F = 0.80).Previously unknown1,10a-dihydro-1-aza-10a-boraphenanthrene and6a,7-dihydro-7-aza-6a-boratetraphene have been efficiently synthesized.Bromination of these BN-PAHs proceeds with complete regioselectivity,resulting in the formation of different substituted derivatives viacross-coupling reactions. These compounds exhibit rather high fluorescencequantum yields (up to ϕ Thus, BN-arenes have beeninvestigated as promising components for improved optoelectronic devices,3 as well as in the search for new pharmacophoresin medicinal chemistry4 and in the developmentof novel ligands for transition metal-based catalysis.5BN/CC-isosterism inaromatic compounds leads to BN-polycyclic aromatichydrocarbons (BN-PAHs),6 Nevertheless, asthese examples cover only a small part of all the possible permutationsof this BN/CC-isosterism, a basic understanding of the simplest ofthese systems is still highly desirable.As a result of the significant progress seen in thefield of BN-PAHsover the past few years, several BN-arenes have been prepared in sufficientquantities, thus facilitating further studies into the propertiesof these heterocycles.7 In particular, with respect to tri- and tetracyclic BN-PAHs, differentanthracene,8 phenanthrene,9 tetracene,10 tetraphene,11 chrysene,12 pyrene,13 benzo[c]phenanthrene,14 and triphenylene15 analogues in whicha C=C unit has been replaced by a B–N bond have beendescribed, showing that the position of the B–N unit has acrucial effect on both their reactivity and photophysical properties.16 Herein, we report an efficientsynthesis for two novel systems, namely, isosteres of phenanthreneand tetraphene -protectinggroup from this substrate, followed by a Suzuki–Miyaura cross-couplingreaction, using chloro[(tri-tert-butylphosphine)-2-(2-aminobiphenyl)]palladium(II) (tBu3P–Pd-G2) asa catalyst and Cs2CO3 as a base,18 afforded biphenyl derivative 5.Finally, a ring-closing metathesis of this intermediate using thesecond-generation Grubbs catalyst gave the desired compound 1. Altogether, this novel BN-phenanthrene was prepared infive steps, with only three purifications, in 46% overall yield.The synthesis of BN-phenanthrene 1 1 started1 (3 and subs2 was prepared in five steps (threepurification processes), using a slightly modified methodology, in27% overall yield ,20 showed a B–N bond length similar to thosereported for other BN-aromatic compounds  and tetraphene (19) isostere analogues of 1 and 2 derivatives,respectively, were monitored in the 250–500 nm range. All spectrashow two main structureless bands ; however, both bands for 18 and 19 areshifted slightly to the blue relative to those for BN-phenanthreneand BN-tetraphene derivatives. Besides, less energetic bands for 18 and 19 are much less intense. The presenceof the fourth aromatic ring in 19 and the BN-tetraphenesfavors ring conjugation, shifting all spectra by about 8–14nm to the red with respect to those obtained for 18 andBN-phenanthrene derivatives. The effect of a larger contribution tothe ring conjugation of some substituents over others (H < Ph <PhC≡C) is also the reason for the observed bathochromic displacementsof the absorption peaks for the less energetic bands in 11 (14) and 12 (15) with respectto their parent 1 (2). As reported previously,the peaks for the morpholinyl-containing derivatives 13 (16) are shifted slightly to the blue with respectto 1 (2) (12a Larger molarabsorptivities were observed for BN-tetraphene derivatives than fortheir BN-phenanthrene counterparts. 18 and 19 PAH models deviate from this trend.UV–vis absorption spectra for BN-phenanthrenes to 1 (2) 1.12a Lar18 and BN-tetraphene derivatives with respect to the 19 and BN-phenanthrene ones and the substituent conjugation also affectsthe emission location in a similar manner by shifting the peaks of 11 (14) and 12 (15)to the red and 13 (16) to the blue relativeto 1 (2). However, both morpholinyl-containingderivatives 13 and 16 displayed additionalbroad fluorescence bands centered at 521 and 545 nm, respectively.Both of these bands, the intensity of which depends on the natureof the solvent and which were previously observed in a morpholinyl-functionalized4a-aza-12a-borachrysene,12a were attributedto the presence of rather stable π–π stacking aggregatesin solution .The emission spectra show features similar to theabsorption spectra4. Thus, 13 and 16), the rest of the BN-PAHs studiedshowed relatively high quantum yields (ϕF > 0.33),much higher than the PAHs 18 and 19, whosefluorescence is very weak. In particular, 12, 2, 14, and 15 exhibited rather high fluorescence,with quantum yields of 0.63, 0.68, 0.80, and 0.66, respectively. Theeffect of phenylalkynyl substituents on the fluorescence increasein BN-aromatic compounds has been reported previously.16a Fluorescence intensity profiles for 18 and 19 and BN-phenanthrene and tetraphene derivatives  were reasonably adjusted to monoexponential decays.Lifetimes  as the p-orbital of the boron center in BN-PAHs can acceptan electron pair from Lewis bases such as F–.22 To that end, fluorescence titrationexperiments were carried out on 1 and 2 withTBAF23 . The addition of aliquots of fluoride led to a monotonic quenchingof the fluorescence intensity in both cases, which can presumablybe attributed to the formation of 1 and 2 fluoroborate complexes. The titrations were verified by 19F, 11B, and 10B NMR measurements. Upon additionof 4 equiv of TBAF to BN-phenanthrene 1, a new signalappeared at −144 ppm in 19F NMR and 0 ppm in 11B and 10B NMR spectra, which could indicate theformation of a fluoroborate complex .9e On the other hand, the comparativeanalysis of the results from the titrations by TBAF of 2 and the methylated BN-tetraphene derivative 17  led us to discard that quenchingwas due to the F– binding to the NH via hydrogenbonding. The Stern–Volmer plots of fluorescence intensities and lifetimes (τ0/τ = 1 at any [TBAF]) also confirmed that the decreasein fluorescence intensity was due to the likely formation of ground-statefluoroborate complexes. However, these complexes seem to be significantlyless stable  than thosereported previously by us for 4a-aza-12a-borachrysene whose complexationconstant was a magnitude order larger.12aAdditionally, we studied the ability of 3 proceeded with completeregioselectivity, thus allowing subsequent derivatization based onpalladium-catalyzed cross-coupling reactions under standard conditions.The fluorescence of these BN-PAHs was also tested, showing ratherhigh fluorescence quantum yields (up to ϕF = 0.80).Syntheses of the previously unknown compounds1,10a-dihydro-1-aza-10a-boraphenanthreneand 6a,7-dihydro-7-aza-6a-boratetraphene have been described in fivesteps (three purification processes). The reactivity of these BN-PAHswith brominating agents was explored in order to obtain functionalizedderivatives. Treatment with NBS/AlCl1H, 13C{1H}, and 11B{1H} NMR spectrawere recorded using either a Varian Mercury VX-300, Varian Unity 300,or Varian Unity 500 MHz spectrometer at room temperature. Chemicalshifts are given in ppm (δ) downfield from TMS, with calibrationwith respect to the residual protonated solvent used (δH = 7.24 ppm and δC = 77.0 ppm for CDCl3). 11B{1H} NMR spectra were referencedexternally to BF3·OEt2 (δB = 0 ppm). Coupling constants (J) are in hertz (Hz),and signals are described as follows: s, singlet; d, doublet; t, triplet;q, quadruplet; m, multiplet; br, broad; ap, apparent. High-resolutionanalysis (HRMS) was performed using an Agilent 6210 time-of-flightLC/MS. Absorption spectra were recorded using a Uvikon 941 (KontronInstruments) UV–vis spectrophotometer. Steady-state fluorescencemeasurements were carried out using a PTI Quanta Master spectrofluorimeterequipped with a Xenon flash lamp as a light source, single concavegrating monochromators, and Glan-Thompson polarizers in the excitationand emission paths. Detection was allowed by a photomultiplier cooledby a Peltier system. Slit widths were selected at 6 nm for both excitationand emission paths, and polarizers were fixed at the “magicangle” condition. Right angle geometry and rectangular 10 mmpath cells were used for the fluorescence measurements.Reagents were acquired from commercialsources and used without further purification. When required, solventswere dried using an MBRAUN MB-SPS-800 apparatus. In general, reactionswere carried out under an argon atmosphere using oven-dried glasswarewith magnetic stirring and dry solvents. For reactions that