Privileged Quinolylnitrones For The Combined Therapy Of Ischemic Stroke And Alzheimer’s DiseaseⅠ

Abstract: Cerebrovascular diseases such as ischemic stroke are known to exacerbate dementia caused by neurodegenerative pathologies such as Alzheimer’s disease (AD). Besides, the increasing number of patients surviving stroke makes it necessary to treat the co-occurrence of these two diseases with a single and combined therapy. For the development of new dual therapeutic agents, eight hybrid quinolylnitrones have been designed and synthesized by the juxtaposition of selected pharmacophores from our most advanced lead compounds for ischemic stroke and AD treatment. 

Click to cistanche tubulosa powder for Alzheimer's disease

Biological analyses looking for efficient neuroprotective effects in suitable phenotypic assays led us to identify MC903 as a new small quinolylnitrone for the potential dual therapy of stroke and AD, showing strong neuroprotection on (i) primary cortical neurons under oxygen–glucose deprivation/normoglycemic reoxygenation as an experimental ischemia model; (ii), neuronal line cells treated with rotenone/oligomycin A, okadaic acid or β-amyloid peptide Aβ25–35, modeling toxic insults found among the effects of AD. 

Keywords: Alzheimer’s disease; ischemic stroke; multipotent drugs; neuroprotection; quinolylnitrones

1 Introduction 

Due to the high quality of life in our developed countries, and the increased life expectancy of the eldest, aging diseases such as stroke and Alzheimer’s disease (AD) have become first-order individual, socio-economic and medical problems with unmet and limited therapies [1]. Consequently, the search for new and more efficient therapies for stroke and AD is urgent and one of the most active areas of research in the current neuroscience domain [2]. Furthermore, the interconnection and co-occurrence of stroke and AD in the same patients is known and has been largely documented [3], although the implicated biological mechanisms have not yet been established [4]. 

Cerebrovascular diseases are not only the basis of a vascular-related cognitive impairment, referred to as vascular dementia (VD) but they are also known to exacerbate dementia caused by other factors such as AD and other degenerative diseases [5]. As noted recently by Macrae and Allan, “ . . . With increasing numbers of patients surviving a stroke, there is also a need to focus more on post-stroke complications that affect the quality of life. These include not only motor and speech impairments but also depression, dementia, epilepsy, and anxiety, among other things . . . ” [6]. Hence, the identification of new drugs for the combined therapy of both ischemic stroke and AD would be an ideal therapeutic issue seldom put into practice, but that currently concentrates our most recent efforts, taking advantage of two of our most advanced lead compounds. 

Recently, we identified (Z)-N-tert-butyl-1-(2-chloro-6-methoxyquinolin-3-yl)-methenamine oxide (QN23) (Figure 1) as a very effective neuroprotective quinolylnitrone (QN), showing neuroprotection induction in two in vivo models of global and focal cerebral ischemia [7], resulting in a new lead-compound for ischemic stroke treatment. On the other hand, we have described that Contilisant (Figure 1) is a permeable and highly neuroprotective agent against several AD-relevant toxic insults, showing in vitro inhibitory properties for neurotransmitter-catabolizing enzymes (ChE, MAO) [8] or G protein-coupled receptors (H3R [8], S1R [9]), and in vivo better protective effect than our previous hit-compound ASS234 [10] or donepezil on the Y-maze and radial arm maze task against cognitive decline induced by the β-amyloid peptide Aβ1−42 oligomers [9]. Remarkably, QN23 and Contilisant are also potent antioxidants, a beneficial feature for the potential treatment of ischemic stroke and AD taking into account the noxious effect that oxidative stress and reactive oxygen species (ROS) exert on both pathologies [11].

With this knowledge, we have designed the new N-tert-butyl and N-benzyl hybrid QNs of types I and II shown in Figure 1, as different combinations of those privileged structural features present in QN23 and Contilisant over the QN core. QNs of type I, JMA98C, and JMA101A, are the result of the insertion of the key piperidinopropyloxy motif—a polyvalent pharmacophore present in Contilisant for the effective ChE, H3R, and S1R binding [8,9]—at C2 of QN23, keeping the methoxy group at C6, a feature that seems critical for efficient neuroprotection [7]. 

An analogous analysis resulted in QNs DDI88 and DDI89, where we have installed an additional N-propargyl motif—a typical MAO pharmacophore—in a piperazinopropyloxy group—a proven pharmacophore for ChE inhibition [10]. A similar analysis has resulted in four more QNs of type II, such as JMA11A, JMA12A, MC902, and MC903, where we have kept the chlorine atom at C2 as in QN23, and the functionalized cycloalkylaminopropyloxy moiety linked at C6 instead of the methoxy group. Based on these designs, which maintain the structural features responsible for the neurotransmitter level regulation impaired in AD [12], on a QN core, the QNs reported here are expected to act as multipotent small molecules potentially suitable for the treatment of AD and ischemic stroke [13]. 

