Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability

Bioorganic Chemistry 117 (2021) 105472 Available online 6 November 2021 0045-2068/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Design, synthesis and structure-activity evaluation of novel 2-pyridone-- based inhibitors of α -synuclein aggregation with potentially improved BBB permeability Alejandro Mahía a , b , Samuel Pe˜ na-Díaz c , d , Susanna Navarro c , d , Juan Jos´ e Galano-Frutos b , e , Irantzu Pallar´ es c , d , Jordi Pujols c , d , María D. Díaz-de-Villegas a , f , Jos´ e A. G´ alvez a , f , * , Salvador Ventura c , d , g , * , Javier Sancho b , e , h , * a Departamento de Química Org´ anica, Facultad de Ciencias, University of Zaragoza, 50009 Zaragoza, Spain b Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50018 Zaragoza, Spain c Departament de Bioquímica i Biologia Molecular, Universitat Aut` onoma de Barcelona, 08193 Bellaterra, Spain d Institut de Biotecnologia i Biomedicina, Universitat Aut` onoma de Barcelona, 08193 Bellaterra, Spain e Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, University of Zaragoza, 50009 Zaragoza, Spain f Instituto de Síntesis Química y Cat´ alisis Homog´ enea (ISQCH), CSIC-University of Zaragoza, 50009 Zaragoza, Spain g ICREA, 08010 Barcelona, Spain h Aragon Health Research Institute (IIS Arag´ on), 50009 Zaragoza, Spain ARTICLE INFO Keywords: Parkinson’s disease Anti-aggregative Inhibitor 2-Pyridone BBB permeability Structure-activity ABSTRACT The treatment of Parkinson’s disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α -synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/ pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α -synuclein aggregation. Several of the derivatives obtained retain the parent’s compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD. 1. Introduction Parkinson’s disease (PD) is, after Alzheimer’s disease (AD), the second most common neurodegenerative disorder in humans and its incidence is on the rise [1]. PD is highly dependent on age, affecting 1–2% of people older than 65 [2]. Classic symptoms of PD are Abbreviations: α -syn, α -synuclein; AD, Alzheimer’s disease; AUC, area under the curve; BBB, blood-brain barrier; CC50, 50% cytotoxic concentration; CSF, cerebrospinal fluid; IP, intraperitoneal; PD, Parkinson’s disease; P-gp, P glycoprotein; PSA, polar surface area; Th-T, Thioflavin-T; TLC, thin-layer chromatography. * Corresponding authors at: Departamento de Química Org´ anica, Facultad de Ciencias, University of Zaragoza, 50009 Zaragoza, Spain (J.A. G´ alvez). Departament de Bioquímica i Biologia Molecular, Universitat Aut` onoma de Barcelona, 08193 Bellaterra, Spain (S. Ventura). Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50018 Zaragoza, Spain (J. Sancho). E-mail addresses: [email protected] (J.A. G´ alvez), [email protected] (S. Ventura), [email protected] (J. Sancho). Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg https://doi.org/10.1016/j.bioorg.2021.105472 Received 3 June 2021; Received in revised form 20 October 2021; Accepted 3 November 2021 Bioorganic Chemistry 117 (2021) 105472 2 bradykinesia or slowness of movement, muscle rigidity, resting tremor and postural and gait detriment [3]. In addition, PD patients usually have other non-motor symptoms that negatively affect their quality of life and may lead to cognitive impairment and, finally, dementia in the last stages of the disease [4]. From a physiopathological point of view, PD is characterised by the death of dopaminergic neurons in the midbrain, specifically in the substantia nigra, and by the presence of intraneuronal cytoplasmic inclusions named Lewy bodies [5]. Lewy bodies are mainly composed of amyloid fibrils as a result of the aggregation of α -synuclein ( α -syn), a 140-amino-acid intrinsically disordered protein whose specific physiological roles are not completely understood [6]. On the other hand, the great variety of genes involved in the evolution of PD, some of which are responsible for the aggregation and proteostasis of α -syn [5,7], highlights the complexity of the pathology and the urgent need to develop novel, precise and effective diagnostic and therapeutic tools. The therapy currently used to treat PD is a symptomatic treatment based on palliating the symptoms and not acting directly on their molecular mechanisms. Thus, the development of drugs that target the main biomarkers of PD, e.g. the aggregation of α -syn, is a research topic of high interest [8]. In this direction, high-throughput experimental screening methods previously used to discover antimicrobials or aggregation inhibitors [9,10], have been successfully adapted [11] to identify inhibitors of α -syn aggregation [12–14]. Among them, SynuClean-D (Fig. 1) stands out. This small metabolically stable 2-pyridonebased compound is not only able to inhibit the aggregation of wild type α -syn and some variants related to familial cases of PD, but also to disrupt pre-formed α -syn fibrils and to avoid aggregate amplification and template seeding processes in vitro. Furthermore, SynuClean-D reduces α -syn inclusion formation in human neuroglioma cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD [12]. All these properties make SynuClean-D a promising chemical scaffold to develop and optimise novel therapeutic compounds for the treatment of PD. On the other hand, a major challenge in designing drugs targeting the central nervous system (CNS) is to develop molecules able to cross the blood–brain barrier (BBB) and reach optimal concentrations in brain cells [15]. Most CNS drugs are small molecules that cross the BBB by passive diffusion, so the knowledge and purposely optimisation of their physicochemical properties, mainly polarity and lipophilicity, play a crucial role in the design of optimal drug candidates [16]. SynuClean-D was discovered through the screening of a commercially available chemical library. Given the facts that no synthetic procedure has been reported so far to obtain SynuClean-D, and that many-gram amounts of this compound are required for pharmacokinetic and pharmacodynamic (PK/PD) assays, and for efficacy testing in mammals, we have developed a synthetic methodology that allows the multi-gram scale synthesis of this molecule in a rapid, efficient and economical manner. Importantly, the designed synthetic strategy can be used for the synthesis of novel derivatives with optimised pharmacological properties. Straightforward assessment of some SynuClean D physicochemical features, such as its topological polar surface area (tPSA), suggests that this compound may benefit from rationally improvement in order to increase its BBB permeability [17]. With this purpose, we report the synthesis and in vitro efficacy testing of SynuClean-D derivatives 6, 8a-8d (Fig. 1), 8e-f and 9 that have been designed to identify the functionalities related to SynuClean-D inhibitory activity on α -syn aggregation and to improve its BBB permeation properties. 2. Results and discussion 2.1. Synthesis of SynuClean-D The synthetic strategy initially designed to obtain SynuClean-D is depicted in Scheme 1 and was based on the construction of the 2-pyridone ring through cyclisation between 2-nitro-1,3-dicarbonylic compound 4 and cyanoacetamide as the final step of the reaction sequence. This cyclisation reaction has been previously reported in scientific literature to obtain 3-cyano-6-phenyl-4-trifluoromethyl-2(1H)-pyridone (compound 1) [18,19]. α -Nitro ketone 3 was synthesised from 3-nitrobenzaldehyde in two reaction steps with an overall yield of 81% [20]. However, the synthesis of 4 through acylation of compound 3 did not succeed. Unfortunately, the reaction did not provide the desired 1,3dicarbonylic compound 4 in any of the reaction conditions tested; ethyl trifluoroacetate or trifluoroacetic anhydride as acylating agents and different basic reagents. This is probably due to the high stability of the enolate of 3, which leads to a very low or null reactivity. Taking into account these results, a new synthetic strategy in which the heterocyclic nitro group is incorporated to the bicycle scaffold as the final reaction step was designed. Thus, the synthesis of SynuClean-D was attempted by means of the initial construction of the 2-pyridone ring followed by regioselective nitration, as is shown in Scheme 2. Fig. 1. Chemical structures of SynuClean-D and active derivatives with potentially improved BBB permeation. A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 3 The first step of the synthesis was the acylation of m-nitroacetophenone with ethyl trifluoroacetate [21] to obtain 1-trifluoromethyl-1,3-dicarbonylic key intermediate 5, which was used in the next step without further purification. Compound 6 was obtained through cyclisation reaction between intermediate 5 and cyanoacetamide in the presence of piperidine as catalyst [19], in 42% overall yield for the two steps. The challenge in the synthesis of SynuClean-D from compound 6 lies in its regioselective nitration at 5-position of the 2-pyridone ring due to the high number of positions susceptible to be nitrated, which could hinder a clean synthesis of the desired compound. In order to evaluate the chance of the regioselective nitration, we had previously calculated the most nucleophilic position of substrate 6 by two computational methods. The first one, reported by Jørgensen et al. in 2016, uses the prediction of 1 H NMR and/or 13 C NMR chemical shifts to propose the regioselectivity of S E Ar (specially on heteroaromatic systems) in a quick, easy and reliable way using simple computer programs such as ChemDraw [22]. This method is based on the fact that the position with the lowest chemical shift will be that with more electron density and, consequently, more prone to the electrophilic attack. 