requiredheating, the heat source was a sand bath. Reactions were monitoredusing analytical TLC plates , and compoundswere visualized with UV radiation. Silica gel grade 60 (70–230mesh) was used for column chromatography. All melting points weredetermined in open capillary tubes using a Stuart Scientific SMP3melting point apparatus (uncorrected). IR spectra were obtained usinga PerkinElmer FTIR spectrum 2000 spectrophotometer. tert-butyldimethylsilyl)-2-chloro-1,2-dihydro-1,2-azaborine 3  was added anhydrous CH2Cl2 , and the resulting solutionwas cooled to 0 °C. A recently prepared bromine solution  in anhydrous CH2Cl2  was added under argon at a rate of 1.1 mmol/h.The reaction was stirred for 15 additional minutes at 0 °C andwas allowed to warm to room temperature for an hour and a half. Themixture was concentrated under reduced pressure to afford the correspondingintermediate 3-bromo-1-(tert-butyldimethylsilyl)-2-chloro-1,2-dihydro-1,2-azaborineas an air- and moisture-sensitive oil, which could be used as is inthe next step without further purification. To the Schlenk containingthe 3-bromo-1-(tert-butyldimethylsilyl)-2-chloro-1,2-dihydro-1,2-azaborinewas added anhydrous THF , and the resulting solutionwas cooled to −30 °C. The vinylmagnesium bromide solution was addeddropwise using a syringe, and then the reaction mixture was allowedto warm to room temperature and stirred for 18 h. At the end of thereaction, the mixture was concentrated under reduced pressure, andthe remaining residue was purified by flash column chromatography(hexane) to afford the corresponding 3-bromo-2-vinyl-1-(tert-butyldimethylsilyl)-1,2-dihydro-1,2-azaborine 4  as a yellow oil. 1H NMR : δ (ppm) 7.90 ,7.29 , 6.46 , 6.17 ,5.91 , 5.80 , 0.92 , 0.45 . 13C{1H} NMR : δ(ppm) 145.6 (CH), 138.1 (CH), 131.0 (C**), 130.0 (CH), 111.3 (CH),26.7 (3CH3), 1.1 (2CH3). **Carbon not observedin 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 35.86.HRMS (APCI) calcd for C12H21BBrNSi [M + H]+: 297.0829. Found [M + H]+: 297.0828.To the Schlenk containing the 1-was dissolved in 1.7 mL of THF. A TBAF solution  was added, and the mixture was stirred for 10 minat room temperature. At the conclusion of the reaction, the solventwas removed under reduced pressure. The resulting crude material wasfiltered through a pad of silica gel  to afford the corresponding 3-bromo-2-vinyl-1,2-dihydro-1,2-azaborineas a white oil, which could be used as is in the next step withoutfurther purification. In an oven-dried Biotage microwave vial equippedwith a stir bar, the 3-bromo-2-vinyl-1,2-dihydro-1,2-azaborine  and 2-vinylphenylboronic acid  were dissolved in dioxane (1.5 mL). The resultingsolution was treated with a suspension of cesium carbonate  in distilled water (1.0 mL) before the additionof tBu3P–Pd-G2 . The vial was sealed with a cap lined with a disposableTeflon septum, and the reaction was stirred at 60 °C for 18 h.At the end of the reaction, the mixture was quenched with distilledwater (2.5 mL), and the aqueous layer was extracted with EtOAc (3× 2.5 mL). The combined organic layers were dried over anhydroussodium sulfate, filtered, and evaporated under reduced pressure. Theremaining residue was purified by flash column chromatography on silicagel (1% EtOAc/hexane) to afford the corresponding coupled product 5  as a yellow oil. 1H NMR: δ (ppm) 8.16 , 7.65 , 7.43 , 7.35 , 7.29–7.27, 7.15–7.13 , 6.74 , 6.42 ,6.31 , 5.71–5.63, 5.11 . 13C{1H} NMR : δ (ppm) 144.1(C), 143.8 (CH), 143.7 (C**), 136.5 (CH), 135.2 (C), 133.1 (CH), 130.0(CH), 128.3 (CH2), 127.9 (CH*), 127.5 (CH), 126.2 (CH),125.0 (CH), 113.5 (CH2), 110.5 (CH). *Carbon not observedin 13C{1H} NMR, assigned by gHSQC. **Carbonnot observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ(ppm) 31.60. HRMS (EI-TOF) calculated for C14H14BN [M]+: 207.1228. Found [M]+: 207.1219.Compound 2Cl2 was added to a solution of the diene 5  in CH2Cl2  under argon.The reaction mixture was heated at reflux for 24 h. The crude productwas cooled to room temperature, diluted with dichloromethane (8 mL),and filtered through a pad of silica gel. The filtrate was concentrated in vacuo, and the remaining residue was purified by flashcolumn chromatography on silica gel (5% EtOAc/Hex) to give the corresponding1,10a-dihydro-1-aza-10a-boraphenantrene 1  as a brown solid. Mp: 80–82 °C. 1H NMR : δ (ppm) 8.98 , 8.79 , 8.54 , 8.12 ,7.78 , 7.73 , 7.56 , 7.47 , 7.10 , 6.87 . 13C{1H}NMR : δ (ppm) 145.5 (CH), 137.9 (CH),134.8 (CH), 134.5 (C), 134.2 (C**), 134.1 (C), 130.8 (CH), 126.8 (CH*),126.5 (CH), 125.5 (CH), 121.6 (CH), 111.2 (CH). *Carbon not observedin 13C{1H} NMR, assigned by gHSQC. **Carbonnot observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ(ppm) 27.89. HRMS (APCI) calcd for C12H10BN[M + H]+: 179.1015. Found [M + H]+: 179.1011.The ruthenium catalyst Grubbs Second Generation G-II  in CH6  was dissolved in anhydrous toluene  in a Schlenkflask. Boron trichloride solution  was added dropwise via syringe to the vigorouslystirring solution of amine in toluene. At the conclusion of the addition,the reaction mixture was heated at reflux for 3 h. At the end of thereaction, volatiles were removed under reduced pressure to affordthe corresponding B–Cl intermediate 2-chloro-1-aza-2-boranaphthaleneas an air- and moisture-sensitive oil, which could be used as is inthe next step without further purification. To the Schlenk containingthe 2-chloro-2,1-borazaronaphthalene was added anhydrous CH2Cl2 , and the resulting solution was cooledto −30 °C. A recently prepared bromine solution  in anhydrous CH2Cl2  was added under argon at a rate of 1.1 mmol/h. After theaddition, the reaction mixture was slowly warmed to −10 °Cfor an hour and a half, and the mixture was concentrated under reducedpressure to afford the corresponding intermediate 3-bromo-2-chloro-1-aza-2-boranaphthaleneas an air- and moisture-sensitive oil, which could be used as is inthe next step without further purification. To the Schlenk containingthe 3-bromo-2-chloro-1-aza-2-boranaphthalene was added anhydrous THF, and the resulting solution was cooled to −30°C. The vinylmagnesium bromide solution  was added dropwise using a syringe,and then the reaction mixture was allowed to warm to room temperatureand stirred for 18 h. At the end of the reaction, the mixture wasconcentrated under reduced pressure, and the remaining residue waspurified by flash column chromatography (1% EtOAc/hexane) to affordthe corresponding 3-bromo-2-vinyl-1,2-dihydro-1-aza-2-boranaphthalene 7  as a brown oil. 1H NMR: δ (ppm) 8.30 , 7.91 , 7.55 , 7.44 , 7.27 ,7.21–7.17 , 6.68 , 6.17 , 6.03 . 13C{1H} NMR: δ (ppm) 145.9 (CH), 139.1 (C),131.4 (CH*), 131.4 (CH2), 128.9 (CH), 128.8 (CH), 127.9(C**), 125.3 (C), 121.9 (CH), 118.2 (CH). *Carbon not observed in 13C{1H} NMR, assigned by gHSQC. **Carbon not observedin 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 31.52.HRMS (EI-TOF) calculated for C10H9BBrN [M]+: 233.0012. Found [M]+: 233.0011.2-Vinylaniline 7  and 2-vinylphenylboronicacid  were dissolved in dioxane (2.87mL). The resulting solution was treated with a suspension of cesiumcarbonate  in distilled water (2.87mL), before addition of tBu3P–Pd-G2. The vial was sealed with a cap linedwith a disposable Teflon septum, and the reaction was stirred at 60°C for 18 h. At the end of the reaction, the mixture was quenchedwith distilled water (5 mL), and the aqueous layer was extracted withEtOAc (3 × 5 mL). The combined organic layers were dried overanhydrous sodium sulfate, filtered, and evaporated under reduced pressure.The remaining residue was purified by flash column chromatographyon silica gel (hexane) to afford the corresponding coupled product 8  as a brown oil. 1H NMR: δ (ppm) 7.99 , 7.78 , 7.66–7.62 , 7.45 , 7.34–7.28 , 7.21–7.17 , 6.72, 6.34 , 5.81–5.73, 5.65 , 5.09 . 13C{1H} NMR: δ (ppm) 143.9 (CH), 143.4 (C),139.5 (C), 137.5 (C**), 136.2 (CH), 135.4 (C), 130.4 (CH*), 130.3(CH2), 129.8 (CH), 129.6 (CH), 128.5 (CH), 127.6 (CH),126.6 (CH), 125.2 (C), 125.1 (CH), 121.4 (CH), 117.9 (CH), 114.0 (CH2). *Carbon not observed in 13C{1H} NMR,assigned by gHSQC. **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 31.98. HRMS (APCI) calcd forC18H17BN [M + H]+: 258.1452. Found[M + H]+: 258,1451.In an oven-dried Biotage microwave vialequipped with a stir bar the 3-bromo-2-vinyl-2,1-borazaronaphthalene 2Cl2 was added to a solution of the diene 8  in CH2Cl2  underargon. The reaction mixture was heated at reflux for 24 h. The crudeproduct was cooled to room temperature, diluted with dichloromethane(10 mL), and filtered through a pad of silica gel. The filtrate wasconcentrated in vacuo, and the remaining residuewas purified by flash column chromatography on silica gel (2% EtOAc/Hex)to give the corresponding 6a,7-dihydro-7-aza-6a-boratetraphene 2  as a white solid. Mp: 130–132°C. 1H NMR : δ (ppm)9.19 , 8.56 , 8.46 , 8.03 , 7.99 , 7.68 , 7.60–7.53 , 7.51–7.46 , 7.34, 7.01 . 