Introduction

The term prion, coined by Stanley Prusiner, was originated from the expression proteinaceous infectious particle (Prusiner 1998), denoting its putative property of acting as an unusual causative infectious agent in the pathogenesis of transmissible spongiform encephalopathies (TSEs), also known as prion diseases (Prusiner 1998). TSEs comprise a collection of genetic, infectious, or sporadic neurodegenerative disorders that affect humans and other mammals and are triggered by the conversion of the native cellular prion protein (PrPC) into its pathogenic, misfolded form, which is commonly referred as prion scrapie (PrPSc) (Caughey et al. 2009).

The constitutive, α-helix-rich PrPC is specially abundant in the nervous and immune systems and is typically located on the cell surface via a glycosyl-phosphatidyl inositol (GPI) anchor (Caughey et al. 2009). According to the protein-only hypothesis, the aberrant, β-sheet-rich PrPSc induces PrPC misfolding and promotes the propagation and amplification of PrPSc aggregates, which, in turn, induce widespread neurotoxicity (Caughey et al. 2009). While PrPC is protease-sensitive, PrPSc is partially protease-resistant and, for that reason, is also referred as PrPRes (Prusiner 1998; Caughey et al. 2009).

The precise mechanism of transmission and propagation of TSEs is still controversial, especially regarding the potential involvement of cofactors assisting PrPC to PrPSc conversion (Abid et al. 2010; Supattapone 2014; Macedo and Cordeiro 2017). In addition, evidences indicate that subversion of PrPC normal function by PrPSc is a key process in the pathophysiology of TSEs (Westergard et al. 2007). In normal conditions, PrPC is thought to play a physiological role as a scaffold protein in cell signaling (Linden 2017) and to take part in cell survival, cell differentiation, cell adhesion, Cu2+ homeostasis, synaptic transmission, myelin maintenance, and neuroprotection (Castle and Gill 2017).

Prion diseases are usually associated with long silent incubation periods, ranging from years to decades. However, when the clinical signs become detectable, disease usually progresses in a fast and aggressive manner, leading to severe disability and death (Prusiner 1998). Unfortunately, there is no effective therapy available in the clinics, highlighting the relevance of searching for novel alternatives for the treatment of TSEs.

Several promising bioactive compounds from different structural classes, like 4-amino-quinolines and trimethoxy-chalcones, have been described in the literature for their anti-prion effects in in vitro and/or in vivo assays. However, none of them have proven to be effective in preventing the progress of TSEs in clinical trials so far (reviewed in Cordeiro and Ferreira 2015; Forloni et al. 2013). Among the previously identified anti-prion drug candidates, some already approved drugs—e.g., quinacrine (1) (Geschwind et al. 2013), flupirtine (2) (Otto et al. 2004), and doxycycline (3) (Haïk et al. 2014)—stood out during preclinical studies, suggesting the possibility of their repositioning for TSE treatment (Fig. 1). However, these drugs were also not effective in further clinical stages of development (Forloni et al. 2013; Ghaemmaghami et al. 2014).

Fig. 1
figure 1

Anti-prion prototypes previously described in the literature

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A hotspot region within PrPC structure, which is susceptible to undergo conformational fluctuations, comprises specific amino acid residues, such as Asn159 at the α1–β2 loop; His187, Val189, Thr192, and Lys194 at the α2; and Glu196 at the α2–α3 loop (Langella et al. 2004; Kuwata et al. 2007). In an attempt to identify novel PrPC-stabilizing ligands, one of the putative molecular mechanisms for small anti-prion molecules, these authors conducted in vitro, in silico, and in vivo assays and have reported the anti-prion activity of prototype GN8 (4) (Kuwata et al. 2007; Yamamoto and Kuwata 2009; Ishikawa et al. 2009) (Fig. 1).

Kuwata and coworkers reported that GN8 (4) was able to reduce the PrPRes content in prion-infected cells and prolong the survival of TSE-infected mice. Using nuclear magnetic resonance (NMR) and computational simulations, this molecule was shown to interact with the hotspot region within the major cavity of PrP globular domain. The reporting of this key hotspot pocket emphasized its relevance for the structural stabilization of PrPC and thus offered the basis for further efforts in order to find novel anti-prion drug candidates (Hyeon et al. 2015; Ishibashi et al. 2016).

Our research group has contributed to this field with the description of anti-prion derivatives bearing different chemical scaffolds, including 4-amino-quinolines (Macedo et al. 2010), trimethoxy-chalcones (Ferreira et al. 2014, 2018), and oxadiazoles (Ferreira et al. 2014), as exemplified by compounds 5–7 depicted in Fig. 1. Herein we describe the synthesis and the in silico and in vitro evaluation of new trimethoxy-benzamide analogs, designed by molecular hybridization from the 4-amino-quinoline 5 and the trimethoxy-chalcone 6, as novel anti-prion candidates (Fig. 2).

Fig. 2
figure 2

Structural design of the target trimethoxy-benzamide analogs, designed by molecular hybridization from the 4-amino-quinoline 5 and the trimethoxy-chalcone 6, as novel anti-prion candidates

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Material and methods

Chemistry

All commercially available reagents and solvents were used without further purification. Reactions were routinely monitored by thin-layer chromatography (TLC) in silica gel (F245 Merck plates), and the products were visualized with ultraviolet light (254 and 365 nm). 1H and 13C NMR spectra were determined in DMSO-d6, D2O, TFA-d, acetone-d6, or CDCl3 solutions using a Varian 400-MR (IPPN-UFRJ, BR) spectrometer. The chemical shifts are given in parts per million (δ) from solvent residual peaks, and the coupling constant values (J) are given in Hz. Signal multiplicities are represented by s (singlet), d (doublet), dd (double doublet), t (triplet), dt (double triplet), q (quadruplet), qu (quintuplet), m (multiplet) and/or br (broad signal). Saturation transfer difference NMR (STD-NMR) spectra were collected at 25 °C on a 600 MHz Bruker Avance III spectrometer equipped with a room temperature inverse-detection 5-mm TXI probe.