Thus, prompted by the recent communication by Sun et al. [14] on the pharmacological profile of the neuro immunomodulator agent AD110 for the therapeutic management of AD and stroke, and the results reported by Liu et al. [15] for MT-20R—a substituted α-phenyltert-butyl nitrone bearing MAO and ChE pharmacophores for Parkinson’s disease—we describe here our preliminary results on this area. We have identified MC903 as a new small QN for the potential combined therapy of ischemic stroke and AD, showing strong neuroprotective properties on primary cortical neurons under hypoxia/reoxygenation as an experimental ischemia model, and on neuronal line cells treated with rotenone plus oligomycin A, okadaic acid or Aβ25–35, modeling three toxic insults found in AD.

2. Results and Discussion 

2.1. Synthesis 

The synthesis of the target QNs was carried out starting from readily available precursors in short synthetic sequences, affording the desired ligands as stereochemically single Z-isomers in pure form, good overall yields, and multigram amounts (Supplementary Material). Next, the QNs were submitted to biological analysis to determine their neuroprotective capacity

2.2. Evaluation of Neuroprotection in an Experimental Model of Ischemia in Primary Neuronal Cultures 

QNs JMA101A, JMA98C, DDI89, DDI88, JMA12A, JMA11A, MC903, and MC902 were tested in an experimental model of ischemia to evaluate their potential neuroprotective effect after an ischemic insult. To do so, primary neuronal cultures were subjected to oxygen and glucose deprivation (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, NXY-059, QN23, or Contilisant. 

Neuroprotection was monitored by the measurement of cell viability, as determined by the MTT assay (Figure 2), in which the control group was set as 100%. The unrecovered experimental group (OGD4h) showed a severe decrease in cell viability (64.4 ± 1.4%, p < 0.0001 compared with 100% control, one-sample t-Test), which was only partially reversed after reoxygenation for 24 h (R24h; 77.2 ± 1.3%, p < 0.0001, by Student’s t-Test). The addition of 100 µM citicoline, a well-known neuroprotective agent, prompted a significant increase in cell viability (82.8 ± 1.1%) compared with the untreated (vehicle) R24h group (Figure 1). When standards NXY-059 [7], QN23 [7], or Contilisant [8] were added instead of citicoline, higher levels of cell viability were achieved (88.9 ± 3.7%, 95.1 ± 1.4% and 88.7 ± 1.9% for 250 µM NXY-059, 100 µM QN23 and 50 µM Contilisant, respectively) (Figure 2).

Treatment with the new QNs provided different effects on the recovery of cell metabolism after the OGD insult. In the first place, and remarkably, almost the complete set of QNs provided higher cell viability than untreated R24h cells in the whole concentration range tested (0.1–250 µM). Based on our previous experience, we initially explored the concentration range of 1–250 µM, which was expanded to 0.1 µM in those cases still showing a good response at low concentrations. Compounds DDI88 and MC902 were not assayed at 250 µM despite exerting relatively good cell viability values because of solubility issues. Only QNs DDI89, JMA12A, and JMA11A exerted a toxic effect—i.e., lower cell viability than the untreated group, R24h—at the highest tested concentration of 250 µM. Best-performing QNs JMA101A at 10 and 100 µM, JMA98C at 10 µM and MC903 at 10 µM and 100 µM, achieved higher cell viability than citicoline (100 µM), reference nitrone NXY-059 (250 µM) and Contilisant (10–50 µM). Remarkably, the MC903 effect at 10 µM and 100 µM (93.7 ± 2.1% and 93.8 ± 1.7%, respectively) reached values similar to the ones observed for QN23 (100 µM), our most potent QN found to date [7] (Figure 2).

From the previous results of cell viability, we defined neuroprotection activity as the effect that achieved cellular viability higher than the one produced by the normoxic recovery alone, determined by the untreated R24h group, which was set as 0%. The cell viability of the control group was set as 100% of neuroprotection. 

Neuroprotection induced by QNs JMA101A, JMA98C, DDI89, DDI88, JMA12A, JMA11A, MC903 and MC902, and Contilisant, was compared with that induced by standards NXY-059 and QN23 (Table 1). Not surprisingly, and according to the highest cell viability values mentioned above, QNs JMA101A (10–100 µM), JMA98C (10 µM), and MC903 (10–100 µM) exerted the highest neuroprotective effect, being even higher than NXY-059. Again, 10 µM or 100 µM MC903 achieved the neuroprotection values most similar to our ischemic stroke lead candidate QN23. These findings confirm the selection of the QN as a proper scaffold in the search for neuroprotective ligands. 

Modification of the QN core is not only easily feasible but also able to provide new functionalities and properties without being detrimental to the OGDprotecting effect. Especially, for the cell viability results after OGD presented above, the preliminary structure–activity relationship (SAR) reported here has afforded no significant deleterious combination of structural moieties for an OGD-protecting new entity, and only four compounds were found neurotoxic at the highest concentration tested. On the other hand, one specific QN, MC903, has been revealed as the best-performing of the set, with an OGD-protecting activity similar to QN23.

To evaluate the suitability of these structures as multipotent hits, an assessment of their neuroprotective effect on the specific pathological contributions of AD was required. These results are described below.