13C{1H} NMR : δ (ppm) 147.9 (CH), 139.8 (C), 138.3(CH), 134.8 (C), 134.2 (C), 134.0 (C**), 130.9 (CH), 130.7 (CH), 129.0(CH), 127.1 (CH), 127.1 (CH*), 126.7 (CH), 125.1 (C), 122.2 (CH),121.2 (CH), 118.5 (CH). *Carbon not observed in 13C{1H} NMR, assigned by gHSQC. **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 28.74. HRMS (APCI)calcd for C16H13BN [M + H]+: 230.1138.Found [M + H]+: 230.1138.The ruthenium catalyst Grubbs Second GenerationG-II  in CH3 and N-bromosuccinimide (NBS)  was loaded in a Schlenk flask under argon. Dichloromethane(12 mL) was added, and the mixture was stirred at 25 °C for 30min and then cooled to −35 °C. The resulting solutionwas treated with a solution of 1  in 12 mL of dichloromethane, and the reaction mixture was allowedto warm to room temperature over 6 h. At the end of the reaction,a saturated sodium thiosulfate solution (20 mL) was added, and theaqueous layer was extracted with dichloromethane (3 × 20 mL).The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness. Purification of the resultingresidue by flash column chromatography on silica gel (hexanes) affordedthe product 9 as a yellow pale solid . Mp: 133–135 °C. 1H NMR : δ (ppm) 9.12 , 8.94 , 8.44 , 8.27 , 7.85 , 7.66 , 7.54 , 7.43 , 6.94 . 13C{1H} NMR : δ (ppm) 144.9 (CH), 138.9 (CH), 135.5 (CH), 134.5(C**), 134.1 (C), 133.9 (C), 130.2 (CH), 126.9 (CH), 126.1 (CH), 125.3(C**), 121.9 (CH), 112.4 (CH). **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 26.62. HRMS (EI-TOF)calculated for C12H9BBrN [M]+: 257.0015.Found [M]+: 257.0011.A mixture of AlCl3  and N-bromosuccinimide (NBS)  was loaded in a Schlenk flask under argon. Dichloromethane(16 mL) was added, and the mixture was stirred at 25 °C for 30min and then cooled to −35 °C. The resulting solutionwas treated with a solution of 2  in 16 mL of dichloromethane, and the reaction mixture wasallowed to warm to room temperature over 6 h. At the end of the reaction,a saturated sodium thiosulfate solution (30 mL) was added, and theaqueous layer was extracted with dichloromethane (3 × 30 mL).The combined organic layers were dried over Na2SO4, filtered, and concentrated to dryness. Purification of the resultingresidue by flash column chromatography on silica gel (hexanes) affordedthe product 10 as a yellow pale solid . Mp: 159–161 °C. 1H NMR : δ (ppm) 9.11 , 8.64 , 8.45, 8.16 , 7.96 , 7.62 , 7.58 , 7.56–7.53, 7.45–7.42 , 7.37 . 13C{1H} NMR : δ (ppm) 147.0 (CH), 139.6 (C), 139.4 (CH),134.5 (C), 133.5 (C), 130.7 (CH), 130.4 (CH), 130.3 (C**), 129.6 (CH),127.5 (CH), 127.0 (CH), 125.7 (C**), 125.5 (C), 122.5 (CH), 121.8(CH), 118.8 (CH). **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 26.96. HRMS (EI-TOF) calculatedfor C16H11BBrN [M]+: 307.0158. Found[M]+: 307.0168.A mixture of AlCl9  and phenylboronic acid  weredissolved in 0.32 mL of toluene and 0.08 mL of methanol and treatedwith a suspension of Na2CO3 (190.0 mg) in 0.76mL of water. Then Pd(PPh3)4  was added, and the mixture was heated to 70 °C and stirredovernight. After the addition of water (3.5 mL) and extraction withdichloromethane (3 × 3.5 mL), the combined organic layers weredried over Na2SO4, filtered, and concentratedunder a vacuum. The crude organic product was purified by flash columnchromatography on silica gel (5% AcOEt/hexane) to give 11 as a white solid . Mp: 152–154 °C. 1H NMR : δ (ppm) 9.02 , 9.02 , 8.53 , 7.98 , 7.83–7.78, 7.58–7.51 , 7.48 , 7.41–7.37 , 6.91 . 13C{1H}NMR : δ (ppm) 144.0 (C), 142.2 (CH),140.9 (C**), 138.2 (CH), 135.0 (CH), 134.9 (C**), 134.2 (C), 133.8(C), 131.1 (CH), 129.2 (2CH), 128.3 (2CH), 126.6 (CH), 126.4 (CH),125.8 (CH), 121.5 (CH), 111.4 (CH). **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 27.49. HRMS(APCI) calcd for C18H14BN [M + H]+: 256.1295. Found [M + H]+: 256.1289.In a round-bottom flask equipped with astir bar, thebrominated BN-phenantrene 9 , phenylacetylene, Pd(PPh3)2Cl2 , and CuI  was added triethylamine  and DMF (0.9 mL). The mixture was heated and stirred at 80°C for 24 h. The resulting mixture was successively washed withwater (5 mL) and extracted with dichloromethane (3 × 5 mL). Thecombined organic layers were dried over Na2SO4, filtered, and concentrated under a vacuum. The resulting productwas purified by flash column chromatography on silica gel (hexanes/EtOAc95:5) to give 12 as a brown oil . 1H NMR : δ (ppm)9.25 , 8.97 ,8.47 , 8.28 , 7.90–7.86, 7.75 , 7.64–7.61, 7.55 , 7.45, 7.42–7.34, 6.93 . 13C{1H} NMR : δ(ppm) 147.8 (CH), 138.7 (CH), 135.3 (CH), 135.0 (C**), 134.6 (C),133.4 (C), 131.7 (2CH), 131.1 (CH), 128.5 (2CH), 128.0 (CH), 127.4(CH), 125.9 (CH), 124.4 (C), 121.6 (CH), 119.9 (C**), 112.0 (CH),94.9 (C), 90.6 (C). **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 27.47. HRMS (APCI) calcd forC20H14BN [M + H]+: 279.1328. Found[M + H]+: 279.1316.To an oven-dried Schlenk flask chargedwith 2 , JohnPhos , and t-BuONa .The vial was sealed with a cap lined with a disposable Teflon septum,evacuated under vacuum, and purged with argon three times. Toluene(0.25 mL) was added, followed by brominated BN-phenanthrene 9  and morpholine . The resulting mixture was heated to 80 °Cand stirred until full consumption of 9 was observedby TLC (24 h). The reaction mixture was cooled to room temperature,diluted with Et2O (5 mL), and filtered over Celite. Thesolvent was removed in vacuo, and the resulting product was purifiedby flash column chromatography on silica gel (hexanes/EtOAc 8:2).The product 13 was obtained as yellow solid . Mp: 197–199 °C. 1H NMR : δ (ppm) 8.96 , 8.87 , 8.36–8.34 , 7.74 , 7.60–7.58 , 7.40–7.21, 7.21 , 6.85 , 4.00 , 3.20 . 13C{1H} NMR : δ (ppm) 137.9 (CH), 135.8 (C**) 134,4(CH), 134,1 (C**), 133.6 (CH), 131.9 (C), 129.5 (CH), 125.9 (CH),125.1 (CH), 124.8 (CH), 121.3 (CH), 111.4 (CH), 67.4 (2CH2), 53.1 (2CH2).**Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR: δ (ppm) 26.93. HRMS (APCI) calcdfor C16H17BN2O [M + H] +: 264.1543. Found [M + H]+: 264.1549.To an oven-dried Biotage microwave vialequipped with a stir bar were added [PdCl]10  andphenylboronic acid  were dissolvedin 0.40 mL of toluene and 0.10 mL of methanol and treated with a suspensionof Na2CO3 (238.0 mg) in 1.0 mL of water. ThenPd(PPh3)4  wasadded, and the mixture was heated to 70 °C and stirred overnight.After the addition of water (4 mL) and extraction with dichloromethane(3 × 4 mL), the combined organic layers were dried over Na2SO4, filtered, and concentrated under a vacuum.The crude organic product was purified by flash column chromatographyon silica gel (1% AcOEt/hexane) to give 14 as a whitesolid . Mp: 131–133 °C. 1H NMR : δ (ppm) 9.24 , 8.59 , 8.57 , 8.01, 7.90 , 7.74 , 7.62–7.55 , 7.50–7.47, 7.43 , 7.35 . 13C{1H} NMR : δ (ppm) 144.4 (CH), 143.8 (C), 141.0(C**), 139.7 (C), 138.6 (CH), 134.6 (C), 133.9 (C), 131.2 (CH), 130.6(CH), 129.3 (2CH), 129.0 (CH), 128.1 (2CH), 127.1 (CH), 126.9 (CH),126.7 (CH), 125.1 (C), 122.1 (CH), 121.4 (CH), 118.7 (CH). **Carbonnot observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ(ppm) 28.28. HRMS (APCI) calcd for C22H17BN[M + H]+: 306.1453. Found [M + H]+: 306.1462.In a round-bottom flask equipped with astir bar, the brominatedBN-tetraphene 10 , phenylacetylene , Pd(PPh3)2Cl2 , and CuI  was addedtriethylamine  and DMF (1.0 mL).The mixture was heated and stirred at 80 °C for 24 h. The resultingmixture was successively washed with water (5 mL) and extracted withdichloromethane (3 × 5 mL). The combined organic layers weredried over Na2SO4, filtered, and concentratedunder a vacuum. The resulting product was purified by flash columnchromatography on silica gel (hexanes) to give 15 asa pale yellow solid . Mp: 156–158°C. 1H NMR : δ (ppm)9.19 , 8.78 , 8.52 , 8.21 , 8.01 , 7.69–7.66, 7.63–7.62 , 7.57–7.54 , 7.48–7.36. 13C{1H} NMR : δ (ppm) 149.9 (CH), 139.6 (C), 139.2 (CH), 134.1 (C), 134.1(C), 132.8 (C**), 131.8 (2CH), 131.2 (CH), 130.7 (CH), 129.4 (CH),128.6 (2CH), 128.2 (CH), 127.9 (CH), 126.9 (CH), 125.4 (C), 124.3(C), 122.3 (CH), 121.5 (CH), 120,0 (C**), 118.8 (CH), 95.2 (C), 90.4(C). **Carbon not observed in 13C{1H} NMR, assignedby gHMBC. 