Enantioselective synthesis of 4-amino-3,4-dihydrocoumarins and their non-cyclic hydroxyester precursors: Biological evaluation for the treatment of glioblastoma multiforme

European Journal of Medicinal Chemistry 243 (2022) 114730 Available online 1 September 2022 0223-5234/© 2022 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Research paper Enantioselective synthesis of 4-amino-3,4-dihydrocoumarins and their non-cyclic hydroxyester precursors: Biological evaluation for the treatment of glioblastoma multiforme Lorenzo G. Borrego a , Rocío Recio a , * , Nazaret Moreno a , Ahmed Chelouan a , Eleuterio ´ Alvarez b , Antonio S´ anchez-Coronilla c , Carlos Caro d , John R. Pearson d , Maria Luisa García-Martín d , e , Noureddine Khiar b , ** , Inmaculada Fern´ andez a , *** a Departamento de Química Org´ anica y Farmac´ eutica, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor García Gonz´ alez, 2, 41012, Sevilla, Spain b Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Avda. Am´ erico Vespucio, 49, 41092, Sevilla, Spain c Departamento de Química Física, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor García Gonz´ alez, 2, 41012, Sevilla, Spain d Instituto de Investigaci´ on Biom´ edica de M´ alaga y Plataforma en Nanomedicina (IBIMA Plataforma BIONAND), C/ Severo Ochoa, 35, 29590, M´ alaga, Spain e Biomedical Research Networking Center in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN), Spain ARTICLE INFO Keywords: Enantiopure 4-amino-3,4-dihydrocoumarins N-sulfinylarylimines Glioblastoma multiforme β-hydroxyesters ABSTRACT The stereoselective addition of ethyl acetate enolate to the C═N bond of N-tert-butylsulfinylimines has been investigated in depth. A significant effect of the LHMDS amount and the N-sulfinylimine nature on the stereoselectivity of the process was observed. Conditions were found where sulfinylimines of differently substituted salicylaldehydes derivatives, ethyl acetate, and LHMDS afforded the corresponding addition products as a single diastereomer in good yields. The developed protocol was successfully applied to the first stereoselective synthesis of differently substituted 4-amino-3,4-dihydrocoumarin derivatives. Computational models confirmed the prominent role of the ortho aryl substituent in the stereoselectivity of the process. A significant and selective cytotoxic activity against Glioblastoma Multiforme (GBM) cancer line has been determined for the noncyclic hydroxy ester derivative. 1. Introduction Synthetic and natural coumarins have received great interest due to their interesting biological activities [1]. Particularly attractive are 3, 4-dihydrocoumarins, as they hold a wide range of pharmacological activities including antimicrobial [2], estrogenic [3], antioxidant [4], antitumor [4a], immunomodulatory [4b], antihypertensive [1], antiplatelet [5], or anti-inflammatory [6] (Fig. 1). Given the significance of these chiral molecules, great effort has been devoted to their synthesis in an enantiopure form [7]. However, in the case of 3- and 4-amino-3, 4-dihydrocoumarins, which can be considered as α or β-amino acid derivatives, respectively, only a few approximations have been developed for the former [8], and to our knowledge, no asymmetric approximation has yet been developed for the preparation of the latter in enantiopure form. This is surprising as this motif is present, among others, in the general structure of the family of inhibitors of platelet aggregation 1 (Fig. 1). The interest of these derivatives lies in their activity as inhibitors of the integrin α v β 3 [9], a transmembrane adhesion glycoprotein that, in addition to being involved in platelet aggregation, participates in other physiopathological processes such as inflammation and angiogenesis [10]. Note that the active product is the hydroxy acid that results from the hydrolysis of the coumarin derivative. Therefore, the non-cyclic hydroxy esters of analogous structure could also be of interest as they can act as prodrugs in a similar way. In addition, in recent years, coumarin and its derivatives have gained attention since they have demonstrated antitumor activity [11] by promoting many different cellular mechanisms, such as cell cycle arrest, kinase inhibition, or antimitotic activity [12]. Based on these premises and as part of our ongoing interest in the enantioselective synthesis of biologically relevant chiral amines [13], here we report an efficient and * Corresponding author. ** Corresponding author. *** Corresponding author. E-mail address: [email protected] (R. Recio). