5; (b) a fluorine atom, resulting in QNs 6,7; (c) arylsulfones such as in QNs 8-11; and (d) a carbon atom implying an aryl group such as those in QNs 12-15; (Figure 1) or an ethyl α,β-unsaturated carboxylic ester, QN16 ( Figure 1). The synthesis of QNs 1-16 has been carried out as shown in Scheme 2. We have applied the general methodology shown in Scheme 1, starting from commercial or readily available carbaldehydes and the appropriate N-alkylhydroxylamine hydrochlorides, as described in the Supplementary Material. The synthesis of diversely functionalized QNs 5-16 has been carried out as shown in Scheme 2. Fluorine-and arylsulfonyl-substituted intermediates 6, 7 and 8 (Scheme 2) were prepared by nucleophilic substitution of 2-chloro-6-methoxyquinoline-3-carbaldehyde (1) using KF and arylsulfonyl chlorides, respectively, and following modifications of the reported procedures [20,21]. Thus, reaction of carbaldehydes 6-8 with the appropriate N-alkyl hydroxylamine hydrochloride led to the expected QNs 6 and 7 (Scheme 2), and QNs 8-11 (Scheme 2), in moderate to excellent yields. QNs 12-15 substituted with 2-aryl were envisioned through a short reaction sequence including C-C coupling process under Suzuki conditions. Thus, as The synthesis of diversely functionalized QNs 5-16 has been carried out as shown in Scheme 2. Fluorine-and arylsulfonyl-substituted intermediates 6, 7 and 8 (Scheme 2) were prepared by nucleophilic substitution of 2-chloro-6-methoxyquinoline-3-carbaldehyde (1) using KF and arylsulfonyl chlorides, respectively, and following modifications of the reported procedures [20,21]. Thus, reaction of carbaldehydes 6-8 with the appropriate N-alkyl hydroxylamine hydrochloride led to the expected QNs 6 and 7 (Scheme 2), and QNs 8-11 (Scheme 2), in moderate to excellent yields. QNs 12-15 substituted with 2-aryl were envisioned through a short reaction sequence including C-C coupling process under Suzuki conditions. Thus, as 2-chloroquinoline 1 was unreactive under the experimental conditions, we tried (Pd 2 (dba) 3 /PPh 3 /tert-BuOK) on the more reactive 2-iodo-6-methoxyquinoline-3-carbaldehyde (9), affording the expected aryl-substituted quinolines 10 and 11 with good to excellent yields (Scheme 2). A modification of reported Heck-type reaction on heteroaromatic systems [22], also allowed us the preparation of 2-ethoxypropenyl quinoline carbaldehyde 12 in good yield (Scheme 2). Unfortunately, the reaction of compound 12 with N-benzyl hydroxylamine did not afford the corresponding benzyl-substituted nitrone, probably due to intramolecular cycloaddition reactions leading to complex reaction mixtures. Similar side reactions possibly took place in the reaction of intermediate 12 with N-tertbutyl hydroxylamine, although in this case, we succeeded to isolate 2-ethoxyoxopropenyl quinoline nitrone QN16 in moderate yield after purification by column chromatography (Scheme 2), as the pure and single E-isomer, showing the vinyl protons at δ 7.95 and 7.07 as doublets with a vicinal coupling constant of 15.4 Hz. Finally, but unfortunately, other experiments targeted to get quinoline carbaldehydes decorated with carbonitrile or sulfonic acid groups at C2-position were unsuccessful, providing only decomposition products or extremely insoluble reaction mixtures.
QNs 1-16 have been isolated as single and pure Z-isomers, whose structures and stereochemistry are in good agreement with the observed analytical and spectroscopic data (Supplementary Material), as well as with those described in the literature for analogous nitrones [15].