11B{1H} NMR : δ (ppm) 28.06. HRMS (APCI) calcd for C24H17BN [M + H]+: 330.1453. Found [M + H]+: 330.1461.To an oven-dried Schlenk flask charged with 2 , JohnPhos , and t-BuONa . The vial was sealedwith a cap lined with a disposable Teflon septum, evacuated undervacuum, and purged with argon three times. Toluene (0.30 mL) was added,followed by brominated BN-tetraphene 1  and morpholine .The resulting mixture was heated to 80 °C and stirred until fullconsumption of 1 was observed by TLC (24 h). The reactionmixture was cooled to room temperature, diluted with Et2O (5 mL), and filtered over Celite. The solvent was removed in vacuo,and the resulting product was purified by flash column chromatographyon silica gel (hexanes/EtOAc 9:1). The product 16 wasobtained as a yellow solid . Mp: 138–140°C. 1H NMR : δ (ppm)9.10 , 8.44 , 8.42–8.40 , 7.96 , 7.59–7.56 , 7.54–7.52, 7.40–7.38 , 7.36–7.33 , 7.12, 4.03–4.01 , 3.21–3.19 . 13C{1H} NMR : δ(ppm) 152.4 (C**), 138.9 (C), 138.3 (CH), 135.4 (C), 134.1 (C**),131.7 (C), 130.5 (CH), 129.6 (CH), 129.0 (CH), 127.1 (CH), 126.3 (CH),125.3 (CH), 125.1 (C), 122.0 (CH), 121.5 (CH), 118.6 (CH), 67.4 (2CH2), 52.7 (2CH2). **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 27.51. HRMS (APCI)calcd for C20H20BN2O [M + H] +: 315.1667. Found [M + H]+: 315.1675.To an oven-dried Biotage microwave vialequippedwith a stir bar were added [PdCl]2  followed by THF (0.18 mL). Thevial was sealed and placed under an Ar atmosphere. LiHMDS  was added dropwise via a syringe. The solutionwas stirred for 4 h at room temperature. After this time, the reactionmixture was cooled to 0 °C, and iodomethane  was added. The reaction mixture was allowed to stirat 0 °C for 10 min, then warmed to room temperature. The solutionwas stirred at this temperature overnight. A second addition of LiHMDS and iodomethane  were added at this time, and the resulting solutionwas stirred for 6 h at room temperature. The reaction was quenchedwith deionized H2O, and the aqueous layer was extractedwith Et2O. The organic layer was dried (Na2SO4), filtered, and concentrated under a vacuum. The resultingproduct was purified by flash column chromatography on silica gel(hexanes) to give the desired product 17 as a white solid. Mp: 114–116 °C. 1H NMR : δ (ppm) 9.19 , 8.57, 8.07–80.2 , 7.80, 7.71–7.67 , 7.56–53, 7.47–7.41 , 7.39 , 7.24 ,4.06 . 13C{1H} NMR : δ (ppm) 147.7 (CH), 141.8 (C), 138.5 (CH), 134.4(C), 134.2 (C), 133.3 (C**), 131.5 (CH), 130.7 (CH), 129.4 (CH), 127.1(CH), 127.1 (CH*), 126.6 (CH), 125.8 (C), 122.2 (CH), 120.8 (CH),115.0 (CH), 35.2 (CH3). *Carbon not observed in 13C{1H} NMR, assigned by gHSQC. **Carbon not observed in 13C{1H} NMR, assigned by gHMBC. 11B{1H} NMR : δ (ppm) 29.51. HRMS(APCI) calcd for C17H15BN [M + H]+: 244.1295. Found [M + H]+: 244.1296.Toa 4 mL reaction vial equipped with a stir bar was added 7-aza-6a-boratetraphene


Neodidymelliopsis sp., led to the isolation of a novel polyketide, (2Z)-cillifuranone (1) and five previously reported natural products, (2E)-cillifuranone (2), taiwapyrone (3), xylariolide D (4), pachybasin (5), and N-(5-hydroxypentyl)acetamide (6). It was discovered that (2Z)-cillifuranone (1) was particularly sensitive to ambient temperature and light resulting in isomerisation to (2E)-cillifuranone (2). Structure elucidation of all the natural products were conducted by NMR spectroscopic techniques. The antimicrobial activity of 2, 3, and 5 were evaluated against a variety of bacterial and fungal pathogens. A sodium [1-13C] acetate labelling study was conducted on Neodidymelliopsis sp. and confirmed that pachybasin is biosynthesised through the acetate polyketide pathway.Fungi have become an invaluable source of bioactive natural products, with more than 5 million species of fungi spanning the globe. Fractionation of crude extract of  Neodidymelliopsis sp. (ICMP 11463) to show antibacterial activity against Staphylococcus aureus and Mycobacterium abscessus but not Escherichia coli or Pseudomonas aeruginosa. Fractionation of the crude extract of Neodidymelliopsis sp. led to the isolation of two furanones, (2Z)-cillifuranone (1) and (2E)-cillifuranone (2); two α-pyrones, taiwapyrone (3) and xylariolide D (4); one anthraquinone, pachybasin (5); and one acetamide, N-(5-hydroxypentyl)acetamide (6) (Z)-cillifuranone (1) was novel, while compounds 2–6 have been previously reported . It . It E)-c the two ,8. HereiNeodidymelliopsis sp. (ICMP 11463) were extracted with a combination of MeOH and CH2Cl2. Preliminary fractionation of the brown gum was carried out using C8 reversed-phase flash chromatography, eluting with a MeOH/H2O gradient, to generate five fractions (F1–F5). Further purification of F3 and F4 by a combination of Sephadex LH20 and silica gel column chromatography afforded compounds 1–6 (Freeze-dried plates (dry weight 108.6 g) inoculated with unds 1–6 .1, isolated as pale-yellow needles, showed a sodiated adduct of m/z 219.0638 [M + Na+] which established a molecular formula of C10H12O4. The 1H-NMR spectrum of 1 exhibited one set of alkene protons  and 6.01 ), three methylenes , 2.60  and 1.75–1.70 ), and one methyl group ) , 193.4 (C-5), and 169.5 (C-1)), three olefinic carbons (δC 114.9 (C-4), 124.4 (C-2), 130.0 (C-3)), three methylenes (δC 76.1 (C-7), 32.9 (C-1′), and 20.3 (C-2′)), and, finally, one methyl carbon (δC 14.0 (C-3′)). Analysis of COSY NMR data established the presence of two distinct proton spin-systems; two protons  were attributed to a Z-alkene as they had a JHH coupling constant of 11.6 Hz, while a second spin-system was attributed to an alkyl chain  (H 6.49 (H-3) and δC 201.2 (C-8) and 169.5 (C-1) combined with correlations from δH 6.01 (H-2) to δC 114.9 (C-4) established the position of a carboxylic acid, two carbonyl carbons and the attachment point of the Z-alkene at position C-4. In addition, HMBC correlations between δH 1.75–1.70 (H2-2′) and 193.4 (C-5) established the position of the alkyl chain at C-5. Issues arose with placement of the remaining methylene carbon at C-7. COSY correlations between δH 4.62 (H2-7) and δH 2.60 (H2-1′) suggested the remaining methylene must be a near H2-1′; however, no HMBC correlations to the alkyl chain were observed. An HMBC correlation from δH 4.62 to δC 193.4 suggested a furanone ring. Thus, compound 1 was determined to be (2Z)-cillifuranone. The spectroscopic data showed many similarities to those observed for (2E)-cillifuranone with differences centred on the alkene, where compound 1 contains a Z-alkene  and  and 2 contains an E-alkene  and . The most notable similarity with the present study was an unusual 5JHH COSY correlation observed between H2-7 and H2-1′ which was also reported by Weise et al. during the structure elucidation of 2 . Rec. Rec7–9)–C (7–9) . An uncoe fungus .Z)-cillifuranone and taiwapyrone using the previously proposed intermediate 11 as a common biosynthetic intermediate  begins with attack of the ketone by the allylic alcohol to form the furanone ring followed by expulsion of water, the resulting product is (2Z)-cillifuranone (1). The second possible cyclization (blue) pathway involves the vinyl alcohol attacking the carbonyl of the acid forming the six membered α-pyrone ring with the expulsion of water. Subsequent reduction of the ketone forms taiwapyrone (3).We speculate that it is possible to achieve the biosynthesis of both -cillifuranone (1) or taiwapyrone (3). Given that some investigations of the chemistry of Neodidymelliopsis sp. yielded no (2E/Z)-cillifuranone, it is plausible that the pathway is sensitive to growing conditions.Having such commonly related intermediates allows E)-cillifuranone, a sodium [1-13C] acetate labelling study was conducted to test the hypothesis that these compounds were produced through the acetate pathway. The organism was regrown in solid culture with 1 mg/mL sodium [1-13C] acetate incorporated in the agar. Standard extraction and fractionation unfortunately only yielded pachybasin (5), with no chromatographic evidence observed for 1–4 and 6.Due to the lack of biosynthetic knowledge for compounds, such as taiwapyrone and (213C-NMR spectra of labelled (5) (5)   were ovelled (5) . The ace (5) (5) , providi5)  previously assigned incorrectly in isolation reports of pachybasin (5) .13C sodium acetate. In the established F folding mode, the octaketide cyclization point lies between C-2 and C-3 + .Pale yellow needles; Rf  0.17; IR (ATR) vmax 3055, 1704, 1625, 1571, 1421, 1265, 1186, 982, 