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: www.elsevier.com/locate/ejmech https://doi.org/10.1016/j.ejmech.2022.114730 Received 17 June 2022; Received in revised form 24 August 2022; Accepted 26 August 2022 European Journal of Medicinal Chemistry 243 (2022) 114730 2 straightforward method for the stereoselective synthesis of 4-amino-3, 4-dihydrocoumarin derivatives from low-cost raw materials and their hydroxy aminoesters precursors. An exhaustive in vitro screening has been conducted in order to determine their cytotoxicity in cancer and noncancer cell lines. The preliminary results on their selective cytotoxic activity against the Glioblastoma Multiforme (GBM) cancer line and an in vivo test with an orthotopic model of GBM after intratumoral administration are presented below. 2. Results and discussion 2.1. Diastereoselective synthesis of 4-amino-3,4-dihydrocoumarins and their noncyclic hydroxy ester analogues A retrosynthetic scheme of 4-amino-3,4-dihydrocoumarins shows that they could be easily prepared using tert-butylsulfinylimines as a starting chiral substrate (Fig. 2). Indeed, sulfinylimines have been shown to be the starting substrates of choice for the stereoselective synthesis of various structurally diverse chiral amines [14]. A variety of nucleophiles of different nature have been added in a stereoselective manner to the CN double bond of tert-butylsulfinyli- mines, with high chemical and stereochemical yields, owing to the stereodirecting effect of the tert-butylsulfinyl group. According to the data found in the literature, the diastereoselectivity of this type of additions is strongly influenced by the nature of the nucleophile and the solvent [15]. The pioneering work of Ellman and Tang demonstrated that the addition of enolate to N-tert-butylsulfinylimines was a very effective methodology for the asymmetric synthesis of β-aminoacids [16]. No difference in the stereochemical outcome was observed in the addition of lithium, sodium, and titanium enolates, generated by LDA, NaHMDS, and ClTi(OiPr) 3 , respectively (Fig. 3). However, the diastereoselectivity was only moderate in the case of the lithium and sodium enolates, while titanium enolates gave the highest chemical yields and diastereoselectivities. Additionally, these results were improved by increasing the amount of the otherwise highly toxic ClTi(OiPr) 3 (from 100 mol% to 200 mol%), providing the addition products in better yields (70–94%) and enhanced diastereoselectivities (90–98% de). With the goal of developing the first approximation to enantiopure 4- amino-3,4-dihydrocoumarins, we decided to study the stereoselective addition of ethyl acetate lithium enolate to the corresponding ortho hydroxilated N-sulfinylarylimine (R =o-hydroxyphenyl, Fig. 3). Therefore, we focused on optimizing the reaction conditions to ensure a totally stereoselective process, using N-sulfinylimine 7a-(R S ), derived from salicylaldehyde, as substrate and LHMDS as base. Interestingly, it was found that it was not necessary to pregenerate the lithium enolate of ethyl acetate, which could be generated in situ, greatly simplifying the experimental conditions. Thus, by simply adding the base to a solution of sulfinylimine 7a-e and ethyl acetate in THF at −78 ◦C afforded the corresponding sulfinamides 8a-e. The stereoselectivity was strongly dependent on the amount of the base and ethyl acetate used. No reaction was observed when an equivalent amount of reagents (imine, ester, and base) were used (Entry 1, Table 1), and it proceeded without stereoselectivity when 100 mol% of ethyl acetate and an excess of base were used (entry 3, Table 1). The reaction took place in a totally stereoselective manner when the amount of base was increased to 200 mol% but with a moderate 50% yield (entry 2, Table 1). To our delight, when the amount of both