Neuroprotection Evaluation of QNs 1-16 in an Experimental Model of Ischemia on Primary Neuronal Cultures
QNs 1-16 were tested in an experimental model of ischemia in order to assess their potential effect on cell viability preservation after an ischemic insult [16,23,24]. To do so, primary neuronal cultures were subjected to OGD conditions for 4 h and treated at the onset of the reoxygenation period with the previously mentioned QNs, or reference compounds such as citicoline-a well-known neuroprotective agent-, NXY-059 or QN23. Cell viability was determined by the MTT assay, for which control group value accounted for 100% ( Figure 2). As a consequence of the OGD period, a severe decrease in cell viability was observed, as measured in the unrecovered experimental group (OGD 4h, 64.4 ± 1.4%, p < 0.0001 compared with 100% control, one-sample t-test). This impairment in cell metabolism was partially reversed after recovery of normal oxygen and glucose levels for 24 h (R24h; 77.2 ± 1.3%, p < 0.0001 vs. control, by Student's t-test). The addition of citicoline (100 µM) at the onset of the recovery period after OGD prompted a significant increase in cell viability (82.8 ± 1.1%, p < 0.01 vs. R24h, by Student's t-test) compared with the untreated (vehicle) R24h group ( Figure 2). When reference compounds NXY-059 or QN23 were added instead of citicoline, higher levels of cell viability were achieved (88.9 ± 3.7% and 95.1 ± 1.4% for 250 µM NXY-059 and 100 µM QN23, respectively) ( Figure 2). QNs 1-16 were initially tested on the range 1-250 µM, which has proved adequate as a first approach into the dose-effect assessment of these kind of compounds in our experimental model. For some compounds, however, poor solubility made it impossible to test the highest concentrations. Thus, based on the effect on cell viability obtained for this concentration range and its solubility, we expanded the concentrations tested accordingly. The observed results, shown in Figure 2, were gathered into a structure-activity relationship (SAR) study that is described below.
According to the different structural pattern studied, exploration of O-propargyl motif at C6 position on ligands QN1 and QN2 rendered no remarkable overall protective effect in the concentration range tested. Only the N-tert-Bu-derived QN2 at 250 µM afforded a significant increase in cell viability when compared to R24h, although unfortunately, no higher concentration could be tested due to solubility issues ( Figure 2A).
A superior performance of the N-tert-Bu derivative in contrast to the N-tert-Bn substitute was observed for the compounds QN4 and QN3, respectively, both substituted at C6 with the O-Bn group. In fact, QN4 showed a good protective profile with a safe According to the different structural pattern studied, exploration of O-propargyl motif at C6 position on ligands QN1 and QN2 rendered no remarkable overall protective effect in the concentration range tested. Only the N-tert-Bu-derived QN2 at 250 μM afforded a significant increase in cell viability when compared to R24h, although unfortunately, no higher concentration could be tested due to solubility issues (Figure 2A).
A superior performance of the N-tert-Bu derivative in contrast to the N-tert-Bn substitute was observed for the compounds QN4 and QN3, respectively, both substituted at C6 with the O-Bn group. In fact, QN4 showed a good protective profile with a safe con- The next group of QNs tested included different combinations over the quinoline core substituted with a methoxy group at C6 (Figure 2A). The simplest and the only one of the entire set carrying an electron-donating group at C2 (-OH, QN5) did not give a remarkable protective effect at the concentrations tested. Likewise, molecules QN6 and QN7 bearing a C(2)F substituent did not show significant differences to the value obtained for the vehicle treated group (R24h), except for QN7 at 10 µM.
The results of the substitution at C2 with other electron-withdrawing groups, different from Cl as in QN23, such as an arylsulfone (QN8-11), a substituted phenyl group (QN12-15) or a α,β-unsaturated carboxylic ester (QN16), are shown in Figure 2B. For the first group, only the N-tert-Bn nitrone QN10 showed a significantly higher effect than R24h, both at 0.1 µM and 1 µM doses. Phenyl-substituted ligands QN12 and QN13 with a trifluoromethyl group in para position showed toxic effects in the concentration range tested, whilst the p-NO 2 -substituted QN14 and QN15 were, in contrast, safe in the whole concentrations range tested. Remarkably, QN15 showed interesting cell viability results when applied in the range 1-50 µM, significantly higher than vehicle-treated group (R24h; 70% at 50 µM). Finally, α,β-unsaturated carboxylic ester QN16 showed interesting results at 10 µM, even though it sharply decreased the beneficial effect with higher concentrations.
With these results, in order to express cell viability as a more useful concept in our search for protective compounds in the ischemic disease, we defined neuroprotection activity as the effect that achieved a cellular viability higher than the untreated group (R24h), which set our basal neuroprotection value (0%). Cell viability observed in the control group was set as 100% of neuroprotection. Consequently, the neuroprotection values obtained for standards NXY-059, QN23 and the new ligands QNs 1-16 have been gathered together in Table S1 (Supplementary Material), while in Table 2 we have only shown the most active QNs at the diverse tested doses. 70.26 ± 0.63 * According to these results, QN4 and QN15 are the compounds that exert the highest neuroprotection among the QN1-16 set ( Figure 2, Table 2). When compared to our lead compound QN23, neither QN4 nor QN15 reach the cell viability value obtained for QN23 at its best-performing concentration, meaning a slightly decreased activity (67.9% and 70.3% as maximal neuroprotection values for QN4 and QN15, respectively, vs. 79.7% for QN23). On the other hand, both QN4 and QN15 still exert higher cell viability values than reference compound NXY-059, and their effective concentrations are lower than the ones needed for QN23 (1 µM and 50 µM for QN4 and QN15, respectively, vs. 100 µM for QN23), meaning increased pharmacological potency. Finally, solubility and tolerability range must be considered. QN4 showed good solubility and no toxic effects in the range 0.01 µM to 100 µM (100-fold higher than the most effective concentration 1 µM), whereas QN15 was not soluble when assayed at any concentration higher than 50 µM, its most effective concentration. For QN23, its safe concentration range was restricted to 10-500 µM (5-fold higher than its most effective concentration 100 µM).