896, 734, 704 cm−1; 1H-NMR  δ 7.32 , 6.83 , 4.67 , 2.76 , 1.81–1.72 , 1.02 ; 13C-NMR  δ 201.9 (C-8), 196.7 (C-5), 170.9 (C-1), 132.6 (C-3), 118.7 (C-2), 112.6 (C-4), 76.3 (C-7), 31.6 (C-1’), 21.1 (C-2’), 14.0 (C-3’) (1H- and 13C-NMR data agreed with literature) [m/z 219.0638 [M + Na]+ .Yellow oil; Rf  0.23; IR (ATR) vmax 3333, 2946, 2835, 2164, 1648, 1450, 1412, 1111, 1019 cm−1; [α]D21 = −44.8   literature) + .Yellow oil; Rf  0.43; 1H-NMR  δ 7.51 , 6.23 , 4.65 , 2.27 , 1.80–1.72 , 1.52–1.42 , 1.40–1.35 , 1.29–1.24 , 0.95  (1H-NMR data agreed with literature) + .Colourless oil; Rf  0.87; IR (ATR) vmax 1672, 1637, 1591, 1361, 1276, 1223, 757, 710 cm−1; 1H-NMR  δ 12.58 , 8.32–8.29 , 7.82–7.79 , 7.67 , 7.12–7.10 , 2.47 ; 13C-NMR  δ 188.3 (C-9), 182.9 (C-10), 162.9 (C-1), 148.8 (C-3), 134.6 (C-6), 134.3 (C-7), 133.8 (C-10a), 133.5 (C-8a), 133.3 (C-4a), 127.5 (C-5), 127.0 (C-8), 124.3 (C-2), 121.0 (C-4), 114.3 (C-9a), 22.4 (C-11) (1H-NMR data agreed with literature) [m/z 261.0529 [M + Na]+ .Yellow solid; Rf  0.46; IR (ATR) vmax 3286, 3097, 3062, 2937, 2864, 1633, 1559, 1437, 1370, 1266, 1042, 1005, 896, 733, 702 cm−1; 1H-NMR  δ 3.64 , 3.25 , 1.97 , 1.62–1.50 , 1.45–1.39 ; 13C-NMR  δ 170.4 (C-6), 62.6 (C-1), 39.6 (C-5), 32.3 (C-2), 29.4 (C-4), 23.4 (C-3), 23.2 (C-7); (+)−HRESIMS m/z 168.0989 [M + Na]+ .Clear oil; R13C] acetate/PDA agar) were prepared. The sodium [1-13C] acetate agar plates were inoculated with 1 cm3 cubes of pre-prepared Neodidymelliopsis sp. ICMP 11463 PDA plates. Plates were sealed with parafilm, inverted, and incubated at 37 °C for 18 h.Agar plates with a concentration of 1 mg/mL (sodium  acetate incorporation experiment was performed to determine the biosynthesises of these natural products. Only pachybasin (5) was isolated from the labelled extract of Neodidymelliopsis sp., with the pattern of label incorporation being consistent with biosynthesis via cyclization of an acetate polyketide pathway-derived octaketide.A study of


N,N-diallyltryptamine (5-MeO-DALT) is reported.The synthesis and solid-state structure of the fumarate salt of the synthetic psychedelic 5-methoxy- N,N-diallyl-5-methoxytryptammonium) (5-MeO-DALT) fumarate (systematic name: bis{N-[2-(5-methoxy-1H-indol-3-yl)ethyl]- N-(prop-2-en-1-yl)prop-2-en-1-aminium} (E)-but-2-enedioate), 2C17H23N2O+·C4H2O42−, has a single tryptammonium cation and half of a fumarate dianion in the asymmetric unit. The tryptammonium and fumarate ions are held together in one-dimensional chains by a series of N—H⋯O hydrogen bonds. These chains are combinations of R44(22) rings, and C22(14) and C44(28) parallel chains along [111].The title compound, bis( N,N-dimethyltryptamine (5-MeO-DMT) is a naturally occurring tryptamine found in the parotid gland of some toads, and this compound has been explored for its clinical effects in treating mood disorders  is a synthetic analogue of 5-MeO-DMT, which was synthesized in 2004 by Alexander Shulgin  fumarate herein.5-Methoxy-N,N-diallyltryptammonium) fumarate contains one tryptammonium cation and one half of a fumarate dianion     and 4-HO-MiPT  and N-methyl-N-allyltryptamine (MALT) : δ 7.44 , 7.27 , 7.10 , 6.94 , 6.67 , 5.91–5.81 , 5.62–5.56 , 3.87 , 3.79 , 3.42–3.38 , 3.17–3.13 ; 13C NMR : δ 172.1 (COO), 152.7 (CH), 135.3 (ArC), 132.5 (ArC), 127.22 (ArC), 127.20 (ArC), 126.2 (ArC), 125.8 (ArC), 113.7 (ArC), 112.6 (ArC), 108.9 (CH=CH2), 101.3 (CH=CH2), 56.8 (AkC), 55.7 (AkC), 52.2 (AkC), 20.4 (AkC).110 mg of 5-MeO-DALT freebase were dissolved in 10 mL of methanol and 47 mg of fumaric acid was added and refluxed overnight. 129 mg (82% yield) of white powder was obtained upon removal of solvent Ueq of the parent nitrogen atom. All other hydrogen atoms were placed in calculated positions (C—H = 0.93–0.97 Å). Isotropic displacement parameters were set to 1.2Ueq  or 1.5Ueq (CH3).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021002838/ey2005sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002838/ey2005Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021002838/ey2005Isup3.cmlSupporting information file. DOI: 2070873CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Compound 14 showed the most robust anticonvulsant activity  = 31.3 mg/kg, ED50scPTZ = 67.4 mg/kg). Notably, it was also effective in the 6 Hz (44 mA) model of drug-resistant epilepsy (ED50 = 63.2 mg/kg). Apart from favorable anticonvulsant properties, compound 14 revealed a high efficacy against pain responses in the formalin-induced tonic pain, the capsaicin-induced neurogenic pain, as well as in the oxaliplatin-induced neuropathic pain in mice. Moreover, compound 14 showed distinct anti-inflammatory activity in the model of carrageenan-induced aseptic inflammation. The mechanism of action of compound 14 is likely complex and may result from the inhibition of peripheral and central sodium and calcium currents, as well as the TRPV1 receptor antagonism as observed in the in vitro studies. This lead compound also revealed beneficial in vitro ADME-Tox properties and an in vivo pharmacokinetic profile, making it a potential candidate for future preclinical development. Interestingly, the in vitro studies also showed a favorable induction effect of compound 14 on the viability of neuroblastoma SH-SY5Y cells.We report herein a series of water-soluble analogues of previously described anticonvulsants and their detailed in vivo and in vitro characterization. The majority of these compounds demonstrated broad-spectrum anticonvulsant properties in animal seizure models, including the maximal electroshock (MES) test, the pentylenetetrazole-induced seizure model ( Water solubility is one of the essential physicochemical properties from the perspective of formulation development for orally or parenterally  administrated drug candidates . Thus, iscPTZ), as well as the 6 Hz seizure model (32 and 44 mA) in mice. Importantly, compound II (R-enantiomer) vs. I (racemate) was characterized by a distinctly more beneficial safety profile in the rotarod test, a higher metabolic stability on human liver microsomes (HLMs), as well as a less pronounced inhibition of CYP2C9 [Our most recent studies led to the identification of several hybrid compounds belonging to a group of pyrrolidine-2,5-dione derivatives, which proved to be promising candidates for further preclinical development in epilepsy and neuropathic pain indications ,7,8. It Despite the beneficial pharmacological properties of I and II, as well as their close analogues described before ,13,14,15scPTZ, and 6 Hz (32 mA) seizure models. In addition, for the most promising anticonvulsant, its efficacy in the 6 Hz (44 mA) seizure model of drug resistant epilepsy, an influence on the seizure threshold in the ivPTZ test, as well the as anti-nociceptive and anti-inflammatory activity were evaluated. Additionally, as part of the safety profiling for the lead molecule, we determined its influence on motor coordination in the rotarod test, neuromuscular strength, and rectal temperature in mice. We also assessed its in vivo pharmacokinetic profile and several in vitro ADME-Tox parameters, such as the hepatotoxicity, neurotoxicity, and influence on the function of several cytochrome P-450 isoforms (CYP3A4 and CYP2D6), to support the early development of a potential new drug candidate. Finally, we attempted to elucidate the possible mechanisms of action in the binding and functional  studies.All substances obtained in the current studies were tested in vivo for their anticonvulsant activity in the MES, 13–18 were prepared according to the method depicted in N-(tert-butoxycarbonyl)-DL-phenylglycine was coupled in the presence of DCC  with an appropriate phenylpiperazine to give Boc-protected phenylglycine derivatives 1–3. The removal of the Boc-protecting group in the presence of trifluoroacetic acid (TFA) yielded amines 4–6. Next, amines 4–6 were converted to amido-acid  derivatives 7–9 in the condensation reaction with maleic anhydride. The monounsaturated pyrrolidine-2,5-dione derivatives 10–12 were obtained applying the hexamethyldisilazane (HMDS)-promoted cyclization reaction of derivatives 7–9. The final compounds were obtained in the addition reaction of the primary or secondary amine with derivatives 10–12. This reaction was performed in benzene at room temperature. Finally, the water-soluble salts 13–18 were converted into water-soluble hydrochlorides by treating the compound with a 2M methanolic hydrochloric acid solution. The desired compounds