1 H NMR and 13 C NMR chemical shifts predicted by ChemDraw for compound 6 are collected in Fig. 2. As can be observed, the lowest value in both cases was obtained for the 5-position of the 2-pyridone ring, what indicates that the synthesis of SynuClean-D by regioselective nitration of compound 6 might be viable. Besides, the real 1 H NMR spectrum of 6 also showed the lowest chemical shift on that position. More recently, RegioSQM has been reported by the same authors as a novel, accurate and free-access computational tool to predict the regioselectivity of S E Ar reactions [23]. RegioSQM is based on semiempirical quantum mechanical (SQM) methods that predict the arenium ion with lowest free energy and, therefore, the most reactive position. When RegioSQM Scheme 1. Initially proposed synthetic route to SynuClean-D. The red cross indicates the impossibility of synthesising compound 4 through acylation of 3. Scheme 2. Successful synthetic route to SynuClean-D. Fig. 2. Computational predictions for the regioselectivity nitration of compound 6. The values of 1 H NMR and 13 C NMR chemical shifts predicted by ChemDraw for compound 6 are shown in blue. The green circle indicates the most reactive aromatic position of 6 (in S E Ar reactions) predicted by RegioSQM online tool. A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 4 computational tool was tested with compound 6, the 5-position of the 2pyridone cycle was predicted again as the most reactive (Fig. 2), providing new evidence on the feasibility of synthesising SynuClean-D by regioselective nitration of 6. In effect, the nitration of 6 in the presence of nitric acid and sulphuric acid at low temperature [24] afforded SynuClean-D as the sole reaction product with an excellent yield of 93% (Scheme 2). This result confirmed the goodness of the assayed predictive computational methods for our particular issue. Therefore, an efficient and inexpensive synthetic procedure has been developed that allows us to obtain SynuClean-D in as few as three steps with a global yield of 39%. Furthermore, this methodology offers the possibility of synthesising novel derivatives of SynuClean-D by varying substituents at different positions. By comparing the anti α -syn aggregation activity of SynuClean-D and derivatives so synthesised we have identified structural determinants of SynuClean-D inhibitory activity as well as introduced functionalities that may increase the BBB permeability of the parent compound (see below). 2.2. SynuClean-D concentration in cerebrospinal fluid (CSF) and in plasma. Computational prediction of BBB permeability To preliminarily examine SynuClean-D BBB permeability, its pharmacokinetic profile was evaluated. Table 1 summarises the pharmacokinetic parameters for SynuClean-D in plasma and cerebrospinal fluid (CSF). According to the obtained results, SynuClean-D mean concentration in CSF is significantly lower than in plasma (Fig. 3): C max values of 30.9 ng/mL and 44.2 μ g/mL, respectively. A terminal elimination half-life (T 1/2 ) of 3.6 h and 3.4 h was determined in plasma and CSF, respectively. The T max in plasma and CSF was reached in both cases 0.5 h after intraperitoneal (IP) administration, and SynuClean-D concentrations were quantifiable for up to 24 h post-dosing in plasma and 6 h in CSF. The CSF exposure (96.7 h∙ng/mL) was very low compared with plasma (372.8 h∙ μ g/mL). The Ratio CSF/plasma calculated by means of the AUCs was 0.0003 (0.03%). Although this low CSF/plasma ratio could arise −in principle −from very intense SynuClean-D uptake by plasma proteins or by brain tissue, or even from very active efflux of SynuClean-D molecules after having crossed the BBB, it suggests that SynuClean-D may have a low BBB penetration. To further investigate this possibility we have obtained a consensus computational evaluation of SynuClean-D BBB crossing potential using three web servers based on different models. BOILED-Egg predicts the passive gastrointestinal absorption and BBB permeation of small organic molecules, from their lipophilicity (octanol–water partition coefficient: logP) and polarity (polar surface area: PSA) [25]. This model computes and graphically represents lipophilicity versus polarity of the studied compound. In the generated plot the compound may lay in one of three regions: the outer one corresponding to compounds that are not even absorbed by the gastrointestinal