Noteworthy, when attending to their structural patterns and differences to QN23, QN4 bears an O-benzyl group at C6 instead of a methoxy group, whereas QN15 shows a 4-nitroaryl substituent at C2 instead of a chlorine atom, both with a N-tert-butyl substituent at the nitrone motif ( Figure 3).
According to these results, QN4 and QN15 are the compounds that exert the highest neuroprotection among the QN1-16 set ( Figure 2, Table 2). When compared to our lead compound QN23, neither QN4 nor QN15 reach the cell viability value obtained for QN23 at its best-performing concentration, meaning a slightly decreased activity (67.9% and 70.3% as maximal neuroprotection values for QN4 and QN15, respectively, vs. 79.7% for QN23). On the other hand, both QN4 and QN15 still exert higher cell viability values than reference compound NXY-059, and their effective concentrations are lower than the ones needed for QN23 (1 μM and 50 μM for QN4 and QN15, respectively, vs. 100 μM for QN23), meaning increased pharmacological potency. Finally, solubility and tolerability range must be considered. QN4 showed good solubility and no toxic effects in the range 0.01 μM to 100 μM (100-fold higher than the most effective concentration 1 μM), whereas QN15 was not soluble when assayed at any concentration higher than 50 μM, its most effective concentration. For QN23, its safe concentration range was restricted to 10-500 μM (5-fold higher than its most effective concentration 100 μM).
Noteworthy, when attending to their structural patterns and differences to QN23, QN4 bears an O-benzyl group at C6 instead of a methoxy group, whereas QN15 shows a 4-nitroaryl substituent at C2 instead of a chlorine atom, both with a N-tert-butyl substituent at the nitrone motif ( Figure 3).

Density Functional Theory (DFT) Calculations
Density Functional Theory (DFT) calculations were carried out at the dispersion corrected B3LYP-D3/def2-SVP level to gain more insight into the reactivity of QN23 and related species with HO • , as a model radical species. Four different processes, shown in Figure 4, can be envisaged, namely, (i) the addition of the radical to the electrophilic carbon atom of the nitrone, or alternatively, (ii) to the conjugated double-bond involving the pyridine fragment; and the hydrogen atom abstractions involving either (iii) the tert-Bu group of the nitrone, or (iv) the Me group of the methoxy substituent ( Figure 4).

Density Functional Theory (DFT) Calculations
Density Functional Theory (DFT) calculations were carried out at the dispersion corrected B3LYP-D3/def2-SVP level to gain more insight into the reactivity of QN23 and related species with HO • , as a model radical species. Four different processes, shown in Figure 4, can be envisaged, namely, (i) the addition of the radical to the electrophilic carbon atom of the nitrone, or alternatively, (ii) to the conjugated double-bond involving the pyridine fragment; and the hydrogen atom abstractions involving either (iii) the tert-Bu group of the nitrone, or (iv) the Me group of the methoxy substituent ( Figure 4).