were obtained as white solids, followed by the concentration of organic solvents under reduced pressure and wash-up with diethyl ether.The (hydrochlorides) R-configuration of the asymmetric carbon atom (C1-R) located in the phenylacetamide moiety. Notably, this series was restricted only to three diastereoisomers R)-31-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid was used as a starting material -31C1-–33 were diastereoisomers generated by the introduction of amine at the C3 carbon atom of the imide ring ; the assessment of the enantiomeric purity at the C1 carbon atom was determined for the monounsaturated pyrrolidine-2,5-dione derivatives R)-28-31-28-31-31(C1-) was synthesized by applying a similar procedure as for the R-stereoisomers. In this case, the (S)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid was used as the starting material  was performed for all final compounds (these data confirmed the formation of monohydrochlorides). The purity of target compounds determined by the use of the chromatographic UPLC method was ≥99%.The target hydrochlorides were obtained in good yields (>70%). The structures of the final molecules were confirmed by scPTZ tests, remain a routine tool in the search for new antiseizure drugs (ASDs) in view of the high predictability of the assessment of the potential clinical efficacy of candidates for new anticonvulsants [i.p.) administration at a screening dose of 100 mg/kg in mice  and the protection against MES seizures was observed at the pretreatment time point of 0.5 h, similarly to our previous studies [The preclinical animal seizure models, such as the MES, 6 Hz (32 mA or 44 mA), and vulsants . Therefovulsants . In cons studies .14, 15, 17, R)-31(C1- and R)-32-31(C1-) and 3-OCF3 (R)-32(C1-) derivatives provided maximal (100%) protection. Other substances showed weak (25%) or no activity at a dose of 100 mg/kg. An equally important, well-established, and commonly used preclinical seizure model in the discovery of new ASDs effective in human focal epilepsy is the psychomotor 6 Hz (32 mA) test [14, 15, R)-31-32 displayed a potent anticonvulsant efficacy providing 75% protection, whereas 13 and 17 protected 50% of the animals. The further pharmacological investigations were focused on the efficacy assessment in the scPTZ seizure model, which is known to be an animal model of general absence epilepsy [17, R)-31-32) were the most potent compounds and displayed 75% protection at the time point of 0.5 h. Importantly, the detailed in vivo characterization of racemate 14 and its C1 stereoisomers revealed a distinctly weaker protection in all seizure models for compound S)-31-31 diastereoisomer. These observations were in line with data obtained for its close analogues described previously [According to the results obtained in the MES test, at least 75% of protection (three mice protected out of four tested) was demonstrated for compounds C1-R)-32 . NotablymA) test . Therefoepilepsy . In thiseviously ,13.3 -31(C1-) or 3-OCF3 -32(C1-) substituents at the phenylpiperazine analogues revealed broad-spectrum anti-convulsant activity and potent protection in the MES, scPTZ, and 6 Hz (32 mA) seizure models. The replacement of the dimethylamine group attached to the succinimide ring into diethylamine (15) yielded a compound that was predominantly active in the MES and 6 Hz (32 mA) tests. All other structural modifications of this place such as the introduction of methylamine (13) or morpholine (16), the insertion of the trifluoromethyl-thio group -33) at the meta-position of the phenylpiperazine moiety in place of trifluoromethyl or trifluoromethoxy substituents, as well as the inversion of the absolute configuration of the C1 carbon atom located in the phenylacetamide linker (from R to S), decreased the anticonvulsant activity (or caused the compounds to become inactive).In summary, based on the above screening data, four compounds with the dimethylamine group at the pyrrolidine-2,5-dione ring and 3-CF50) in the MES, 6 Hz (32 mA), and scPTZ tests and the median neurotoxic doses (TD50) in the rotarod test were determined for the most effective compounds .In the next step, based on the above screening data, the median effective doses (ED14 (3-CF3 derivative), 17 , and their R stereoisomers at the phenylglycine fragment R)-31(C1- and R)-32 and compounds with the R configuration in the phenylglycine fragment—R)-31(C1- and R)-32 test and, of note, there was a distinct improvement of activity in the scPTZ seizure model for 17 and R)-32(C1- (both 3-OCF3 derivatives) compared to the parent and hardly soluble compounds reported previously .The quantitative data summarized in d before . The hig14, which demonstrated the most beneficial anticonvulsant and safety profile, proved its activity in the 6 Hz (44 mA) test. It should be emphasized that the 6 Hz (44 mA) test is currently one of the most important screening models for the identification and characterization of novel compounds with a potential efficacy in pharmacoresistant epilepsy [14, chemical prototype I , LEV, a14 in the 6 Hz (44 mA) test. It should be stressed that compound 14 was more effective and displayed a better safety profile  than that of VPA, which is one of the most clinically relevant broad-spectrum ASDs [The results obtained revealed a relatively high activity for compound rum ASDs . It is nrum ASDs .14 as a potent and broad-spectrum anticonvulsant for future preclinical development . Notably, compound 14 showed distinctly more potent anticonvulsant activity in the MES, 6 Hz (32/44 mA), and scPTZ models compared to the broad-spectrum ASD, that is, VPA. It should be emphasized that the key chemical modification performed in the current studies allowed to obtain water-soluble salts which were close analogues of hybrid anticonvulsants reported previously [scPTZ seizure models, but, unfortunately, compounds reported herein were simultaneously characterized by a higher motor impairment in the rotarod test that resulted in a lower safety margin expressed as PIs in relation to their chemical precursors I and II  the first myoclonic twitch, (2) generalized clonic seizure with the loss of the righting reflex, and (3) forelimb tonus. The experimental procedure has been described in detail elsewhere [The timed lsewhere .14 on the seizure thresholds in the ivPTZ test is shown in p = 0.041, for myoclonic twitch; F = 7.04, p = 0.003, for generalized clonus; F = 0.72, p = 0.50, for forelimb tonus). Compound 14 administered at a dose of 30 mg/kg did not significantly affect the thresholds for the first myoclonic twitch and generalized clonus. However, when injected at a dose of 60 mg/kg, it slightly increased the thresholds for both the first myoclonic twitch and generalized clonic seizure . No statistically significant changes in the threshold for forelimb tonic extension after compound 14 administration (at 30 and 60 mg/kg) were observed.The acute effect of compound p = 0.053) showed no statistically significant differences between group means, the Dunnett’s post hoc test revealed that compound 14 at a dose of 60 mg/kg significantly decreased the grip strength (p < 0.05). At a lower dose, i.e., 30 mg/kg, compound 14 did not significantly affect the neuromuscular strength in mice  = 3.28,  in mice .14 at doses of 30 and 60 mg/kg did not produce statistically significant changes in body temperature, as determined by the comparison of the differences between the pre-injection and post-injection values of rectal temperature in mice  = 2.00, p = 0.155; Compound 14), the in vivo studies were conducted to confirm its antinociceptive activity in various animal models of pain.Taking into account the fact that ASDs are most often used in the treatment of pain resulting from the damage/dysfunction of the central or peripheral nervous system, for the most potent anticonvulsant  followed by a quiescent period (10 min) and then phase two (15 min). C-fibers’ initial and direct stimulation in phase one led to peripheral inflammation accompanied by ongoing peripheral nerve activity and central sensitization during phase two. Moreover, a formalin injection induces persistent changes similar to those observed in nerve injury and subsequent neuropathic pain . Our pre14. As shown in i.p. administration of compound 14 before the subcutaneous injection of formalin significantly attenuated the nociceptive response in mice in both phases of the test. Its ED50 value in phase