tract, the white area of the egg-shaped diagram corresponding to compounds absorbed but unable to cross efficiently the BBB, and the yolk area, for those that are expected to cross the barrier. SynuClean-D locates in the outer region (Fig. 4) and is − therefore −predicted as displaying a poor gastrointestinal absorption and being unable to cross the BBB. AlzPlatform [26] is another freeaccess server whose BBB predictor provides, for the input molecule, a BBB crossing prediction based on a support vector machine algorithm that uses four types of fingerprints (mainly hydrogen bond-related properties of the molecule) [27]. The BBB crossing prediction for SynuClean-D is also negative. On the other hand, the BBB predictor from the VEGA suite [28] is a free-access server based on a decision tree model built through machine learning that uses nine attributes of the input molecule related to size, shape, charge, lipophilicity and Kier-Hall Estate descriptors. The BBB crossing prediction of this server for SynuClean-D is also negative. Thus, three different predictors using different models provide a clear consensus prediction indicating that the BBB permeability of SynuClean-D is low and, therefore, it is unable to significantly cross the BBB. We have used the P-gp substrate prediction module in SwissADME [29] to evaluate whether a very active efflux of SynuClean-D from CSF associated to the activity of P glycoprotein could contribute to the very low CSF/plasma concentration ratio observed. This seems unlikely as the P-gp substrate module predicts that SynuClean-D does not interact with P glycoprotein. The possibility that a very high plasma protein or brain tissue binding of SynuClean-D could contribute to the very low observed CSF/plasma concentration ratio cannot be discarded yet. 2.3. Computational design and synthesis of novel derivatives of SynuClean-D with predicted better membrane permeability We have used BOILED-Egg, and the BBB crossing predictors in AlzPlatform and in the VEGA suite to guide the design of SynuClean-D derivatives. On one hand, the derivatives have been designed to provide an opportunity to dissect the contribution of SynuClean-D functionalities to its inhibitory activity on α -syn aggregation and, on the other, to improve their predicted BBB permeability. A quick inspection of the BOILED-Egg graph (Fig. 4) indicates that the membrane permeation of SynuClean-D could be easily improved by reducing its PSA. This can be achieved e.g. through the elimination of the phenyl-nitro group and/or heterocyclic nitro group in SynuClean-D or through their replacement by other less polar substituents such as nitrile or halogen functional groups. In addition, the generation of 1Nmethylated derivatives of SynuClean-D can also reduce its PSA. In this respect, the synthetic strategy followed to obtain SynuClean-D enabled the synthesis of derivatives 1, 6 and 8a-f with expected moderate yields in only two reaction steps starting from different ring-substituted acetophenones or employing a N-methylated cyanoacetamide (Scheme 3 and Table 2). The nitration of compound 8f under the same conditions as those applied to 6 afforded dinitrated compound 9 in a regioselective manner and with a very good yield of 72% (Scheme 4). As well as for SynuClean-D, the synthesis of compounds 6, 8a, 8b, 8c, 8d, 8f and 9 is being reported for the first time. With the exception of dinitrated compound 9, all these newly obtained derivatives are located in the BOILED-Egg graph (Fig. 4) in a region with good predicted membrane permeability, all of them offering better polarity parameters and similar or better lipophilicity than SynuClean-D. Additionally, some of them are located inside the region with high probability to permeate across the BBB. The BBB permeability predictors in AlzPlatform and in the VEGA suite are binary. They classify compounds as BBB+or BBB-, not providing indications of their gastrointestinal absorption. These two servers classify SynuClean-D as BBB-, compound 9 as BBB- (AlzPlatform) or BBB+ (VEGA suite), and all the other derivatives as BBB+. The qualitative agreement between the three servers is good as it coincides in the poor BBB permeability of SynuClean-D, and its significant improvement in all the derivatives synthesised with the possible exception of derivative 9. 