Mass spectra were obtained using a QExactive™ Hybrid Quadrupole Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, USA) using electrospray ionization. High-resolution mass spectrometry (HRMS) data were obtained at the Laboratory for the Support of Technological Development of the Federal University of Rio de Janeiro (LADETEC-UFRJ, BR).

The purity of the testing compounds was determined by HPLC on a Shimadzu Prominence HPLC system (Shimadzu, Tokio, Japan) consisting of a vacuum degasser (DGU-20A5), a binary pump (LC-20AD), an autosampler (SIL-20A), UV/VIS Photodiode Array Detector (SPD-M20A) and fitted with a guard column (CLC G-ODS) and a Shimadzu (CLC-ODS, M) column (250 × 4.6 mm ID) running at room temperature. The mobile phase was prepared with Milli-Q water (A) and with acetonitrile (B) for the compounds 8a, 8b, 9b, 10a, and 10b; and with Milli-Q water containing 0.1% formic acid (A) and with acetonitrile (B) for the compound 9a. Isocratic elution was performed with 1:1 or 6:4 acetonitrile:water, at a flow rate set at 1 mL/min, and a 20 µL injection volume was used. The detection was carried out in the range of 294–330 nm wavelengths.

All testing compounds, i.e., trimethoxy-benzamide derivatives (8a, 8b, 9a, 9b, 10a, and 10b), were characterized by 1H and 13C NMR and HRMS, showing spectral data according to the assigned structures. Compounds 8a, 8b, 9a, 9b, 10a, and 10b presented >95% purity, as assessed by HPLC.

We used the following recombinant PrP (rPrP) constructs: hamster rPrP23–231, hamster rPrP90–231, and mouse rPrP90–231. They were expressed in E. coli and purified by nickel affinity chromatography and refolded in-column, as previously described (Wilham et al. 2010).

Synthesis of 2,4,5-trimethoxybenzoyl chloride (14)

In a 100 mL flask, 2,4,5-trimethoxybenzoic acid 15 (0.50 g, 2.36 mmol) was dissolved in dichloromethane (50 mL). Next, oxalyl chloride (0.66 g, 477 μL, 5.21 mmol) and 2 drops of dimethylformamide were added. The resulting solution was stirred at room temperature for 2–4 h and the end of the reaction was detected by TLC (70% ethyl acetate: 30% hexane). The desired acyl-chloride 14 was isolated after solvent evaporation under reduced pressure with a yield of 100% and was used as crude product for the synthesis of benzamide derivatives 10a, 10b, 13a, and 13b (Scheme 2c).

Synthesis of trimethoxy-benzamide intermediates 13a and 13b

A solution of acyl-chloride 14 (0.544 g, 2.36 mmol) in dichloromethane (50 mL) was slowly added dropwise into a 250 mL flask containing a 10 eq solution (23.6 mmol) of the corresponding amine (ethylenediamine or piperazine) in 20 mL of dichloromethane, under constant stirring at room temperature. The resulting reaction mixture was stirred at room temperature for around 20 min, when the end of the reaction was detected by TLC (10% methanol: 90% dichloromethane). The obtained trimethoxy-benzamide intermediates were isolated by the addition of 50 mL distilled water, followed by extraction in a separating funnel. These intermediates could be easily purified by acid–base extraction. Initially, basic derivatives 13a and 13b were extracted from the organic layer with an aqueous solution of HCl (pH = 3). Afterwards, pH of the aqueous layer was adjusted to neutrality and the pure benzamides 13a and 13b were extracted back to the organic layer. The final organic layers containing derivatives 13a and 13b were then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure (Scheme 2d).

N-(2-aminoethyl)-2,4,5-trimethoxybenzamide (13a)

Beige oil; yield: 75%; 1H NMR (DMSO-d6, 400 MHz): δ = 2.74 (2H, t, J = 6.4 Hz, CH2NH2), 3.33 (2H, q, J = 5.96 Hz, CONHCH2), 3.63 (2H, br, NH2), 3.72 (3H, s, ArOCH3), 3.85 (3H, s, ArOCH3), 3.91 (3H, s, ArOCH3), 6.74 (1H, s, H-3), 7.44 (1H, s, H-6), 8.29 (1H, br, CONH); 13C NMR and APT (DMSO-d6, 100 MHz): δ = 40.6 (CH2), 41.0 (CH2), 55.0 (CH3, ArOCH3), 55.9 (CH3, ArOCH3), 56.7 (CH3, ArOCH3), 97.7 (CH, C-3), 112.9 (C, C-1), 113.6 (CH, C-6), 142.4 (C, C-2), 152.1 (C, C-5), 152.5 (C, C-4), 164.5 (C, C=O).

Piperazin-1-yl(2,4,5-trimethoxyphenyl)methanone (13b)

Beige oil; yield: 95%; 1H NMR (DMSO-d6, 400 MHz): δ = 2.61 (2H, br, HNCH2R), 2.72 (2H, t, J = 4.9 Hz, HNCH2R), 3.08 (2H, br, CONCH2R), 3.52 (2H, br, CONCH2R), 3.69 (3H, s, ArOCH3), 3.77 (3H, s, ArOCH3), 3.81 (3H, s, ArOCH3), 6.71 (1H, s, H-3), 6.75 (1H, s, H-6); 13C NMR and APT (DMSO-d6, 100 MHz): δ= 42.1 (CH2), 45.2 (CH2), 45.7 (CH2), 47.4 (CH2), 55.9 (CH3, ArOCH3), 55.9 (CH3, ArOCH3), 56.3 (CH3, ArOCH3), 97.9 (CH, C-3), 112.0 (CH, C-6), 116.4 (C, C-1), 142.8 (C, C-2), 149.5 (C, C-5), 150.3 (C, C-4), 166.5 (C, C=O).

Synthesis of the designed bis-trimethoxy-benzamide analogs 10a and 10b

The acyl-chloride 14 (0.110 g, 0.48 mmol) was initially dissolved in dichloromethane (5 mL) and stirred at room temperature, followed by the gradual addition of 1–4 equivalents of the corresponding amines (ethylenediamine or piperazine) until the end of the reaction, detected by TLC. The work-up consisted of adding 10 mL of dichloromethane to the flask and then washing the organic phase with 10 mL of 10% HCl aqueous solution. Then, the organic phase was dried with sodium sulfate and concentrated under reduced pressure. The resulting residues were purified in chromatographic columns, using dichloromethane as eluent, to provide the desired bis-trimethoxy-benzamide analogs 10a and 10b (Scheme 2e).