ethyl acetate and LHMDS were increased, the nucleophilic addition was completely stereoselective, yielding quantitatively 8a-(R S ,S C ) as a single diastereoisomer (entry 4, Table 1). In order to determine the generality of the process, the enolate ester was added to other sulfinylimines under different conditions, Table 1. The analysis of the data collected in Table 1 allows the drawing of the following conclusions. First, the sulfinylimine/enolate ratio (7-(R S ): AcOEt) determines the nature of the product obtained. Thus, with an excess of sulfinylimine 7b-(R S ) (entry 5, Table 1), the 1,3-bis(sulfinamide) 9b-(R S ,R C ,S C ,S C ,R S ) was obtained as the main product from a double addition process (Scheme 1). Second, with equimolar amounts of both reagents, sulfinylimine and enolate, the monoaddition product is exclusively obtained, Scheme 1, (compare entries 5 vs entries 6 and 7, Table 1). Third, the stereochemical course of the reaction depends strongly not only on the amount of LHMDS used as the base, but also on the nature of the imine. The 2-naph- tyl and p-methoxy substituted phenyl derivatives, 7b-(R S ) and 7d-(R S ), always gave the (R S ,S C ) diasteromer as the major product with high but not complete stereoselectivity (74% and 82% de, respectively) (entries 7 and 12, Table 1). Finally, the stereoselectivity was only modest with the Fig. 1. Pharmacologycally remarkable dihydrocoumarins derivatives. L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 3 alkyl sulfinylimine 7e-(R S ) (entries 13–15, Table 1). In contrast, the orthomethyl substituted derivative 7c-(R S ) yielded sulfinamide 8c-(R S , S C ) as a single diastereoisomer when the amount of base added was significantly increased (de: 100%, entry 10, Table 1). We propose that due to the strong basis conditions, the orthomethyl substituent must be deprotonated, as in the case of the hydroxyderivative 7a-(R S ). The sense of the diastereoselection of the process as well as the absolute configurations of the obtained diasteroismers was determined by X-ray analysis and 1 H NMR spectroscopy. The main isomer 8d-(R S ,S C ) obtained with the addition of ethyl acetate lithium enolate to (R S )-N-(pmethoxybenzylidene)-2-methyl-2-propanesulfinamide 7d-(R S ) (Table 1, entry 12) is crystalline and we could obtain an adequate crystal for the determination of its structure by X-ray (Fig. 4A). As can be seen, the absolute configuration of the newly created chiral centre is S, which allows us to confirm that the major isomer of the addition has the R S ,S C configuration, and the minor isomer has the R S ,R C absolute configuration. The absolute configuration of the rest of nonhydoxylated sulfinamides was assigned based on their 1 H NMR spectra, taking as a reference the p-methoxy diastereomers 8d-(R S ,S C ) and 8d-(R S ,R C ). The 1 H NMR signals corresponding to the ABX system of each pair of diastereoisomers have similar spectroscopic features with well-differentiated coupling patterns. The ABX system of the methylenic protons in the R S ,R C diastereoisomers always present a very pronounced “non-equivalence” (Δ ν =77–110 Hz), while both methylene protons appear at almost the same chemical shift (Δ ν around 20 Hz) in the R S ,S C diastereoisomers. Fig. 2. Retrosynthetic scheme of the 4-amino-3,4-dihydrocoumarins. Fig. 3. Addition of different nucleophiles to tert-butylsulfinylimines. Table 1 Stereoselective addition of ethyl acetate enolate to N-tert-butylsulfinylimines 7a-e-(R S ). Entry R S (Sulfinylimine) AcOEt (mol%) LHMDS a (mol%) Product b Diast. Ratio c (R S ,S C ): (R S ,R C ) de (%) Yield (%) 1 2-(OH)C 6 H 4 (7a) 100 100 8a NR – 0 2 100 200 100:0 100 50 3 100 500 52:48 d 4 76 4 400 400 100:0 100 quant. 5 2-Napht (7b) 50 250 e , f e – 36 6 100 100 8b 70:30 40 61 7 100 500 87:13 74 quant. 8 2-(CH 3 )C 6 H 4 (7c) 100 100 8c 80:20 60 83 10 100 500 100:0 100 quant. 11 4-(CH 3 O)C 6 H 4 (7d) 100 100 8d 85:15 70 64 12 100 500 91:9 82 quant. 