2.4. Anti-aggregative activity of the obtained SynuClean-D derivatives The inhibitory effect of SynuClean-D on the aggregation of α -syn in Table 1 Pharmacokinetic parameters of SynuClean-D in plasma and CSF following an intraperitoneal single dose 10 mg/kg in male Wistar rats. a T max (h) C max (ng/ mL) AUC last (h∙ng/mL) T 1/2 (h) CSF to plasma exposure ratio Plasma 0.5 44 230 372 820 3.6 0.03% CSF 0.5 30.9 96.7 3.4 a Mean concentration values were used for calculation of pharmacokinetic parameters. A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 5 vitro has been carefully analysed previously [11]. It can be conveniently monitored as an attenuation of the Thioflavin-T (Th-T) fluorescence increase observed upon α -syn aggregation in absence of inhibitor. SynuClean-D alone does not quench or absorb Th-T emission fluorescence and the inhibition observed using Th-T has been thoroughly validated using transmission electron microscopy (TEM). TEM analysis indicates that α -syn aggregation in presence of SynuClean-D takes place with an important decrease in both number and size of α -syn fibrils [11] . In order to assess whether SynuClean-D modifications intended to improve its BBB permeability impact its anti-aggregative capacity we have assayed the derivatives using the same protocol implemented to discover SynuClean-D and the active ZPD-2 and ZPDm molecules [11–14]. This protocol attains the formation of α -syn amyloid fibrils in vitro in a ~ 30 h reaction, and uses Th-T fluorescence as a readout for the presence of these structures (Fig. 5). The incubation of 70 μ M of recombinant α -syn in the presence and absence of 100 μ M of the different SynuClean-D derivatives revealed that compound 8e has lost any antiaggregative activity and compound 1 has turned into a pro-aggregative molecule. The rest of compounds could be ranked into three groups according to their activity: (8b and 8c) >(6 and 8d) >(8a and 8f and 9). Remarkably, the anti-aggregative activity of the first two groups is comparable to that of SynuClean-D (Fig. 5, Fig. S1 and Table S1). 2.5. Cellular toxicity of SynuClean-D derivatives The structural modifications introduced in SynuClean-D derivatives may impact their toxicity in human cells. This effect was assayed in the MRC-5 (non-tumor human lung fibroblast) cell line at the SynuClean-D concentration shown to be neuroprotective (10 μ M) [12], and at 5and 10-fold this concentration. All the molecules were innocuous at 10 μ M, except compound 8d which allowed for a 80% cellular viability at this concentration. At the high 100 μ M concentration, all compounds Fig. 3. Plasma and CSF concentration vs time for Synuclean-D (10 mg/kg, IP) in rat. Plasma and CSF samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 24 h postdosing, three Wistar male rats per time point were assessed. 3 rats were not administered and referred as t =0. The values represent mean ±SEM (n =3). Fig. 4. BOILED-Egg graph for compounds 1, 6, 8a-f, 9 and SynuClean-D. SwissADME free web tool was used for graphic representation of lipophilicity (WLOGP) and polarity (tPSA) computationally obtained values [29]. Scheme 3. Successful synthetic route to SynuClean-D derivatives: 1, 6, 8a-f. Table 2 Synthesis of derivatives of SynuClean-D (1, 6, 8a-f) with different substituents by adapting the designed synthetic route. SynuClean-D derivative R 1 R 2 R 3 Yield (%) 1 H H H 75 6 NO 2 H H 42 (2 steps) 8a CN H H 23 (2 steps) 8b F F H 46 (2 steps) 8c Br H H 51 (2 steps) 8d Br Br H 38 (2 steps) 8e H H Me 65 8f NO 2 H Me 36 (2 steps) A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 6 allowed for at least 80% cell viability, except compounds 8b and 8d. Thus, only the compounds containing either two fluorine (8b) or two bromine atoms (8d) were significantly toxic at the highest concentration (Fig. 6). Scheme 4. Regioselective nitration of compound 8f. Fig. 5. In vitro analysis of the inhibitory capacity of SynuClean-D derivatives. α -syn aggregation kinetics followed by Th-T–emitted fluorescence in the absence (black) and presence of SynuClean-D (blue), and (A) derivative 1 (green), (B) derivatives 6 (green) and 8a (orange), (C) derivatives 8b (green) and 8c (orange), (D) derivatives 8d (green) and 8e (orange), and (E) derivatives 8f (green) and 9 (orange). Th-T fluorescence is plotted as normalised means and error bars are represented as SE of mean values. A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 7 2.6. Structure-activity insights from comparison of SynuClean-D and derivatives’ in vitro efficacies Qualitatively, the above results indicate that the ability of SynuClean-D to inhibit the amyloid aggregation of α -syn in vitro is strongly influenced by the absence or presence of some of its functional groups (Figs. 5 and S1). For example, the lack of activity found for non-nitrated compound 1 compared to the activity of mononitrated compound 6 and dinitrated SynuClean-D indicates that the phenyl-nitro group, unlike the heterocyclic one, plays an important role in keeping the antiaggregative activity of the compounds. In fact, non-nitrated compound 1 promotes rather than inhibits α -syn aggregation. On