N,N′-(ethane-1,2-diyl)bis(2,4,5-trimethoxybenzamide) (10a)

White solid; yield: 82%; 1H NMR (DMSO-d6, 400 MHz) (Fig. S1): δ = 3.48 (4H, br NCH2), 3.71 (6H, s, ArOCH3), 3.84 (6H, s, ArOCH3), 3.88 (6H, s, ArOCH3), 6.72 (2H, s, H-3 and H-3′), 7.46 (2H, s, H-6 and H-6′), 8.30 (2H, s, CONH); 13C NMR (DMSO-d6, 100 MHz) (Fig. S2): δ = 39.3 (CH2), 55.9 (CH3, ArOCH3), 55.9 (CH3,ArOCH3), 56.5 (CH3,ArOCH3), 97.8 (CH, C-3), 112.7 (C, C-1), 113.8 (CH, C-6), 142.5 (C, C-2), 152.2 (C, C-5), 152.6 (C, C-4), 164.7 (C, C=O); 1H NMR (CDCl3, 400 MHz): δ = 3.69 (2H, br, CH2), 3.70 (2H, br CH2), 3.89 (6H, s, ArOCH3), 3.90 (6H, s, ArOCH3), 3.92 (6H, s, ArOCH3), 6.49 (2H, s, H-3 and H-3′), 7.75 (2H, s, H-6 and H-6′), 8.18 (2H, s, CONH); 13C NMR and APT (CDCl3, 100 MHz): δ = 39.8 (CH2), 56.2 (CH3, ArOCH3), 56.3 (CH3, ArOCH3), 56.7 (CH3, ArOCH3), 96.6 (CH, C-3), 113.0 (C, C-1), 114.2 (CH, C-6), 143.3 (C, C-2), 152.5 (C, C-5), 152.9 (C, C4), 165.8 (C, C=O); HRESIMS m/z (Fig. S3): [M+Na]+: 471.17368 C22H28N2NaO8 (calcd. 471.17378).

Piperazine-1,4-diylbis((2,4,5-trimethoxyphenyl)methanone) (10b)

White solid; yield: 87%; 1H NMR (DMSO-d6, 400 MHz) (Fig. S4): δ = 3.16 (4H, br, CH2), 3.54 (4H, br, CH2), 3.67–3.82 (18H, m, ArOCH3), 6.67 (1H, s, H-3), 6.73 (1H, s, H-3′), 6.80 (1H, s, H-6 and H-6′); 13C NMR (DMSO-d6, 100 MHz) (Fig. S5): δ = 40.0 (CH2), 40.4 (CH2), 45.2 (CH2), 45.6 (CH2), 53.8 (CH3, ArOCH3), 54.7 (CH3, ArOCH3), 54.9 (CH3, ArOCH3), 55.0 (CH3, ArOCH3), 55.0 (CH3, ArOCH3), 96.5 (CH, C-3), 96.6 (CH, C-3′), 110.9 (CH, C-6), 111.0 (CH, C-6′), 114.7 (C, C-1), 114.7 (C, C-1′), 141.5 (C, C-2), 141.6 (C, C-2′), 148.3 (C, C-5), 148.3 (C, C-5′), 149.2 (C, C-4), 149.2 (C, C-4′), 165.3 (C, C=O); HRESIMS m/z (Fig. S6): [M+Na]+ 497.18888 C24H30N2NaO8 (calcd. 497.18943).

Synthesis of the designed 7-chloro-4-amino-quinoline analogs 8a and 8b

In a 50 mL flask equipped with a reflux condenser, the 4,7-dichloroquinoline 11 (80 mg, 0.40 mmol) was dissolved in 8 mL of isopropyl alcohol. Next, 1.1 equivalents of the corresponding benzamide intermediate (13a or 13b) were added. The resulting reaction mixture was stirred and heated at 82 °C until completion of the reaction, verified by TLC (10% methanol: 90% dichloromethane). The resulting precipitate was directly isolated by vacuum filtration (Scheme 1a).

Scheme 1
scheme 1

Reagents and conditions: (a) isopropyl alcohol, 82 °C, 48–72 h, 45–56%; (b) isopropyl alcohol, Et3N, 82 °C, 48–72 h, 54–72%

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N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-2,4,5-trimethoxy-benzamide (8a)

Beige powder; yield: 45%; 1H NMR (D2O, 600 MHz) (Fig. S7): δ = 3.51 (3H, s, ArOCH3), 3.52 (3H, s, ArOCH3), 3.61 (2H, t, CH2), 3.68 (2H, t, CH2), 3.72 (3H, s, ArOCH3), 6.41 (1H, s, H3′), 6.63 (1H, d, Jortho = 7.4 Hz, H-3), 6.87 (1H, s, H6′), 7.46 (1H, d, Jortho = 9.3 Hz, Jmeta = 2 Hz, H-6), 7.89 (1H, d, Jortho = 7.4 Hz, H-2), 7.90 (1H, d, Jortho = 9,3 Hz, H-5); 1H NMR (TFA, 400 MHz) (Fig. S8): δ = 4.36 (13H, m, CH2 and ArOCH3), 7.13 (1H, s, H-3′), 7.23 (1H, s, H-6′), 7.96 (1H, br, H-3), 8.03 (1H, br, H-6), 8.21 (1H, s, H-8), 8.53 (1H, br, H-5), 8.63 (1H, br, H-2); 13C NMR (TFA, 100 MHz) (Figs. S9 and S10): δ = 39.6 (CH2), 43.8 (CH2), 55.8 (CH3, ArOCH3), 56.5 (CH3, ArOCH3), 56.7 (CH3, ArOCH3), 96.8 (CH, C-3′), 98.3 (CH, C-6′), 106.6 (C, C-1′), 115.4 (CH, C-3), 119.6 (C, C-4a), 123.6 (CH, C-6), 129.4 (CH, C-5), 138.5 (CH, C-8), 142.2 (C, C-7), 142.6 (C, C-2′), 143.3 (CH, C-2), 156.6 (C, C-5′), 156.9 (C, C-4′), 157.2 (C, C-8a), 171.1 (C, C-4), 181.4 (C, C=O); HRESIMS m/z (Fig. S11): [M+H]+ 416.13677 C21H23ClN3O4 (calcd. 416.13716).