13 iPr (7e) 100 100 8e 35:65 30 60 14 300 300 48:52 4 50 15 100 500 44:56 2 52 a The reaction was carried out by adding the base to a solution of the sulfinylimine and ethyl acetate in THF at −78 ◦C. b The products are obtained after a reaction time of 1–2 h. c The diasteromeric ratios are determined by NMR spectroscopy from the crudes. d The product was partially degraded. e LHMDS (500 mol%) is progressively added to a solution of the sulfinylimine (220 mol%) and ethyl acetate (100 mol%) in THF at −78 ◦C. f 1,3-bis(sulfinamide) was obtained as the major compound. L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 4 In the case of 1,3-bis-sulfinamide, the diastereomer obtained 9b-(R S , R C ,S C ,S C ,R S ) lacks C 2 symmetry, according to the 1 H NMR spectrum, and the configuration was confirmed by X-ray analysis (Fig. 4). Therefore, unlike previously described additions of methyl sulfonyl carbanions [17], the stereochemical course of the second addition proceeds differently from the first addition step, due to the chiral carbon present in the intermediate enolate. The peculiar stereochemical behavior of N-sulfinylimine 7a-(R S ) must be attributed to the presence of the phenolic OH group. The complete stereoselectivity of the process prompted us to evaluate its substrate scope by adding lithium enolate from ethyl acetate to a series of N-sulfinylimines derived from differently substituted salicylaldehydes, under the optimized conditions (Entry 4, Table 1) and the results are collected in Table 2. The addition of both electron-deficient and electron-rich aromatic derivatives yielded the desired products in high chemical yields and with total diastereoselectivities, yielding the (R S ,S C ) diastereoisomer as the unique product. Next, the transformation of these sulfinamides into the corresponding enantiopure 4-amino-3,4-dihydrocoumarins was studied. Attempts of lactonization of 8a-(R S ,S C ) and 8f-m-(R S ,S C ) under basic conditions, with LHMDS or CaCO 3 , were unsuccessful. In the presence of tBuOK as a base, the desired products were obtained with very low yields along with cumarin derivatives as elimination products. Fortunately, in the presence of an excess of BF 3 ⋅OEt 2 as Lewis acid and high dilution conditions, the desired lactones 10a-(R S ,S C ), 10f-g-(R S ,S C ) and 10i-m-(R S ,S C ) were obtained with high yields as the sole products (Scheme 2). The X-ray study of compound 10f-(R S ,S C ) confirmed the configurational assignment of the new stereogenic center generated in the addition step. Once again, in this process the tert-butylsulfinyl group can be considered not only as a chiral auxiliary but also as a protecting group of the amine function, which can be smoothly removed by treatment with HCl in dioxane. Therefore, the desulfinylation of 10a-(R S ,S C ) and 10i- m-(R S ,S C ) yields the hydrochlorides of the corresponding enantiopure 4- amino-3,4-dihydrocoumarins, 11a-(S C ) and 11i-m-(S C ), with a high yield as indicated in Scheme 3. Similarly, desulfinylation of the non-cycled hydroxy esters, 8a-(R S , S C ) and 8a-(S S ,R C ), 8h-k-(R S ,S C ) and 8m-(R S ,S C ), produces the corresponding β-aminoester hydrochlorides 12a-(S C ) and 12a-(R C ), 12h-k (S C ) and 12m-(S C ) (Scheme 4). 2.1.1. Justification of the observed stereoselectivity The singular and unusual total stereoselectivities observed in the addition of ester enolate to the imines of salicylaldehyde derivatives (7a- (R S ), 7f-m-(R S )) are indicative of the primordial role of the phenolic Scheme 1. Stereoselective synthesis of sulfinamide 8b-(RS,SC) and 1,3-bis-sulfinamide 9b-(RS, RC,SC,SC,RS). Fig. 4. (A) ORTEP drawing of the major sulfinamide 8d-(R S ,S C ). (Thermal ellipsoids are shown at the 50% probability level, the hydrogen atoms are omitted for clarity). (B) ORTEP drawing of the 1,3-bis(sulfinamide) 9b-(R S ,R C ,S C ,S C ,R S ). L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 5 hydroxyl group in the transition state of the nucleophilic addition. It can be assumed that the phenoxide anion formed under the basic medium is coordinated to the lithium cation of LHMDS, which is also coordinated to the enolate and the sulfinylimine in a Zimmerman-Traxler-type sixmembered transition state. In this transition state, the re face of the imine is shielded by the tert-butyl group (Model A2, Fig. 5), so that the transfer of chirality from the tert-butylsulfinyl group takes place more effectively than in the traditionally proposed coordinated model A1, Fig. 5. A similar situation could be proposed in the case of the ortho tolylderivative 7c-(R S ), which, being deprotonated in the presence of an excess of base, allows for a coordination (CH 2 − … Li + ) similar to that represented in model A2 through the benzylic carbon. Table 2 Stereoselective addition of ethyl acetate enolate to N-tert-butylsulfinylimines of substituted salicylaldehydes 7a-(R S ), 7a-(S S ) and 7f-m-(R S ). a Entry Sulfinylimine R 1 R 2 R 3 Sulfinamide b Yield (%) c 1 7a-(R S ) H H H 8a-(R S ,S C ) quant. 