the other hand, compounds 8e, 8f and 9 are 1N-methylated derivatives of compounds 1, 6 and SynuClean-D, respectively. These 1N-methylated analogues display a reduced PSA compared to the non-methylated corresponding ones, which brings them closer to the yellow ellipse in the graphic BOILED-Egg representation (Fig. 4). Unfortunately, 1N-methylation, which locks the amide-iminol equilibrium of the non-methylated compounds in the amide form, significantly reduces the compounds’ antiaggregation activity. The amide-iminol region of SynuClean-D may play a significant role in the recognition of α -syn aggregates. Actually, the aggregation kinetics of the non-nitrated and 1N-methylated compound 8e cannot be distinguished from control kinetics, suggesting the compound doesn’t bind to α -syn or its aggregates. Another way to decrease the PSA of SynuClean-D, thus potentially improving its membrane permeability, is by replacing nitro groups with other less polar substituents. While the heterocyclic nitro group can be removed without significant activity loss, the phenyl-nitro has to be judiciously replaced. A comparison between nitro compound 6 and nitrile 8a shows that the bioisosteric replacement of the phenyl-nitro by a cyano group retains most of the anti-aggregation activity of compound 6. Neither compound 6 nor 8a have shown toxicity against MRC-5 cells in our assay (Fig. 6). However, nitro aromatic compounds are not infrequently associated with toxicity problems in vivo [30] and, in this respect, compound 8a constitutes an interesting similarly active alternative to nitro compound 6. The non-isosteric substitution of the phenyl-nitro group by halides enables to bring SynuClean-D derivatives into the BBB permeation area within the BOILED-Egg graph (Fig. 4). Compound 8c, bearing a bromine atom at meta position on the benzene ring, exhibits an anti-aggregation activity similar to that of SynuClean-D. Incorporating a second bromine atom at the other meta position in compound 8d slightly reduces the activity. Finally, compound 8b, where the bromine atoms in 8d are replaced by fluorine, slightly improves the in vitro anti-aggregative activity of SynuClean-D. While compounds 8b and 8d may be more toxic towards MRC-5 cells than SynuClean-D, their CC50 are still >100 µM. Importantly, SynuClean-D displays its neuroprotective effect at 10 µM, a concentration at which compounds 8b and 8c are not toxic. Taken together, the anti-aggregative activity of SynuClean-D and tested derivatives points to an important role of the amide-iminol group in the recognition of α -syn aggregates and of electron-withdrawing groups at meta position of the phenyl ring in the anti-aggregative activity. Fig. 6. Toxicity of SynuClean-D derivatives in MRC-5 cell cultures. Normalised MRC-5 cell survival in presence of different concentrations of compound 1 (A), 6 (B), 8a (C), 8b (D), 8c (E), 8d (F), 8e (G), 8f (H) and 9 (I). Survival is plotted as normalised means. Error bars are shown as standard error of means values, where p < 0.05, p <0.01 and p <0.001 were indicated by *, ** and *** respectively. A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 8 As a classical QSAR modeling is not suitable for such a small compound series here shown, a simple structure–activity analysis has been performed to obtain the individual contribution to the aggregative inhibitory potency of the functional groups added to or removed from SynuClean-D. The analysis performed is described in Section 4.6 and the results obtained are summarized in Table 3 and Fig. 7. Positive contributions are obtained for all the electron-withdrawing functional groups added/removed both in the phenyl and 2-pyridone rings, except for the simultaneous addition of two bromines in the phenyl ring. A modest contribution (0.09/0.10) is provided by a nitro group at position 5 of the heterocyclic ring. At position 3 of the phenyl ring, nitro and cyano groups make larger contributions of 0.31/0.32 and 0.22, respectively. The highest positive contribution at this position is made by bromine: 0.44 (derivative 8c). However, incorporation of a second bromine at position 5 (derivative 8d) brings about a negative contribution: −0.14, making the compound with two bromines less active than that with one. Interestingly, the simultaneous incorporation of two fluorine atoms at positions 3 and 5 of the phenyl ring (derivative 8b) comes with a combined positive contribution of 0.48, which is the highest observed in the series. In contrast, a detrimental negative contribution of –0.22 is obtained for the methyl group placed on the nitrogen of the 2-pyridone ring (compounds 8e, 8f and 9). 