(4-(7-chloroquinolin-4-yl)piperazin-1-yl)(2,4,5-trimethoxyphenyl)-methanone (8b)

Beige powder; yield: 56%; 1H NMR (D2O, 400 MHz) (Fig. S12): δ = 3.62 (2H, br, RNCH2R), 3.75 (3H, s, ArOCH3), 3.81 (3H, s, ArOCH3), 3.83 (3H, s, ArOCH3), 3.97 (2H, br, RNCH2R), 6.68 (1H, s, H-3′), 6.88 (1H, s, H-6′), 7.05 (1H, d, Jortho = 7.00 Hz, H-3), 7.53 (1H, dd, Jortho = 9.1 Hz and Jmeta = 1.1 Hz, H-6), 7.82 (1H, d, Jortho = 1.1 Hz, H-8), 8.00 (1H, d, Jortho = 9.1 Hz, H-5), 8.39 (1H, d, Jortho = 7.00 Hz, H-2); 13C NMR (D2O, 100 MHz) (Fig. S13): δ = 41.5 (CH2), 45.8 (CH2), 50.1 (CH2), 51.0 (CH2), 55.7 (CH3, ArOCH3), 56.1 (CH3, ArOCH3), 56.2 (CH3, ArOCH3), 97.6 (CH, C-3′), 105.2 (CH, C-6′), 111.1 (CH, C-3), 114.0 (C, C-1′), 117.5 (C, C-4a), 119.1 (C, C-6), 126.8 (CH, C-5), 127.8 (CH, C-8), 139.2 (C, C-2′), 139.5 (C, C-7), 141.3 (CH, C-2), 142.2 (C, C-5′), 150.0 (C, C-4′), 150.6 (C, C-8a), 161.1 (C, C-4), 169.8 (C, C=O); HRESIMS m/z (Fig. S14): [M+H]+ 442.15255 C23H25ClN3O4 (calcd. 442.15281).

Synthesis of the designed 4-amino-quinazoline analogs 9a and 9b

In a 50 mL flask equipped with a reflux condenser, the 4-chloroquinazoline 12 (180 mg, 1.09 mmol) was dissolved in 20 mL of isopropyl alcohol. Next, 1.1 equivalents of the corresponding benzamide intermediate (13a or 13b) and 5 drops of triethylamine (Et3N) were added. The resulting reaction mixture was stirred and heated at 82 °C until completion of the reaction, verified by TLC (10% methanol: 90% dichloromethane). The reaction medium was concentrated under reduced pressure and redissolved in ethyl acetate. The resulting products were isolated and purified by extraction in a separation funnel. The organic layer was then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure (Scheme 1b).

2,4,5-trimethoxy-N-(2-(quinazolin-4-ylamino)ethyl)benzamide (9a)

This compound was obtained from the benzamide intermediate 13a and isolated after liquid–liquid extraction employing an aqueous phase pH around 6 as a pure white powder; yield: 72%; 1H NMR (DMSO-d6, 400 MHz) (Fig. S15): δ = 3.61 (2H, t, J = 5.50 Hz, ArHNCH2), 3.74 (5H, m, CONHCH2 and ArOCH3), 3.78 (3H, s, ArOCH3), 3.83 (3H, s, ArOCH3), 6.71 (1H, s, H-3′), 7.43 (1H, s, H-6′), 7.52 (1H, t, Jortho1 = 7.5 Hz and Jortho2 = 8.10 Hz, H-6), 7.68 (1H, d, Jortho= 8.10 Hz, H-5), 7.76 (1H, t, Jortho1 = 7.50 Hz and Jortho2 = 8.0 Hz, H-7), 8.23 (1H, t, Jortho = 8.0 Hz, H-8), 8.30 (1H, t, J = 5.50 Hz, NHAr), 8.42 (1H, t, J = 5.20 Hz, CONH), 8.47 (1H, s, H2); 13C NMR (DMSO-d6, 100 MHz) (Fig. S16): δ = 38.7 (CH2), 40.3 (CH2), 55.9 (CH3, ArOCH3), 56.5 (CH3, ArOCH3), 97.7 (CH, C-3′), 112.8 (C, C-1′), 113.6 (CH, C-6′), 115.0 (C, C-8a), 122.6 (CH, C-6), 125.7 (CH, C-8), 127.5 (CH, C-5), 132.6 (CH, C-7), 142.4 (C, C-4a), 149.1 (C, C-2′), 152.1 (C, C-5′), 152.5 (C, C-4′), 155.1 (CH, C-2), 159.6 (C, C-4), 164.7 (C, C=O); HRESIMS m/z (Fig. S17): [M+H]+ 383.17094 C20H23N4O4 (calcd. 383.17138).

(4-(quinazolin-4-yl)piperazin-1-yl)(2,4,5-trimethoxyphenyl)methanone (9b)

Compound 9b was obtained from the benzamide intermediate 13b and isolated after a liquid–liquid extraction employing an aqueous phase pH around 9, followed by an additional purification acid-base extraction for removing an undesired side product. For that, product 9b was extracted from the organic layer with an aqueous solution of HCl (pH = 3). Afterwards, pH of the aqueous layer was adjusted to neutrality and the pure derivative 9b was extracted back to the organic layer. The final organic layers containing 9b were then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure, giving 9b as a pure beige powder; yield: 54%; 1H NMR (acetone-d6, 400 MHz) (Fig. S18): δ = 3.58 (4H, br, CH2), 3.82 (3H, s, ArOCH3), 3.90 (3H, s, ArOCH3), 3.92 (3H, s, ArOCH3), 3.98 (4HH, br, CH2), 6.81 (1H, s, H-3′), 6.91 (1H, s, H-6′), 7.57 (1H, t, Jortho = 7.5 Hz, H-6), 7.87 (2H, m, H-5 and H-7), 8.13 (1H, d, Jortho = 8.3 Hz, H-8), 8.71 (1H, s, H-2); 13C NMR (acetone-d6, 100 MHz) (Fig. S19): δ = 42.1 (CH2, ArNCH2), 47.3 (CH2, ArNCH2), 50.4 (CH2, CONCH2), 50.9 (CH2, CONCH2), 56.3 (CH3, ArOCH3), 56.7 (CH3, ArOCH3), 56.9 (CH3, ArOCH3), 98.6 (CH, C-3′), 113.7 (CH, C-6′), 117.5 (C, C-1′), 117.8 (C, C-4a), 126.1 (CH, C-6), 126.5 (CH, C-8), 129.4 (CH, C-5), 133.4 (CH, C-7), 144.4 (C, C-2′), 151.0 (C, C-5′), 152.0 (C, C-4′), 152.9 (C, C-8a), 154.7 (CH, C-2), 165.4 (C, C-4), 167.6 (C, C=O); HRESIMS m/z (Fig. S20): [M+H]+ 409.18656 C22H25N4O4 (calcd. 409.18703).