2 7a-(S S ) H H H 8a-(S S ,R C ) quant. 3 7f-(R S ) Me H H 8f-(R S ,S C ) 75 4 7g-(R S ) H H Me 8g-(R S ,S C ) 90 5 7h-(R S ) H OMe H 8h-(R S ,S C ) 63 6 7i-(R S ) H H OMe 8i-(R S ,S C ) 58 7 7j-(R S ) OMe H Br 8j-(R S ,S C ) quant. 8 7k-(R S ) H H Br 8k-(R S ,S C ) 91 9 7l-(R S ) H NEt 2 H 8l-(R S ,S C ) 83 10 7m-(R S ) H H NO 2 8m-(R S ,S C ) 92 a The reaction was carried out by adding LHMDS to a solution of the sulfinylimine and ethyl acetate in THF at −78 ◦C. b The products are obtained after a reaction time of 1–2 h. c Isolated yields. Scheme 2. Stereoselective synthesis of 4-tert-butylsulfinamide-3,4-dihydrocoumarins 10a-(RS,SC), 10f-g-(RS,SC) and 10i-m-(RS,SC). L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 6 Scheme 3. Desulfinylation of 4-tert-butylsulfinamide-3,4-dihydrocoumarins 10a-(RS,SC) and 10i-m-(RS,SC). Scheme 4. Synthesis of noncyclic β-aminoester hydrochlorides 12a-(SC), 12a-(RC), 12h-k(SC) and 12m-( SC) by desulfinylation. L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 7 2.2. Computational calculations DFT calculations have been performed to understand the stability of the A1 and A2 transition states. Structures 7n-(R S ) (X =H, Fig. 5) and 7a-(R S ) (X =O − , Fig. 5) were selected as reference for studying the coordinated transition states A1 and A2, respectively. The main difference in those structures is the presence of an ortho hydroxyl group in 7a- (R S ). The optimized geometries of both transition states are shown in Fig. 6. Energetically, it is found that model A2 is c.a. 2 kcal/mol more stable than model A1. With respect to the energetic values, it is understandable that the presence of the oxygen atom from the hydroxyl group may play a relevant role in stabilizing the transition state, leading to the total stereoselectivity of the process. A reasonable explanation for the stability of both A1 and A2 coordinated models is found by applying the Quantum Theory of Atoms in Molecules (QTAIM) to its optimized geometries (Fig. 6) [18]. The theory is based on the topological analysis of the density. According to this theory, for both transition states it is found a bond critical point (BCP) along a bond path between the carbon of Fig. 5. Explanative model for the observed stereoselectivity. Fig. 6. Optimized geometries and molecular graphs for the model A1 and A2 transition states. In the QTAIM molecular graph the bond paths are in red lines, BCP, RCP and CCP in green, red and blue spheres, respectively. L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 8 ethyl acetate and the carbon atom from the CN bond of the sulfinylimine group (Fig. 6). For this BCP in both models, the values of electron density ( ρ ) and its Laplacian (∇ 2 ρ ) are similar, 0.018a.u. and 0.044a.u., respectively. Interestingly, for model A2 the presence of the oxygen atom of the hydroxyl group generates a classic hydrogen bond O–H BCP with the hydrogen atom from the ethyl acetate ( ρ =0.004a.u.,∇ 2 ρ = 0.015a.u.) and a O–N BCP between the oxygen atom (from the hydroxyl) and the N atom from the sulfinylimine group ( ρ =0.012a.u.,∇ 2 ρ = 0.041a.u.). Low ρ and ∇ 2 ρ >0 values are typical of closed-shell interactions such as hydrogen bond, van der Waals, or ionic that stabilize the system [16]. Also, the molecular graph in Fig. 6 shows the presence of ring critical points (red-points in QTAIM analysis in Fig. 6). A critical ring point (RCP) is found in the interior of the bond path that forms a ring and stabilizes the system [18]. Model A1 presents three RCP, whereas the presence of the hydroxyl atom in Model A2 leads to new rearrangement of bond paths