Our calculations using the Z-isomer of QN23, which, in agreement with the experimental observations, is 7.6 kcal/mol more stable than its E-counterpart, indicate that the H-abstraction reaction involving the methoxy group is unfeasible in view of the high barrier (∆G = = 33.2 kcal/mol) computed for this process. In sharp contrast, the other alternative reactions, which begin from INT0 (an initial intermediate where the OH • radical is bonded to the oxygen atom of the nitrone moiety via a hydrogen bond) are nearly barrierless processes (∆G = < 2 kcal/mol) (Figure 4). Despite that, the computed reaction energies of these processes are rather different. Thus, it becomes clear that both addition reactions are thermodynamically more favored than the H-abstraction involving the tert-Bu group of the nitrone. Moreover, the addition to the C=N moiety is much more favored over the process involving the conjugated double-bond, mainly due to two reasons: (a) the significant aromaticity loss in the pyridine ring in INT2, and (b) the stabilization of the radical by the oxygen atom in INT1, as confirmed by the computed spin density (0.49e in the oxygen and nitrogen atoms, see inset of Figure 4). Therefore, and similar to our previous studies involving other nitrone-based neuroprotective agents [8,13], the mode of action of QN23 (and the related systems described herein) against radical species is dictated by a thermodynamic control, where the addition of the radical to the electrophilic C=N moiety of the nitrone is clearly favored. Our calculations using the Z-isomer of QN23, which, in agreement with the experimental observations, is 7.6 kcal/mol more stable than its E-counterpart, indicate that the H-abstraction reaction involving the methoxy group is unfeasible in view of the high barrier (ΔG ≠ = 33.2 kcal/mol) computed for this process. In sharp contrast, the other alternative reactions, which begin from INT0 (an initial intermediate where the OH • radical is bonded to the oxygen atom of the nitrone moiety via a hydrogen bond) are nearly barrierless processes (ΔG ≠ < 2 kcal/mol) (Figure 4). Despite that, the computed reaction energies of these processes are rather different. Thus, it becomes clear that both addition reactions are thermodynamically more favored than the H-abstraction involving the tert-Bu group of the nitrone. Moreover, the addition to the C=N moiety is much more favored over the process involving the conjugated double-bond, mainly due to two reasons: (a) the significant aromaticity loss in the pyridine ring in INT2, and (b) the stabilization of the radical by the oxygen atom in INT1, as confirmed by the computed spin density (0.49e in the oxygen and nitrogen atoms, see inset of Figure 4). Therefore, and similar to our previous studies involving other nitrone-based neuroprotective agents [8,13], the mode of action of QN23 (and the related systems described herein) against radical species is dictated by a thermodynamic control, where the addition of the radical to the electrophilic C=N moiety of the nitrone is clearly favored.
Next, we computationally explored the analogous reactions involving compounds QN4 and QN15 to rationalize their presumed outstanding antioxidant and radical scavenging capacities in comparison to the standard QN23 ( Figure 5). Not surprisingly, rather similar energy values were computed for all the systems as the reactive nitrone moiety remains practically unaltered. Once again, it can be inferred that, similar to QN23, the high observed neuroprotection activity of ligands QN4 and QN15 may be re- Next, we computationally explored the analogous reactions involving compounds QN4 and QN15 to rationalize their presumed outstanding antioxidant and radical scavenging capacities in comparison to the standard QN23 ( Figure 5). Not surprisingly, rather similar energy values were computed for all the systems as the reactive nitrone moiety remains practically unaltered. Once again, it can be inferred that, similar to QN23, the high observed neuroprotection activity of ligands QN4 and QN15 may be related to the high exergonicity of the addition reaction of the HO • radical to the electrophilic C=N moiety present on the nitrones. For QN4 in particular, an alternative reaction involving the Habstraction from the benzylic CH 2 O group could be also envisaged due to the stabilization of the corresponding radical by the adjacent phenyl group (INT4, Figure 5). However, our calculations indicate that this alternative process is thermodynamically not favored over the main reaction pathway involving the addition of the hydroxyl radical to the nitrone. lated to the high exergonicity of the addition reaction of the HO • radical to the electrophilic C=N moiety present on the nitrones. For QN4 in particular, an alternative reaction involving the H-abstraction from the benzylic CH2O group could be also envisaged due to the stabilization of the corresponding radical by the adjacent phenyl group (INT4, Figure 5). However, our calculations indicate that this alternative process is thermodynamically not favored over the main reaction pathway involving the addition of the hydroxyl radical to the nitrone.

Conclusions
In our search for new neuroprotective agents for stroke therapy to improve the neuroprotective efficacy of ligand QN23 [15,19], we have designed, prepared and investigated sixteen new, related, easily available QNs. As a result, we have identified two compounds, QN4 and QN15, showing high neuroprotection capacity, quite similar to that observed for QN23 (Figure 2). At the same time, these results have shown that subtle, but simple functional modifications on the QN scaffold are tolerated, affording new neuroprotective agents, and resulting in a structural pattern that could be of interest for new designs and future developments. In particular, and regarding the structure of compound QN23, QN4 bears a O-benzyl group at C6 instead of a methoxy group, whereas QN15 shows a 4-nitroaryl substituent at C2 instead of a chlorine atom, both bearing the N-tert-Bu substituent at the nitrone motif ( Figure 3).