Molecular docking

Initially, the three-dimensional structures of the compounds were constructed on Avogadro software (version 1.2.0) (Hanwell et al. 2012), and their energy was minimized using Merck molecular force field (MMFF94) (Halgren 1996). We then performed the blind docking between the compounds and mouse PrP globular domain (PDB: 1AG2) using AutoDock software (version 4.2.6) (Morris et al. 2009). The protein was set rigid, and the ligands were set flexible. The search space was centered on x-, y- and z-coordinates of −3.000, 0.752, and −1.572, respectively, and defined in a grid of 126 × 126 × 126 points with default 0.375 Å spacing. We considered the neutral ε-tautomeric state for His177 and His187 and the neutral δ-tautomeric state for His140, according to previous work (Langella et al. 2006; Malevanets et al. 2017). A total of 50 docking runs were performed using the Lamarckian Genetic Algorithm (LGA) and default parameters. In the next step, we performed a site-directed molecular docking based on a previously described method (Ferreira et al. 2018). The search space was centered on x-, y- and z-coordinates of 4.913, 7.305, and 2.540, respectively, and defined in a grid of 64 × 58 × 54 points. LGA parameters were the same used in the blind docking. The selected conformer was the one that presented the lowest Gibbs free energy (ΔG) of binding. GN8 (4) was used to validate the site-directed molecular docking.

Real-time quaking induced conversion (RT-QuIC)

The RT-QuIC assay was performed as previously described (Wilham et al. 2010). The brain homogenate was diluted in PBS containing 0.1% SDS and N2 medium supplement (GIBCO) (SDS–PBS–N2). We loaded each well of a black-walled, clear-bottom 96-well plate (Nunc) with 90 μL of the reaction medium, which had the following composition: 10 mM phosphate buffer (pH 7.4); 170 mM NaCl; 10 μM EDTA; 10 μM ThT; and hamster rPrP90–231 at 0.1 mg/mL (6 μM) or hamster rPrP23–231 at 0.1 mg/mL (4.3 μM). Then, we added 2 μL of brain homogenate (1 × 105 dilution) and 8 μL of stock solutions of the compounds to reach the final concentrations of 40, 50, 100, and 200 μM. The plates were sealed with a plate sealer film (Nalgene Nunc International) and incubated on a BMG FLUOstar plate reader (BMG Biotech), at 42 °C, under continuous cycles of 1 min of double-orbital shaking at 700 rpm and 1 min of rest. ThT fluorescence readings were performed every 15 min (excitation at 450 ± 10 nm and emission at 480 ± 10 nm). Post-mortem-collected sporadic Creutzfeldt-Jakob disease (sCJD) brain tissue was obtained from Prof. Pierluigi Gambetti at Case Western Reserve University and was used under Exemption #11197 from the NIH Office of Human Subjects Research Protections.

Saturation transfer difference nuclear magnetic resonance (STD-NMR)

NMR spectra were collected at 25 °C on a 600 MHz Bruker Avance III spectrometer equipped with a room temperature inverse-detection 5-mm TXI probe. The mixture contained 8a or 8b at 1 mM and murine rPrP90–231 at 10 μM in 100% D2O (1:100 protein:ligand molar ratio). Protein signals were selectively saturated using 60 Gaussian-shaped pulses of 50 ms each, resulting in a total saturation time of 3 s. A relaxation delay of 3 s was used during the STD-NMR experiment, taking into consideration the long T1 values of the ligands. Selective protein saturation occurred at different frequencies. On-resonance saturation occurred at –0.19, 0.12, 0.76, 6.52, 6.62 ppm for 8b–rPrP interaction and at –0.11, 0.21, 0.38, 7.28, 8.45 ppm for 8a–rPrP interaction, while off-resonance saturation occurred at –10 ppm. STD-NMR experiments were acquired with 48 scans and Watergate sequence was used for residual water suppression. A 40 ms spinlock filter was applied to suppress protein background signals. A 3 Hz line broadening was applied to FID prior to Fourier transformation. STD-NMR spectra were obtained by subtracting the on- from the off-resonance spectra.

Isothermal titration calorimetry (ITC)

Binding studies between rPrP90–231 and the compounds were performed at 25 °C on an ITC-200 microcalorimeter (Malvern MicroCal, USA). Both protein and ligand working solutions were prepared in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl. The syringe was filled with compound working solutions, while 200 μL of rPrP working solution was placed into the sample cell. The reference cell was loaded with degassed ultrapure water. Then, we performed a single injection of 20 μL at an interval of 180 s, during which the solution was stirred at 750 rpm with the syringe inside the sample cell with no foaming. For 8a analysis, the 8a solution in the syringe was at 1 mM and the rPrP solution in the sample cell was at 10 μM. For 8b analysis, the 8b solution in the syringe was at 0.5 mM and the rPrP solution in the sample cell was at 5 μM. In both cases, the protein:ligand molar ratio was 1:10. The heat of dilution of the compounds in buffer was subtracted from the heat of dilution of the compounds in rPrP solution. Analysis of ITC data was carried out on Origin 7.0.