3. Conclusions SynuClean-D, a potent inhibitor of α -syn aggregation that protects against α -syn induced dopaminergic neurons damage in cellular and animal models, can be efficiently synthesised in gr-amounts using an economical route that also enables the synthesis of SynuClean-D derivatives. The BBB permeability of SynuClean-D appears to be low. Using the synthetic route, new derivatives have been synthesised and tested that retain the anti-aggregative activity and low toxicity of SynuClean-D and are predicted to display better BBB permeability. Structure-activity comparisons have allowed us to identify specific functionalities in the SynuClean-D molecule that are particularly important for its biological activity, and others that can be removed without activity loss. These findings will facilitate designing, synthesising and testing even more active variants. 4. Experimental section 4.1. Animals and treatments Animal care, including environmental and housing conditions conformed to the applicable Standard Operating Procedures regarding laboratory animals of Draconis Pharma S.L. (Barcelona, Spain). Animal experimental procedures were approved by the Animal Experimentation Ethical Committee of Universitat Aut` onoma de Barcelona (procedure number: 3967) and by Catalan Government Experimentation Commission Board. The animals were maintained and used in accordance with the National rules and the European Community Council Directive (86/ 609/EEC) for the care and handling of laboratory animals. 4.2. Plasma and brain distribution of SynuClean-D Analysis of SynuClean-D exposure in plasma and brain was conducted by Draconis Pharma S.L. BBB permeability studies to evaluate the plasma and brain distribution of SynuClean-D were measured as Table 3 Quantitative structure-activity analysis for evaluating the individual contribution of functional groups added to or removed from SynuClean-D. Cp Change Sub-structure 1 a Sub-structure 2 a Effect upon change b Effect breakdown c Quantitative effect 1 Removed –NO 2 (–x) –NO 2 (–y) –1.30 –x +(–y) =–1.30 (Eq. (1)) Outlier d Added - - 6 Removed - –NO 2 (–y) –0.09 –y =–0.09 (Eq. (2)) y =0.09 Added - - 8a Removed –NO 2 (–x) –NO 2 (–y) –0.21 –x +z +(–y) =–0.21 (Eq. (3)) z =0.20 e Added –CN (z) - 8b Removed –NO 2 (–x) –NO 2 (–y) 0.07 –x +2w +(–y) =0.07 (Eq. (4)) w =0.24 e Added 2 x –F (w) - 8c Removed –NO 2 (–x) –NO 2 (–y) 0.03 –x +v 1 +(–y) =0.03 (Eq. (5)) v 1 =0.44 e Added –Br (v 1 ) - 8d Removed –NO 2 (–x) –NO 2 (–y) –0.11 –x +v 1 +v 2 +(–y) =–0.11 (Eq. (6)) v 2 =–0.14 e Added 2 x –Br (v 2 ) - 8e Removed –NO 2 (–x) –NO 2 (–y) –0.63 –x +u +(–y) =–0.63 (Eq. (7)) x =0.32 (0.31) f Added - –CH 3 (u) 8f Removed - –NO 2 (–y) –0.32 u +(–y) =–0.32 (Eq. (8)) y =0.10 g Added - –CH 3 (u) 9 Removed - - –0.22 u =–0.22 (Eq. (9)) u =–0.22 Added - –CH 3 (u) a Sub-structure 1 corresponds to the phenyl ring whereas sub-structure 2 corresponds to the heterocyclic ring in SynuClean-D derivatives. b Obtained by subtracting SynuClean-D’s aggregative inhibitory potency (Table S1 and Fig. S1) from those of the derivatives. c The effect upon change (functional group added or removed) relying on the individual contributions of the functional groups added/removed. The contribution of the moiety is assumed to be positive when it is added to any sub-structure and negative when it is removed. d Effect upon change for compound 1 considered as atypical (outlier) so that Eq. (1) was not used in the calculation of the quantitative contributions. e Obtained by solving the corresponding Eq. with y being 0.09 (Eq. (2)) and x being 0.32 (Eq. (7)). To solve Eq. (6) the value of v1 used is that obtained from solving Eq. (5). Any other combination of values used (e.g. y =0.1, x =0.31) will lead to very similar results. f Value of 0.32 obtained for x (–NO 2 in sub-structure 1) by solving Eq. (7) with y being 0.09 (Eq. (2)) and u being −0.22 (Eq. (9)). Between parentheses the value obtained for x if used y =0.1 as obtained from Eq. (8). g Obtained by solving Eq. (8) with u being −0.22 (Eq. (9)). A. Mahía et al. Bioorganic Chemistry 117 (2021) 105472 9 comparison of cerebrospinal fluid (CSF) levels and plasma levels and were performed after 10 mg/kg SynuClean-D intraperitoneal (IP) administration. The administered volume was 10 mL/kg. Plasma and CSF samples were collected at 0, 0.25, 0.5, 1, 2, 4, 6 and 24 h post-dosing and three rats per time point were assessed (Wistar rats (HsdHanWIST), 125–150 g approx.). Three rats were not administered and referred as t =0. CSF samples were collected from cisterna magna in anesthetised animals. Samples contaminated with blood were discarded and not included in the analysis. The samples were stored at −20 ◦C until analysis. Blood samples (0.5–0.8 mL) were collected from anesthetised animals with isoflurane by cardiac puncture, in tubes containing K2EDTA 5%. Blood samples were centrifuged at 10 000 rpm for 5 min to obtain plasmas that were stored at −20 ◦C until analysis. Analytical measurements were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The calibration curves were conducted using blank rat plasma and artificial rat blank CSF. The lowest standard concentration on the calibration curve was 8.82 ng/mL for plasma and 4.42 ng/mL for CSF. Pharmacokinetic parameters were calculated with Phoenix 64 8.1 (WinNonlin). 