2.3. Evaluation of Neuroprotection in an Experimental Model of AD in Neuronal Cell Line 

In an independent and almost simultaneous assay, the neuroprotective activity of the target QNs (Figure 1) was carried out at different concentrations (0.3, 1, 3, and 10 µM) against three toxic stimuli related to the neurodegeneration found in AD using melatonin as standard, a well-known neuroprotective agent, for comparative purposes. First of all, we used a model of reactive oxygen species generation using a cocktail of mitochondrial blockers, rotenone plus oligomycin A (R/O). As shown in Table 2, QNs JMA98C and MC903 showed an interesting profile against R/O. At the concentrations of 1, 3, and 10 µM, JMA98C produced a significant increase in cell viability against R/O (40.6%, 70.5%, and 57.2%, respectively). 

Similarly, MC903, at 0.3, 1, and 3 µM, showed a potent increase in cell viability (44.1%, 40.4%, and 74.9%). The maximum protective effect of both QNs was achieved at the concentration of 3 µM. Next, we analyzed our ligands in a model of tau hyperphosphorylation using okadaic acid (OA), a well-known protein Ser/Thr phosphatase inhibitor. In this model, both QNs showed a significant increase in cell viability against OA at the concentrations of 1 and 3 µM (JMA98C: 52.9% and 78.3%, respectively; MC903: 46.7% and 56.2%, respectively). Finally, and based on the precedent results, we investigated the neuroprotective effect of QNs JMA98C and MC903 following a toxic insult given by Aβ25–35, as a largely accepted biological target playing a key role in the progress and development of AD. As shown in Table 2, again QN MC903 showed a significant, potent, and dose–response neuroprotective effect of 47.9 ± 8.9% at 3 µM, decreasing at 10 µM, and reaching similar lower values, around 28%, at 0.3 µM and 1 µM. Thus, from two different laboratories carrying out independent assays on the same QNs to assess their neuroprotective capacities under an experimental ischemia model and against AD-derived toxic insults, QN MC903 was found to provide the best, strong, and consistent neuroprotection, identifying it as a potential hit-compound for further preclinical studies.

2.4. Virtual Absorption, Distribution, Metabolism, and Excretion (ADME) Properties of Compound MC903 

MC903 virtual ADME profile was investigated as a key point for a molecule designed to therapeutically act on the brain. Furthermore, MC903 drug-like properties were predicted using the QikProp program, and violations of Lipinski’s rule of 5 (ROF) and Jorgensen’s rule of 3 (ROT), were estimated. The calculated ADME parameters (Table S1, Supplementary Material) are within the reference ranges, showing no violation for ROF and ROT. This analysis confirms that ligand MC903 possesses the appropriate pharmacokinetic profiles required for distribution in the human body, being permeable to the brain–blood barrier. As such, MC903 has the potential as a lead candidate in AD and stroke drug development.

the mechanism of the Cistanche neuroprotection effect

Cistanche is a traditional Chinese herb that has been reported to have neuroprotective effects. The mechanisms behind these effects are not fully understood, but there are several possible mechanisms proposed by researchers:

1. Antioxidant activity: Cistanche extracts have been found to possess potent antioxidant activity, which can protect against oxidative stress-induced neuronal damage.

2. Anti-inflammatory effects: Inflammation is a common cause of neurodegenerative diseases, and cistanche extracts have been shown to have anti-inflammatory effects that can attenuate neuronal damage.

3. Protection of mitochondrial function: Mitochondrial dysfunction is a key contributor to neuronal damage and cell death, and cistanche extracts have been found to protect against mitochondrial damage and improve mitochondrial function.

4. Activation of neurotrophic factors: Neurotrophic factors are chemicals that promote the growth and survival of neurons, and cistanche extracts have been shown to increase the expression of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).

To be continued...

José M. Alonso 1,†, Alejandro Escobar-Peso 2,† , Alejandra Palomino-Antolín 3,†, Daniel Diez-Iriepa 1,4 , Mourad Chioua 1 , Emma Martínez-Alonso 2 , Isabel Iriepa 4,5 , Javier Egea 3,* , Alberto Alcázar 2,* and José Marco-Contelles 1,*

1 Laboratory of Medicinal Chemistry (IQOG, CSIC), Juan de la Cierva 3, 28006 Madrid, Spain; xosemag@yahoo.es (J.M.A.); daniel.diezi@uah.es (D.D.-I.); mchioua@gmail.com (M.C.) 

2 Department of Research, IRYCIS, Hospital Ramón y Cajal, Ctra. Colmenar Km 9.1, 28034 Madrid, Spain; alejandro.escobar@hrc.es (A.E.-P.); emma.martinez@hrc.es (E.M.-A.) 

3 Molecular Neuroinflammation and Neuronal Plasticity Research Laboratory, Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria-Hospital Universitario de la Princesa, 28009 Madrid, Spain; apantolin@gmail.com 

4 Departamento de Química Orgánica y Química Inorgánica, Universidad de Alcalá, Ctra. Madrid-Barcelona Km 33.6, 28871 Alcalá de Henares, Spain; isabel.iriepa@uah.es 5 Institute of Chemical Research Andrés M. del Río, Alcalá University, 28805 Alcalá de Henares, Spain