Dot blot

The dot blot assay was performed as previously described (Ferreira et al. 2018) with ScN2a cells infected with prion strains RML or 22 L. Each treatment was tested in quadruplicate. The vehicle for the compounds at 0.1, 0.5, 1, and 10 μM corresponded to PBS (pH 7.4) containing DMSO at 0.001%, 0.005%, 0.01%, and 0.1%, respectively. We used the primary antibody 6D11 (1:7,500; sc-58581) and the alkaline phosphatase-conjugated secondary antibody.

MTT reduction

Cell viability in the presence of the compounds was followed by the MTT reduction assay as previously described (Ferreira et al. 2014) using ScN2a cells infected with prion strains RML or 22 L. Each treatment was tested in quadruplicate. The vehicle for the compounds at 0.1, 0.5, 1, and 10 μM corresponded to PBS (pH 7.4) containing DMSO at 0.001%, 0.005%, 0.01%, and 0.1%, respectively.

Statistical analysis

Data from dot blot and MTT reduction assays were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. All statistical evaluations were performed with GraphPad Prism 8 software.

Results and discussion

Structural design

Starting from the anti-prion prototypes 4-amino-quinoline 5 (Macedo et al. 2010) and trimethoxy-chalcone 6 (Ferreira et al. 2014), the first heterocyclic trimethoxy-benzamide analog 8a was designed by molecular hybridization, linking the structural fragments A and B from compound 5 to the fragment C from compound 6 in the novel hybrid structure of 8a, as depicted in Fig. 2. Aiming to evaluate the possible influence of conformational flexibility on the ability to interact with the target, i.e., the prion protein, the conformationally restricted analog 8b was also planned. Moreover, to assess the relevance of 7-chloro-4-amino-quinoline heterocyclic moiety A for the anti-prion activity of this novel hybrid compounds, derivatives 9a and 9b, bearing a 4-amino-quinazoline A’ core, were designed. The heterocyclic nucleus A’ was selected due to its well-known property of acting as a privileged scaffold in Medicinal Chemistry, since the 4-amino-quinazoline nucleus has been extensively studied for its wide range of pharmacological and therapeutic effects (Kamal et al. 2006; Waiker et al. 2014; Khan et al. 2015).

In turn, the bis-trimethoxy-benzamide analog 10a was designed by molecular hybridization, combining the fragment C from trimethoxy-chalcone 6 (Ferreira et al. 2014) with the ethylenediamine B from 4-amino-quinoline prototype 5 (Macedo et al. 2010), furnishing the hybrid derivative 10a, which encloses two trimethoxy-phenyl rings separated by a spacer, in a direct analogy to the structure of the anti-prion prototype 6 (Fig. 2). Once again, the influence of the conformational flexibility was considered for the design of the conformationally restricted piperazine analog 10b.

Chemical synthesis

The target heterocyclic trimethoxy-benzamide derivatives 8a, 8b, 9a, and 9b were synthesized via nucleophilic aromatic substitution (SNAr) by a key condensation step between the 4-chloro-heterocycles (11 or 12) and the corresponding amidic intermediates (13a or 13b) (Sashidhara et al. 2012; Kershaw et al. 2013; Braga et al. 2017), as depicted in Scheme 1. 7-chloro-4-amino-quinoline derivatives 8a and 8b precipitated directly from the reaction media, which was favorable for shifting the reaction balance to their formation. In the case of the synthesis of 4-amino-quinazolines 9a and 9b, no precipitation was observed and the addition of triethylamine (Et3N) was necessary to absorb the hydrochloric acid by-product, favoring product formation.

All heterocyclic trimethoxy-benzamide derivatives 8a, 8b, 9a, and 9b were characterized by 1H and 13C NMR and HRMS and showed spectral data consistent with the assigned structures. As expected, the mass spectra of the 7-chloro-4-amino-quinoline products 8a and 8b revealed the isotopic clusters predicted for monochlorinated molecules. The HRMS spectra of 8a and 8b displayed ions with a 3:1 relative abundance, which is the m/z expected for the 35Cl and 37Cl isotopic ratio (Trivedi et al. 2016).

For the synthesis of trimethoxy-benzamide intermediates 13a and 13b, as well as for obtaining the target bis-trimethoxy-benzamide analogs 10a and 10b, the acyl-chloride 14 was employed as starting material (Ahmad et al. 2010; Mallikarjuna et al. 2016) (Scheme 2). The favored formation of the monoamides 13a and 13b or the bis-amides 10a and 10b was easily managed with the proper adjustments in the synthetic methodology in terms of number of amine equivalents and order of addition of the reagents. In other words, for the synthesis of trimethoxy-benzamide intermediates 13a and 13b, a solution of the acyl-chloride 14 in dichloromethane was slowly added dropwise into a dichloromethane solution containing a great excess of ethylenediamine or piperazine (10 eq). In turn, for the synthesis of the designed bis-trimethoxy-benzamides 10a and 10b, the corresponding amines, i.e., ethylenediamine or piperazine, were slowly added to a solution of the acyl-chloride 14 in dichloromethane until the complete consumption of 14 was observed. In the described conditions, only trace amounts of the undesired products were detected by TLC monitoring. The acyl-chloride 14 was obtained from the corresponding benzoic acid 15 by classical functional group interconversion (Ahmad et al. 2010), as detailed in Scheme 2.

Scheme 2
scheme 2

Reagents and conditions: c oxalyl chloride, CH2Cl2, cat. DMF, r.t., 2 h, 100%; d A solution of the acyl-chloride 14 in CH2Cl2 was slowly added dropwise into a piperazine or ethylenediamine 10eq solution, CH2Cl2, r.t., 20’, 75–95%; e piperazine or ethylenediamine (1–4 eq) were slowly added into a CH2Cl2 solution of 14 until its complete consumption was observed, r.t., 2 h, 82–87%

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All benzamide derivatives 10a, 10b, 13a, and 13b were characterized by 1H and 13C NMR. Moreover, the designed bis-trimethoxy-benzamide analogs 10a and 10b were analyzed by HRMS and showed spectral data consistent with the assigned structures.