with the consequence appearance of new RCPs (Fig. 6). The new ring rearrangement of bond paths in model A2 are connected enclosing interstitial spaces arising one stabilizing cage critical point (CCP, in Fig. 6) that explains the highest stability of model A2 as compared to A1. Finally, it is worth pointing out that the disposition of the bond paths in model A2 obtained by applying QTAIM resembles the preferred Zimmerman-Traxler-type transition state (Fig. 6). These results can be extrapolated to the rest of molecules with the presence of the ortho oxygen atom from hydroxyl group explaining its stereoselectivity. Furthermore, both model A1 and A2 transition states were found to be around 7 and 9 kcal/mol, respectively, more stable than the isopropyl derivative transition state (Fig. 7), which reveals the stabilizing role of phenyl ring in the process. 2.3. Biological activity 2.3.1. In vitro experiments The antitumoral activity of the cyclic coumarins and their acyclic analogues was next investigated. To this we first performed a screening analysis by MTT using a plethora of different cell lines (non-cancer cells: N13/mouse microglial cell, BAEC/bovine aortic endothelial cells, and cancer cells: C6/rat glioblastoma multiforme cell, F98/rat glioblastoma multiforme cell, 4T1/mouse mammary cell, MCF7/human mammary cell, HeLa/human cervix cell) to compare the cytotoxic activity of the hydroxy-ester 12a-(S C ) and the corresponding lactone derivative 11a- (S C ) (Table 3 and Figs. S1–S2, see SI). The results revealed that both compounds had anticancer activity against GBM cells (mainly F98), while cyclic coumarin 11a-(S C ) also showed toxicity in non-cancer cells. Hence, the hydroxy ester derivative 12a-(S C ) was selected for further experiments. The hydroxy esters, 12a-(S C ), its enantiomer 12a-(R C ), 12h-k-(S C ), and 12m-(S C ), were evaluated using F98 as the cancer cell line and microglial N13 as the control, to determine the influence of chirality and chemical substitution of the aromatic ring on anticancer activity [19]. F98 was selected based on the screening results, as 12a-(S C ) compound showed a higher antitumor activity, along with high selectivity for this cell line. The results demonstrated that the anticancer activity of these derivatives strongly depends on the chirality and chemical substitution of the aromatic rings (Table 3 and Figs. S3–S12, see SI), with 12a-(S C ) again the one with the highest selectivity. Consequently, 12a-(S C ) was selected for further in vitro and in vivo experiments. To confirm the selectivity of 12a-(S C ) for tumor cells, we performed assays on co-cultures of F98 and N13 cells. The two types have different morphologies and can be easily distinguished, with N13 cells being rounded and F98 cells having a much more elongated shape. However, as treatment could affect cell morphology, we also prelabeled N13 cells with the CM-Dil, a nontoxic compound that stably marks cellular membranes with red fluorescence [20]. Using Harmony software, we developed an automatic segmentation method that could distinguish between the two cell types with a high degree of confidence (Fig. S13, see SI). After exposure to 12a-(S C ), a dose-dependent decrease in the number of elongated F98 cells was easily detectable, while the number of spherical N13 cells remained approximately the same (Fig. 8A–C). Automatic quantification of the two cell types confirmed that this dose-dependent response to 12a-(S C ) treatment was statistically significant (p <0.05) (Fig. 8D), with a large increase in N13 cells after 24 h of treatment (Fig. 8E). In order to determine the mechanism of cell death pathway, the cell cycle of both N13 and F98 after exposure to 100 μ g/mL of 12a-(S C ) for 24 h was assessed by flow cytometry. Cellular DNA content can be used to estimate the frequency of cell cycle phases (G 0/1 vs. S vs. G 2 M). Apoptotic cells (sub G 0 /G 1 region) are characterized by fractional DNA content and, therefore, can be easily identified by this technique [21] Analysis of the sub G 0 /G 1 fraction revealed no features of apoptosis in N13 cells, with no differences between treated and untreated cells (Fig. S14a, see SI), whereas in F98 cells, a statistically significant increase of 42% in sub G 0 /G 1 fraction was observed in treated cells compared to controls (Fig. S14b, see SI). These results indicate that F98 cells exposed to 12a-(S C ) followed an apoptotic cell death pathway. 