4.3. Inhibition of in vitro α -synuclein aggregation by SynuClean-D derivatives α -syn expression and purification were performed in Escherichia coli BL21 DE3 strain as previously described[11]. The obtained protein was lyophilised and stored at −80 ◦C until employed. The aggregation of α -syn was carried out in a 96-well sealed plate. Each well contained 70 μ M α -syn in PBS 1X, 40 μ M Th-T, a 1/8-inch diameter teflon polyball (Polysciences Europe GmbH, Eppelheim, Germany) and 100 μ M of SynuClean-D, its derivatives, or the corresponding amount of DMSO as a control, in a final volume of 150 μ L. The plate was steadily agitated at 100 rpm and 37 ◦C, fixed into an orbital Max-Q 4000 (ThermoScientific, Waltham, Massachusetts, USA). Th-T fluorescence was measured every 2 h by exciting through a 430–450 nm filter and collecting the emission signal with a 480–510 filter, using a TECAN Spark (Tecan Trading AG, Switzerland). All assays were performed in triplicate. Data was normalised and represented as mean and standard error of mean (SEM), and fitted with GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, California, USA) by using Eq. (10): ∝=1−1 kb(ekat−1) + 1(10) where k b and k a account for the homogeneous nucleation rate constant and the secondary rate constant, respectively [31]. 4.4. Toxicity assays of SynuClean-D derivatives MRC-5 cells (ATCC CCL-171) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum at 37 ◦C in a 5% CO 2 humidified incubator in 75 cm 3 tissue culture-treated flasks. Cells were plated at a concentration of 3500 cells/well in 96-well plates in full medium and incubated at 37 ◦C overnight. Then cells were treated with the SynuClean-D derivatives at concentrations 10, 50 and 100 µM for 72 h, in triplicates. In controls, the equivalent amount of DMSO relative to each concentration of compound diluted in PBS was added. Treated cells were incubated with 10 μ L PrestoBlue® Cell Viability Reagent (ThermoFisher Scientific, Waltham, MA, United States) for 15 min. Cell viability was determined recording fluorescence at 615 nm, with an excitation wavelength of 531 nm in a Victor3 Plate reader (Perkin Elmer, USA). 4.5. Chemistry Unless otherwise specified, all reagents were obtained from commercial suppliers and were used without purification. TLC was performed on precoated silica gel polyester plates, and products were visualised using UV light (254 nm) and ninhydrin, anisaldehyde, or potassium permanganate solutions followed by heating. Column chromatography was performed on silica gel 60 (70–200 µm) with air pressure. Melting points were determined in open glass capillaries with a Gallenkamp apparatus. Infrared spectra were recorded with a Fourier transform infrared spectrometer (Nicolet Avatar 360 FT-IR). NMR spectra were recorded with a Bruker AV400 spectrometer (400 MHz for 1 H NMR experiments, 100 MHz for 13 C NMR experiments and 376 MHz for 19 F NMR experiments) in the stated deuterated solvents. 1 H and 13 C chemical shifts were referenced to internal solvent resonances and reported in ppm relative to tetramethylsilane. J values are given in Hz. High-resolution positive (or negative) electrospray ionisation mass spectra were recorded with a Bruker Daltonics MICROTOF-Q spectrometer with use of ultradilute solutions of the chemical compounds in methanol. All compounds used for biological assays are of ≥95% purity based on elemental analysis (found values are within ±0.4% of the calculated values). 2-Oxo-6-phenyl-4-(trifluoromethyl)-1,2-dihydropyridine-3-carbonitrile (1) . To a mixture of benzoyl-1,1,1-trifluoroacetone (1.73 g, 8.00 mmol) Fig. 7. Individual contribution of functional groups to the aggregative inhibitory activity in a SynuCleanD derivatives series. Removed (highlighted in red and preceded by a ‘−’ sign) and added (highlighted in blue and preceded by a ‘+’ sign) functional groups are indicated close to the thick arrows. Changes occurring in the phenyl ring are placed at the arrows’ left or below, and those occurring in the heterocyclic ring at their right or above. Groups’ individual quantitative contributions are indicated with the same color code. Positive values enhance the aggregative inhibitory potency and negative values decrease it. A. Mahía et al.