Interaction between PrP and trimethoxy-benzamide derivatives assessed by molecular docking

In order to investigate the binding mode of the trimethoxy-benzamide derivatives, we docked them into mouse PrP globular domain (PDB: 1AG2) using AutoDock software. Initially, we performed a blind docking, which reinforced that their most probable site of interaction within PrP globular domain was the hotspot region, previously described (Langella et al. 2004; Kuwata et al. 2007). In order to further explore the specific interaction of these compounds with the PrP hotspot site, we firstly docked GN8 (4) into this pocket and obtained interactions and spatial orientation in accordance with the results found by Kuwata’s group (Kuwata et al. 2007; Yamamoto and Kuwata 2009; Ishikawa et al. 2009) (Fig. S21). The validated docking methodology was then performed with the trimethoxy-benzamide analogs. All these compounds exhibited similar orientation and hydrogen bonding with Lys194 (ranging from 2.6 to 3.3 Å) and Asn159 (ranging from 2.6 to 3.5 Å), as well as hydrophobic interactions with Leu125, Leu130, Gln160, Val161, Tyr162, Ile182, Thr183, and Gln186 (Figs. 3 and S22). Their binding energy to PrP ranged from –6.35 to –7.88 kcal/mol, which was similar to the value found for GN8 binding (–8.83 kcal/mol) (Figs. 3 and S21). In summary, these results suggested that these molecules might act as PrP ligands.

Fig. 3
figure 3

Binding mode analysis of the trimethoxy-benzamide analogs with mouse PrP globular domain (PDB: 1AG2). Residues involved on the interactions are highlighted in dark gray, and hydrogen bonds are colored in yellow dashed lines. The binding energy values were the following: –7.00 kcal/mol for 8a, –7.66 kcal/mol for 8b, –6.78 kcal/mol for 9a, –7.88 kcal/mol for 9b, –6.35 kcal/mol for 10a, and –6.90 kcal/mol for 10b

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Modulation of rPrP aggregation assessed by RT-QuIC

The real-time quaking induced conversion (RT-QuIC) is a cell-free diagnostic tool widely used to detect and amplify minute amounts of PrPSc from different biological samples, such as brain, cerebrospinal fluid, blood, nasal brushing, saliva, urine, and feces (Wilham et al. 2010; Atarashi et al. 2011; Orru et al. 2017). In this assay, soluble, monomeric rPrP is used as substrate, and the infectious particles in the biological samples seed the conversion of monomeric rPrP into rPrP fibrils under specific shaking and temperature conditions. The conversion is monitored in real-time by thioflavin T (ThT), an amyloidophilic dye which binds to newly formed rPrP fibrils (Wilham et al. 2010). Besides its usefulness for diagnostic purposes, this assay has been employed in the last years to identify inhibitors of rPrP aggregation in vitro (Ferreira et al. 2014, 2018; Vieira et al. 2014; Schmitz et al. 2016; Hyeon et al. 2017).

As all trimethoxy-benzamide derivatives were predicted to bind to PrP globular domain, we investigated whether they could inhibit rPrP conversion in the RT-QuIC assay. We performed the reactions using hamster rPrP90–231 as substrate and brain homogenate from a patient with sCJD as a source of PrPSc. At all concentrations tested (50, 100, and 200 μM), the analogs neither inhibited the conversion nor delayed the process, except for 8a, which shortened the lag phase (Figs. 4 and S23). These findings indicate that the putative interaction of the designed compounds with PrPC, as predicted by molecular docking, was not able to prevent monomeric rPrP conversion into rPrP aggregates in vitro. However, the reduced lag phase observed in the presence of 8a suggests that this derivative might interact with PrP globular domain.

Fig. 4
figure 4

RT-QuIC kinetics using hamster rPrP90–231 at 6 μM as the substrate and sCJD agent-infected human brain homogenate as the seed. The reactions were performed in the absence (solid black line) or in the presence of the compounds at 50 μM (dotted gray line), 100 μM (dashed gray line), and 200 μM (solid gray line). Normal brain homogenate (NBH) was used as a negative control of seeding (white circles). Each curve represents the average ThT fluorescence of four replicates. The individual replicates are plotted in Fig. S23

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In order to exclude the possibility that the compounds could be quenching ThT fluorescence rather than inhibiting rPrP fibrillation, the analogs were added to the RT-QuIC reaction media after the conversion reached the plateau (17 h). No decrease in ThT fluorescence was observed (Fig. S24).

Although no inhibitory effect in the prion conversion assay was observed, it is relevant to highlight that we were able to identify a new chemical entity (NCE) bearing a novel 4-amino-quinoline trimethoxy-benzamide structural pattern, i.e., 8a, which was able to modulate rPrP aggregation in vitro. Based on these data, the 4-amino-quinoline heterocyclic moiety was considered as preferential for the proposed interaction with PrPC; thus 8a represents a useful starting point for future structural optimization in the design of novel anti-prion drug candidates.

Interaction between rPrP and trimethoxy-benzamide derivatives assessed by STD-NMR and ITC

Considering the results from the RT-QuIC assay, we next performed a saturation transfer difference nuclear magnetic resonance (STD-NMR) experiment to investigate the direct interaction of compound 8a and its conformationally restricted analog 8b with mouse rPrP90–231.

STD-NMR signals were observed for nearly all hydrogens of 8a (hydrogens 2 and/or 5, 6, 8, 3′, 6′, and methoxyl groups) and 8b (hydrogens 2, 3, 5, 8, 3′, 6′, and methoxyl groups) in the presence of rPrP90–231, suggesting that both ligands bind directly to rPrP90–231 via hydrophobic interactions (Fig. 5). The signals highlighted at approximately 6.6 ppm (Fig. 5c), 7.3 ppm, and 8.5 ppm (Fig. 5b) correspond to protein resonances that were selectively saturated, despite the spinlock filter used during the experiment.