2.3.2. In vivo experiments To evaluate the potential effects of 12a-(S C ) on the healthy brain parenchyma, a local administration inside the caudate nucleus of nontumor-bearing rats was carried out, and the possible effects were assessed by in vivo Magnetic Resonance Imaging (MRI). Furthermore, the survival rate of the animals was also monitored. In addition, PBS was also injected into the same brain region as control. Magnetic resonance imaging did not detect evidence of brain injury, even 30 days after local administration of both PBS and 12a-(S C ) (Fig. 9A). Furthermore, the animals did not show any external evidence of pain or suffering, and the survival rate after 30 days was 100% in all cases (Fig. 9B). Once the safety of normal brain tissue after local exposure to 12a- (S C ) was confirmed, its therapeutic potential was evaluated in vivo in F98 tumor-bearing rats. After tumor implantation, tumor volume was calculated at different time points (3,5,8,10,12, and 15 days) using T 2 - weighted images. When tumor volume reached around 325 mm 3 (day 10), PBS alone or 12a-(S C ) in PBS were intratumorally administered. No noticeable effect on the tumor was observed for rats administered with PBS, whereas two days after the intratumoral administration of 12a- (S C ), tumors exhibited a pronounced darkening inside, which can be attributed to necrotic/apoptotic processes (Fig. 10A). Furthermore, the therapeutic effect was evaluated by measuring the volume of the tumor over time. On day 10 after implantation, the average tumor volume was 325 mm 3 for all animals. At this time, the animals were separated into two groups and injected with PBS or 12a-(S C ), as previously described. Two days later, the average tumor size of the PBS group increased to 845 mm 3 , while tumors of the 12a-(S C ) group maintained Fig. 7. Optimized geometry of isopropyl derivative transition state. L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 9 approximately the same volume (370 mm 3 ). Finally, five days after intratumoral administration, the animals in the PBS group could not survive tumor growth, while the animals in the 12a-(S C ) group were still alive, with an average tumor size of 815 mm 3 (Fig. 10B). Furthermore, the survival rate analysis revealed that while all the animals in the PBS group died 13 days after tumor implantation, animals in the 12a-(S C ) group survived an additional 3 days (Fig. 10C). In conclusion, intratumoral administration of 12a-(S C ), caused appreciable tumor tissue damage, as could be observed in the MR images, leading to a delay in tumor growth and an increase in overall survival of 3 days. Interestingly, these results are similar to those obtained after intratumoral delivery of bortezomib in a GBM model [22]. 3. Conclusions In summary, we have reported the first enantioselective synthesis of 4-amino-3,4-dihydrocoumarins. The method which has allowed us the synthesis of a wide range of enantiopure 4-amino-3,4-dihydrocoumarin derivatives and their noncyclic hydroxy ester analogues is based on the stereoselective addition of ethyl acetate lithium enolate to the corresponding N-tert-butylsulfinylimines. The main advantage of this methodology is double: (i) it is a simple four-step synthesis, from commercially available starting material and (ii) the key step of addition of ethyl acetate lithium enolate to the CN bond takes place in a completely stereoselective manner. The biological activity studies demonstrate that both chirality and chemical substitution of the aromatic ring in the ethyl hydroxy esters derivatives are key aspects for selective toxicity against cancer cells. This selectivity was tested and confirmed in co-culture of cancer and non-cancer cells. Furthermore, the selected non-cyclic derivative 12a-(S C ) exhibited good performance for the treatment of GBM in vivo, after intratumoral administration, without affecting normal brain tissue. Therefore, the results presented