Other nitrones, such as azulenyl bis-nitrones, are spin traps having neuroprotective effects in cerebral ischemia [25]. In subsequent studies, a second-generation of the potent antioxidant azulenyl nitrone confirmed their neuroprotective capacity in transient focal cerebral ischemia [26]. In addition, a nitrone with thrombolytic properties, the compound (Z)-N-tert-butyl-1-(3,5,6-trimethylpyrazin-2-yl)methanimine oxide, has been reported for the treatment of ischemic stroke [27]. This is a novel nitrone that shows simultaneous thrombolytic activity and scavenging ROS power for stroke therapy, in addi-

Conclusions
In our search for new neuroprotective agents for stroke therapy to improve the neuroprotective efficacy of ligand QN23 [15,19], we have designed, prepared and investigated sixteen new, related, easily available QNs. As a result, we have identified two compounds, QN4 and QN15, showing high neuroprotection capacity, quite similar to that observed for QN23 (Figure 2). At the same time, these results have shown that subtle, but simple functional modifications on the QN scaffold are tolerated, affording new neuroprotective agents, and resulting in a structural pattern that could be of interest for new designs and future developments. In particular, and regarding the structure of compound QN23, QN4 bears a O-benzyl group at C6 instead of a methoxy group, whereas QN15 shows a 4-nitroaryl substituent at C2 instead of a chlorine atom, both bearing the N-tert-Bu substituent at the nitrone motif (Figure 3).
Other nitrones, such as azulenyl bis-nitrones, are spin traps having neuroprotective effects in cerebral ischemia [25]. In subsequent studies, a second-generation of the potent antioxidant azulenyl nitrone confirmed their neuroprotective capacity in transient focal cerebral ischemia [26]. In addition, a nitrone with thrombolytic properties, the compound (Z)-N-tert-butyl-1-(3,5,6-trimethylpyrazin-2-yl)methanimine oxide, has been reported for the treatment of ischemic stroke [27]. This is a novel nitrone that shows simultaneous thrombolytic activity and scavenging ROS power for stroke therapy, in addition to nontoxicity and blood−brain barrier permeability. Very interestingly, note that in this case the nitrone motif -the origin of its spin trap capacity-is conjugated with a pyrazine heterocyclic ring, while the nitrones described here are quinolylnitrones and where the nitrone group is conjugated with a related quinoline heterocyclic ring. Furthermore, and not surprisingly, nitrone bears a tert-Bu substituent at the nitrogen of the nitrone, the same preferred substituent present in the most potent neuroprotective agents, QN4 and QN15, identified in this work. In this feature, new para-substituted nitrones of the potent α-phenyl-N-tert-butyl nitrone (PBN) have been reported with good spin-trapping and neuroprotective properties [28].
Remarkably, QN4 and QN15 are non-toxic and safe compounds at all tested doses, despite the toxicological alert described for the nitro group [29]. Finally, and in terms of neuroprotection capacity, the effect induced by QN4 and QN15 is still higher than reference compound NXY-059, the furthest-reaching-but withdrawn in Phase III studies-chemical compound in clinical development for the treatment of CI so far. These data prompt us to keep focusing on the quinolylnitrone core, which proved suitable in the development of not only new neuroprotective compounds, but also able to be easily modified in order to obtain more potent and pharmacologically adequate entities for its further development as potential candidates for ischemic stroke.

General Procedures General Procedure for the Synthesis of Nitrones
A solution of the corresponding carbaldehyde (1 mmol), Na 2 SO 4 (3 mmol), TEA (2 mmol) and the appropriate hydroxylamine hydrochloride (1.5 mmol) in THF/EtOH (5 mL, 4:1) was heated at 90 • C during 1-6 h under microwave irradiation (MWI). After that time, the solvent was evaporated and the crude mixture was purified on column chromatography using the indicated mixtures of solvents.
General Procedure for the Synthesis of 6-Methoxy-2-(arylsulfonyl)quinoline-3-carbaldehydes 7, 8 A solution of 2-chloro-6-methoxyquinoline-3-carbaldehyde (1) (1 mmol), the corresponding arylsulfonyl chloride (1.2 mmol) and Na 2 SO 3 (1.3 mmol) in H 2 O (8 mL) was heated at 80 • C in an oil bath for 5 h. After that time, the mixture was diluted with DCM (15 mL), and extracted with NaHCO 3 (5% water solution), brine, and the organic phase was dried over MgSO 4 . After filtration and evaporation of the solvent, the crude mixture was purified on column chromatography using the indicated mixtures of solvents. Arylsulfonyl quinolines were obtained as mixtures of the expected products and starting material that we were unable to separate, and were directly used in the next step.