Fig. 5
figure 5

STD-NMR spectra of 8a and 8b in complex with mouse rPrP90–231. a Chemical structures of 8a and 8b, with the corresponding hydrogens showing STD-NMR signals highlighted in red. For 8a, hydrogens 2 and 5 are highlighted in blue, since they could not be discriminated by their STD-NMR signals. b STD-NMR spectra of 1 mM 8a in the presence of 10 μM rPrP in 100% D2O. 1H off-resonance, reference spectrum is depicted on top of the figure. Each STD-NMR spectrum was collected in a different on-resonance frequency: –0.11, 0.21, 0.38, 7.28, 8.45 ppm, as indicated. c STD-NMR spectra of 1 mM 8b in the presence of 10 μM rPrP in 100% D2O. 1H reference spectrum is depicted on top of the figure. Each STD-NMR spectrum was collected in a different on-resonance frequency: –0.19, 0.12, 0.76, 6.52, 6.62 ppm, as indicated

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Comparison of the STD-NMR spectra obtained for 8a and 8b in complex with rPrP90–231 suggests that the binding epitope for both ligands are similar, reinforcing the relevance of the 4-amino-quinoline and trimethoxy-benzamide moieties for this complementary molecular recognition. Interestingly, 8b showed higher intensity STD-NMR signals than 8a, suggesting that 8b has a tighter interaction with rPrP90–231. In contrast, 8b yielded no significant change in the RT-QuIC assay.

We also analyzed the interaction of 8a and 8b with rPrP90–231 using single-injection isothermal titration calorimetry (ITC). Addition of either 8a or 8b to rPrP90–231 produced an endothermic heat of reaction (Fig. 6), which indicated that both compounds display mainly hydrophobic interactions with rPrP90–231. This is in accordance with the results from the STD-NMR analysis.

Fig. 6
figure 6

Single-injection ITC binding isotherm of 8a and 8b to mouse rPrP90–231. The curves shown were normalized by subtracting the heat of dilution of the compounds in buffer from the heat of dilution of the compounds in rPrP solution. The experiment was carried out at 25 °C with protein:ligand molar ratio of 1:10

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Cellular efficacy/toxicity evaluation of trimethoxy-benzamide derivatives

We next investigated whether the analogs 8a and 8b could change PrPRes levels in PrPSc-infected murine neuroblastoma (ScN2a) cells. This method has been extensively used to assess the anti-prion activity of molecules, as it employs a robust and relatively economical model and allows the evaluation of prion strain-related effects (Kocisko et al. 2003). We treated both RML-strain ScN2a (ScN2a-RML) and 22L-strain ScN2a (ScN2a-22L) cell lines with 8a and 8b at concentrations ranging from 0.1 to 10 μM. In ScN2a-RML cells, 8b induced a significant decrease in PrPRes content at 1 and 10 μM, while 8a promoted a similar outcome only at 10 μM (Fig. 7). In ScN2a-22L cells, both 8a and 8b prompted a significant decrease in PrPRes content only at 10 μM (Fig. 7).

Fig. 7
figure 7

Effect of 8a and 8b on PrPRes content and cell viability in PrPSc-infected murine neuroblastoma (ScN2a) cells. ScN2a cells infected with RML strain (left) or 22L-strain (right) were treated with 8a (black) or 8b (gray) at the concentrations of 0.1 μM (•), 0.5 μM (▪), 1 μM (▴), and 10 μM (▾). The vehicle for the compounds at 0.1, 0.5, 1, and 10 μM corresponded to PBS (pH 7.4) containing DMSO at 0.001%, 0.005%, 0.01%, and 0.1%, respectively. The PrPRes content was determined by dot blot assay using anti-PrP antibody 6D11. Cell viability was determined by MTT reduction assay

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Simultaneously, we examined whether the compounds were affecting cell viability. Both 8a and 8b significantly reduced ScN2a-RML and ScN2a-22L cell viability at 10 μM, while 8b also significantly decreased ScN2a-RML cell viability at 1 μM (Fig. 7). The PrPRes content reduction profiles were reasonably correlated with the cell viability reduction profiles (Fig. 7). These results indicated that these compounds were decreasing the final PrPRes levels by inducing cell death rather than by effectively inhibiting PrPRes formation and/or promoting PrPRes clearance.

The analogs 9a and 9b exhibited similar profiles to those displayed by 8a and 8b, while 10b was neither effective in decreasing PrPRes content nor significantly cytotoxic (Fig. S25). The derivative 10a was also non-toxic and promoted moderate reduction in PrPRes levels at the concentration of 10 μM (Fig. S25). These results suggested that the 7-chloro-4-amino-quinoline and 4-amino-quinazoline portions are toxicophoric in the cell lines used, while the trimethoxy-benzamide region is not.

Although the analogs 8a, 8b, 9a, and 9b were toxic to ScN2a cells, they were not equally toxic to other mammalian cell lines, including human cell lines (data not shown). These findings indicated that the 7-chloro-4-amino-quinoline and 4-amino-quinazoline rings are not universally toxicophoric, thus these compounds might even be tested in other PrPSc-infected cell lines.

Conclusion

This study has described the synthesis and biological testing of a novel series of trimethoxy-benzamide derivatives (8a, 8b, 9a, 9b, 10a, and 10b) designed as anti-prion compounds. Molecular docking studies predicted that all the compounds were expected to bind to a hotspot region in PrP globular domain with very similar spatial orientation and interaction mode. The analogs were then further evaluated by RT-QuIC and, although none of them prevented the conversion of monomeric rPrP90–231 into rPrP fibrils, the analog 8a shortened the lag phase for conversion, suggesting interaction with rPrP. As partial denaturation is supposed to precede PrP aggregation into PrPSc-like species (Kocisko et al. 1994), we speculate whether 8a can promote unfolding of PrPC, thus accelerating fibrillation. However, additional experiments are necessary to sustain this hypothesis. STD-NMR and ITC analyses indicated that the derivative 8a and its conformationally restricted analog 8b bind to rPrP90–231 and do so via hydrophobic interactions. These results point the novel 4-amino-quinoline trimethoxy-benzamide structure as an original chemical pattern for PrP complementary recognition, although affinity improvement should be aimed in a further structural optimization step. Insertion of additional polar interaction sites with the hotspot region might represent an attractive approach to overcome this issue. The accurate evaluation of the anti-prion activity of 8a and 8b in cell models was hindered by their toxicity to the PrPSc-infected cell lines used herein. Notwithstanding, this toxicity was cell line dependent. Derivatives 8a and 8b thus represent Novel Chemical Entities useful as starting points for future structural modifications in the design of novel anti-prion drug candidates with optimized efficacy and safety profiles.