here show the great potential of the hydroxy ester derivative 12a-(S C ) as an antitumor agent and could open new avenues in the treatment of GBM. Table 3 Summary of MTT assays for 12a-(S C ), 12a-(R C ), 12h-k-(S C ) and 12m-(S C ). Grading of cytotoxicity according to ISO 10993-5. Entry Compound Highest Concentration Cytotoxicity on N13 IC50 (mM) Grading of cytotoxicity N13 Cytotoxicity on F98 IC50 (mM) Grading of cytotoxicity F98 μ g/ ml μ M 1 12a-(S C ) 100 0.40 0 ±3% – None 60 ±2% 0.14 Moderate 2 12a-(R C ) 100 0.40 40 ±5% 0.45 Mild 35 ±10% 0.78 Mild 3 11a-(S C ) 100 0.50 15 ±5% 1.57 Slight 55 ±2% 0.48 Moderate 6 12h-(S C ) 100 0.36 55 ±2% 0.34 Moderate 45 ±5% 0.47 Moderate 7 12i-(S C ) 100 0.36 70 ±2% 0.04 Moderate 5 ±3% – Slight 8 12j-(S C ) 100 0.28 60 ±2% 0.22 Moderate 60 ±3% 0.17 Moderate 9 12k-(S C ) 100 0.31 40 ±3% 0.5 Mild 60 ±2% 0.21 Moderate 10 12m-(S C ) 100 0.34 30 ±5% 0.66 Mild 20 ±5% 1.14 Slight Fig. 8. Representative brightfield microscopy images of the co-culture of N13 and F98 cells exposed to different concentration of 12a-(S C ): (A) 0 μ g/mL (B) 10 μ g/mL (C) 100 μ g/mL. Green arrows indicate N13 cells, whereas red arrows indicate F98 cells. Scale bar corresponds to 50 μ m. Total number of cell (D) and cell percentage (E) of N13 and F98 cells after exposure to different concentrations of 12a-(S C ). L.G. Borrego et al. European Journal of Medicinal Chemistry 243 (2022) 114730 16 4.3. Biological studies 4.3.1. In vitro experiments Cell Culture. All cells were cultured at 37 ◦C in an incubator with a humidified atmosphere containing 5% CO 2 , with either Roswell Park Memorial Institute (RPMI) for N13 and 4T1, Bovine Endothelial Growth Medium (BEGM) for BAEC or (DMEM) for F98, C6, MCF7, and HeLa. Cytotoxicity assessment. Cytotoxicity was evaluated in all cells using the MTT assay, which provides an evaluation of mitochondrial activity. The protocol is described in detail in the Supporting Information. Co-Culture assay. N13 was first labeled with a red fluorescent dye. Then, both N13 and F98 cells were seeded in a 96-well plate. Image acquisition was performed using an Operetta High Content Screening system (PerkinElmer). Further details are given in the Supporting Information. 4.3.2. In vivo experiments Animal experiments. Animal experiments were performed in accordance with the ethical guidelines of our local ethical committee and consistent with national regulations for the care and use of laboratory animals (R.D. 53/2013). Tumor implantation. Tumor implantation was conducted following a previous protocol described by some of us [31]. This protocol is described in detail in the Supporting Information. In vivo Magnetic Resonance Imaging (MRI). MRI experiments were carried out on a Bruker Biospec 9.4 T system equipped with 400 mT/m gradients, using a 20 mm-inner-diameter surface coil placed on top of the rat head for signal reception and a 72 mm volume resonator for excitation. High-resolution T 2 -weighted images encompassing the entire tumor were acquired using a turbo-RARE sequence (TE =33 ms, TR = 2500 ms, 2 averages, 384 μ m in-plane resolution, and 1 mm slice thickness). 10 days after tumor implantation, coumarin derivatives were intratumorally administered at a concentration of 5 mg/kg, and their effect on tumor growth was evaluated by measuring the tumor volume using the aforementioned T 2 -weighted MR images. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgment This work was supported by the Ministerio de Ciencia, Innovaci´ on y Universidades (grant number PID2019-104767RB-I00/AEI/10.13039/ 501100011033), and by the Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (grant number:US- 1381590). We gratefully thank CITIUS for NMR facilities. Calculations were performed through CICA - Centro Inform´ atico Científico de Andalucía (Spain). ASC thanks funds from Junta de Andalucía (2019/ FQM-106). MRI studies were performed in the ICTS “NANBIOSIS”, specifically in the U28 Unit at the “Instituto de Investigaci´ on Biom´ edica de M´ alaga y Plataforma en Nanomedicina (IBIMA Plataforma BIONAND)”. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ejmech.2022.114730. 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