I was 85.1 mg/kg, whereas the ED50 value in phase II was 56.1 mg/kg. These results suggested promising analgesic properties of compound 14, which were subsequently confirmed in other pain models.As a continuation of those works, we investigated the analgesic properties of the lead compound 14 on neurogenic pain. In this model, the activation of TRPV1 receptors on C-type primary afferents led to the release of inflammatory neuropeptides, contributing to the local and spinal sensitization and excitability. We found that the duration of pain behaviors in the vehicle-treated mice was 64.36 ± 5.2 s. As shown in 14 attenuated the nociceptive response in a dose-dependent manner with an ED50 value of 54.1 mg/kg. These results showed that compound 14 attenuated the early neurogenic phase of nerve response for the noxious stimulus. It contrasted to VPA, which was active in this test only in a very high dose such as 200 mg/kg, as we showed in our previous paper [We used the capsaicin-induced pain model to test the impact of compound us paper .14 in neuropathic pain, we used the OXPT-induced peripheral neuropathy model. OXPT directly affects neurons, which results in the upregulation of TRPM8 and TRPA1 and the downregulation of Kv4.3 neuronal channels. Moreover, OXPT forms adduct with mitochondrial DNA and, in this way, change the expression of membrane-associated protein such as channels. All these effects finally lead to peripheral and central sensitization [14 on tactile allodynia using the von Frey method 3 h and 7 days after the induction of neuropathy [14, which elevated the pain sensitivity threshold in the early phase in a dose-dependent manner.  and should be further tested in that direction.To investigate the analgesic activity of compound tization ,29. The uropathy . In the  manner.   channels, Cav1.2 calcium channels, as well as the transient receptor potential cation channel vanilloid type 1 (TRPV1) [14 and R)-32, calcium channel Cav1.2 (dihydropyridine site), as well as functional studies including the antagonist effect on Cav1.2 calcium channels and TRPV1 receptors  ,8. Thereeceptors . It shou14 and R)-32 at a concentration of 50 μM. Furthermore, functional assays showed that compound 14 and R)-32-32, and in a generally lower concentration of compound 14 in the brain  vs. serum  calculated for the free base, it may be concluded that anti-seizure activity was probably related with the inhibition of sodium and probably calcium conductance in the CNS. This assumption was confirmed especially by the patch-clamp studies, in which compound 14 significantly inhibited, and in a dose-depended manner, the fast voltage-gated sodium currents starting from the concentration of 0.1 μM . Furthermore, the efficacy of compound 14 in the scPTZ seizure test indicated its inhibitory influence on calcium currents mediated by Cav1.2 channels, as the sodium channels blockers  were ineffective in this seizure model compared to multi-targeted ASDs characterized by interaction with, i.e., calcium channels, e.g., VPA [14, it seems that this activity may have both central (Navx channels) and peripheral  origin that may result from ca. four-fold higher concentration of compound in serum. It should be stressed that the most recent studies identify Cav1.2 channel as a promising molecular target for new analgesics [1.3, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 subtypes) and TRPV1 receptors in nociception and pain stimuli conductance remains unquestionable [According to the results obtained, compounds .g., VPA . Taking algesics ,40. The tionable ,41.14 in the electrically induced seizure models ) and the results of binding studies, suggested its influence on neuronal sodium currents. Consequently, we determined the influence of compound 14 on the fast activating and fast inactivating voltage-gated sodium currents in rat prefrontal cortex pyramidal neurons using the patch-clamp technique. Maximal currents were evoked by rectangular voltage steps to 0 mV from a physiological holding potential of -65 mV. After recording a stable control, compound 14 was applied to the whole bath for three minutes. We found that compound 14 at a concentration of 1 µM inhibited the maximal amplitude of sodium currents , Tukey post hoc test, p < 0.01, n = 5) reported previously [14 similar to compound II was one of the mechanisms providing a similar level of anticonvulsant activity. Moreover, the influence of several additional concentrations of compound 14 on voltage-gated sodium currents were tested to confirm the dose–response relationship. In sum, the following concentrations were applied; 0.01 µM, 0.1 µM, 1 µM, 10 µM, and the results obtained were as follows: 0.95 ± 0.05 , 0.84 ± 0.03 , 0.82 ± 0.05 , and 0.77 ± 0.02 , respectively  seizure models. The amount of the target compound in the serum and brain was determined by an LC-MS/MS system. The concentrations versus time profiles of compound 14 are presented in We performed this study to investigate the pharmacokinetic profile of compound 14 reached the highest concentration at the site of action 15 min after its administration (5.32 ± 0.54 µg/g tissue) and the brain levels slightly dropped to 3.50 ± 0.99 µg/g at the time of in vivo pharmacological testing, i.e., 30 min post-dose.These observations were further confirmed by the values of pharmacokinetic parameters presented in 14. To this purpose, we examined the effect on the activity of CYP3A4 and 2D6 isoforms using commercial luminescent procedures, and on the viability of hepatoma HepG2 and neuroblastoma SH-SY5Y cell lines after a long 72 h treatment with several concentrations of compound 14.The in vitro determination of toxicity of new promising molecules with a therapeutic perspective is a crucial part of the assessment of the drug candidate quality. Drug–drug interactions (DDIs), as well as hepatotoxicity and neurotoxicity, are reported relatively frequently for various clinically used ASDs. Various CYP isoforms were studies, including the induction of CYP3A4, CYP2C9, CYP1A2, and CYP2B6  or the inhibition of CYP2C9 (VPA) ,43,44,45p < 0.0001) inhibited CYP3A4 activity, but only at the highest used doses of 10 µM and 25 µM. Moreover, its activity at 10 µM was higher than 50% , but at much lower concentrations of 1 and 10 µM, respectively . What is more, the similar beneficial effect on neurogenesis was determined previously for compound C11, the pyrrolidine-2,5-dione derivative, which effectively stimulated the viability of astrocytes up to 139.1% of the control [Interestingly, the MTS test demonstrated that compound to 50 µM A. Moreovto 50 µM B. The in control .254 pre-coated aluminum sheets , using the following developing systems: S1–DCM:MeOH ; S2–DCM:MeOH ; S3–DCM:MeOH ). Spots detection: UV light (λ = 254 nm). The UPLC and mass spectra (LC-MS) were obtained on Waters ACQUITY™ TQD system  with the MS-TQ detector and UV–Vis-DAD eλ detector. The ACQUITY UPLC BEH C18, 1.7 μm (2.1 × 100 mm) column was used with the VanGuard Acquity UPLC BEH C18, 1.7 μm (2.1 × 5 mm) . Standard solutions (1 mg/mL) of each compound were prepared in analytical grade MeCN/water mixture . Conditions applied were as follows: eluent A (water/0.1% HCOOH), eluent B (MeCN/0.1% HCOOH), a flow rate of 0.3 mL/min, a gradient of 5–100% B over 10 min, and an injection volume of 10 μL. The UPLC retention times (tR) were given in minutes. Preparative column chromatography was performed using silica gel 60  purchased from Merck . Elemental analyses  for final compounds were carried out by a micro method using the elemental Vario EI III Elemental analyzer . The results of elemental analyses were within ± 0.4% of the theoretical values. The 1H NMR and 13C NMR spectra were obtained in a JEOL-500 spectrometer , in CDCl3 operating at 500 MHz (1H NMR) and 126 MHz (13C NMR). Chemical shifts were reported in δ values (ppm) relative to TMS δ = 0 (1H), as internal standard. The J values were expressed in Hertz (Hz). Signal multiplicities were represented by the following abbreviations: s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), dt (doublet of triplets), t (triplet), td (triplet of doublets), q (quartet), m (multiplet). Enantiomeric purity was determined using a chiral HPLC technique on a Shimadzu Prominence LC-2030C SD Plus system  equipped with an Amylose-C (250 × 4.6 mm) chiral column. The analysis was performed under the following conditions: column temperature: 35 °C; mixture of eluents: hexane/i-PrOH = 95/5 (v/v); flow rate: 1 mL/min; injection volume: 10 μL; analysis time: 100 min. (isocratic); detection at the wavelength λ = 207 nm. Enantiomeric purity was expressed in %. For diastereomers, analyses were performed applying the following conditions: column temperature: 54 °C; mixture of eluents: hexane/i-PrOH = 95.2/4.8 (v/v); flow rate: 2.1 mL/min; injection volume: 10 μL; analysis time: 70 min. (isocratic); detection at the wavelength λ = 207 nm.All chemicals and solvents were purchased from commercial suppliers and were used without further purification. Melting points (mp.) were determined in open capillaries on a BÜCHI 353 melting point apparatus . TLC and the gradient UPLC chromatography were used to assess the purity and homogeneity of the compounds. TLC was carried out on silica gel 60 FR,S), (R) or (S)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid  was successively added DCC  dissolved in 5 mL of DCM. After stirring (15 min), the appropriate phenylpiperazine  dissolved in 5 mL of DCM was added dropwise and the reaction was stirred at room temperature for 4 h. The DCM was evaporated in vacuo, and the product purified by column chromatography using a DCM:MeOH–9:0.3 (v/v) mixture as a solvent system.To the DCM (20 mL) solution of -(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)carbamate (1).f = 0.54 (S1); UPLC (purity: >99%): tR = 8.41 min. C24H28F3N3O3 (463.50). LC-MS (ESI): m/z calcd for C24H28F3N3O3 (M+H)+ 464.21, found 464.2.Light oil. Yield: 85% (1.97 g); TLC: RTert-butyl--(2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)carbamate (2).f = 0.55 (S1); UPLC (purity: >99%): tR = 8.85 min. C24H28F3N3O4 (479.50). LC-MS (ESI): m/z calcd for C24H28F3N3O4 (M+H)+ 480.20, found 480.1.Light oil. Yield: 82% (1.86 g); TLC: RTert-butyl--(2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)carbamate (3). f = 0.68 (S2); UPLC (purity: >99%): tR = 8.67 min. C24H28F3N3O3S (495.56). LC-MS (ESI): m/z calcd for C24H28F3N3O3S (M+H)+ 496.18, found 496.2.Light oil. Yield: 84% (1.89 g); TLC: RTert-butyl-(R)-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) carbamate ((RC1-)-19). f = 0.62 (S2); UPLC (purity: >99%): tR = 8.40 min. C24H28F3N3O3 (463.50). LC-MS (ESI): m/z calcd for C24H28F3N3O3 (M+H)+ 464.21, found 464.2.Light oil. Yield: 87% (2.02 g); TLC: RTert-butyl-(S)-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) carbamate ((SC1-)-19). f = 0.62 (S2); UPLC (purity: 100%): tR = 8.40 min. C24H28F3N3O3 (463.50). LC-MS (ESI): m/z calcd for C24H28F3N3O3 (M+H)+ 464.21, found 464.1.Light oil. Yield: 87% (2.12 g); TLC: RTert-butyl-(R)-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)carbamate ((RC1-)-20). f = 0.65 (S2); UPLC (purity: >99%): tR = 8.51 min. C24H28F3N3O4 (479.50). LC-MS (ESI): m/z calcd for C24H28F3N3O4 (M+H)+ 480.20, found 480.2.Light oil. Yield: 87% (2.07 g); TLC: RTert-butyl-(R)-(2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)carbamate ((RC1-)-21).f = 0.70 (S2); UPLC (purity: >99%): tR = 8.65 min. C24H28F3N3O3S (495.56). LC-MS (ESI): m/z calcd for C24H28F3N3O3S (M+H)+ 496.18, found 496.2.Light oil. Yield: 79% (1.68 g); TLC: R1–3, (RC1-)-19–(RC1-)S-21 or (C1-)-19  was treated with TFA  and stirred at room temperature for 5 h. Next, DCM was evaporated in vacuo. The oil residue obtained was suspended in water (20 mL) and 25% ammonium hydroxide was carefully added to pH = 8. The aqueous phase was extracted using DCM (3 × 20 mL), dried over anhydrous Na2SO4, and concentrated to dryness. Compounds 4–6, (R)-22C1-–(R)-24C1- and (S)-22C1- were obtained as an oil and used for further reactions without purification.The DCM (5 mL) solution of R,S)-2-Amino-2-phenyl-1-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethan-1-one (4). (tR = 4.97 min. C19H20F3N3O (363.38). LC-MS (ESI): m/z calcd for C19H20F3N3O (M+H)+ 364.16, found 364.1.Light oil. Yield: 96% (1.12 g); UPLC (purity: >99%): R,S)-2-Amino-2-phenyl-1-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethan-1-one (5). (tR = 4.63 min. C19H20F3N3O2 (379.38). LC-MS (ESI): m/z calcd for C19H20F3N3O2 (M+H)+ 380.15, found 380.2.Light oil. Yield: 96% (1.15 g); UPLC (purity: >99%): R,S)-2-Amino-2-phenyl-1-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethan-1-one (6). (tR = 5.05 min. C19H20F3N3OS (395.44). LC-MS (ESI): m/z calcd for C19H20F3N3OS (M+H)+ 369.13, found 396.2.Light oil. Yield: 94% (1.03 g); UPLC (purity: >99%): R)-2-Amino-2-phenyl-1-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethan-1-one (RC1-)-22. (tR = 4.98 min. C19H20F3N3O (363.38). LC-MS (ESI): m/z calcd for C19H20F3N3O (M+H)+ 364.16, found 364.2.Light oil. Yield: 94% (1.09 g); UPLC (purity: >99%): S)-2-Amino-2-phenyl-1-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethan-1-one (SC1-)-22. (tR = 4.97 min. C19H20F3N3O (363.38). LC-MS (ESI): m/z calcd for C19H20F3N3O (M+H)+ 364.16, found 364.3.Light oil. Yield: 95% (1.10 g); UPLC (purity: >99%): R)-2-Amino-2-phenyl-1-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethan-1-one (RC1-)-23. (tR = 4.63 min. C19H20F3N3O2 (379.38). LC-MS (ESI): m/z calcd for C19H20F3N3O2 (M+H)+ 380.15, found 380.2.Light oil. Yield: 95% (1.13 g); UPLC (purity: >99%): R)-2-Amino-2-phenyl-1-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethan-1-one (RC1-)-24. (tR = 5.03 min. C19H20F3N3OS (395.44). LC-MS (ESI): m/z calcd for C19H20F3N3OS (M+H)+ 396.13, found 396.2.Light oil. Yield: 96% (1.16 g); UPLC (purity: >99%): 4–6, (RC1-)-22–(RC1-)-24 or (SC1-)-22  in AcOEt (50 mL) and stirred for 30 min. After this time, the solvent was distilled off to dryness. The compound was obtained as solid after washing with diethyl ether (Et2O).Maleic anhydride  was added to a solution of R,S)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) amino)but-2-enoic acid (7). (tR = 6.90 min. LC-MS (ESI): m/z calcd for C23H22F3N3O4 (M+H) + 462.44, found 462.2.White solid. Yield: 82% (3.62 g); UPLC (purity: 98%): R,S)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)amino)but-2-enoic acid (8). (tR = 7.04 min. LC-MS (ESI): m/z calcd for C23H22F3N3O5 (M+H) + 478.15, found 478.2.White solid. Yield: 84% (3.68 g); UPLC (purity: 99%): R,S)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)amino)but-2-enoic acid (9). (tR = 7.12 min. LC-MS (ESI): m/z calcd for C23H22F3N3O4S (M+H) + 494.13, found 494.1.White solid. Yield: 86% (3.71 g); UPLC (purity: 98%): R)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) amino)but-2-enoic acid (RC1-)-25. (tR = 6.92 min. LC-MS (ESI): m/z calcd for C23H22F3N3O4 (M+H) + 462.44, found 462.2.White solid. Yield: 85% (3.76 g); UPLC (purity: 96%): S)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) amino)but-2-enoic acid (SC1-)-25. (tR = 6.92 min. LC-MS (ESI): m/z calcd for C23H22F3N3O4 (M+H) + 462.44, found 462.1.White solid. Yield: 87% (3.89 g); UPLC (purity: 98%): R)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)amino)but-2-enoic acid (RC1-)-26. (tR = 7.05 min. LC-MS (ESI): m/z calcd for C23H22F3N3O5 (M+H) + 478.15, found 478.2.White solid. Yield: 82% (3.43 g); UPLC (purity: 99%): R)-4-Oxo-4-((2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)amino)but-2-enoic acid (RC1-)-27. (tR = 7.10 min. LC-MS (ESI): m/z calcd for C23H22F3N3O4S (M+H) + 494.13, found 494.1.White solid. Yield: 87% (3.75 g); UPLC (purity: 97%): 2  was added to the suspension of 7–9, (RC1-)-25–(RC1-)-27 or (SC1-)-25  in dry benzene (100 mL), and the mixture was heated to 80 °C. Then, the solution of HMDS  in dry benzene (10 mL) was added dropwise over 30 minutes. The reaction was continued with stirring in reflux for about 24 h, then cooled and concentrated under reduced pressure. After distilling off the solvent, the oily residue was dissolved in DCM and extracted with 0.1 M HCl (3 × 50 mL), water (3 × 50 mL), and saturated NaCl solution (3 × 50 mL). The organic layer was dried over anhydrous Na2SO4 and then evaporated to dryness. The crude product was purified by column chromatography. The compound was obtained as solid after washing with Et2O.ZnClR,S)-1-(2-Oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (10). (tR = 7.45 min. LC-MS (ESI): m/z calcd for C23H20F3N3O3 (M+H)+ 444.15 found 444.1.White solid. Yield: 79% (3.34 g); UPLC (purity: >99%) R,S)-1-(2-Oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (11). (tR = 7.51 min. LC-MS (ESI): m/z calcd for C23H20F3N3O4 (M+H)+ 460.14 found 460.2.White solid. Yield: 75% (3.11 g); UPLC (purity: >99%) R,S)-1-(2-Oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (12). (tR = 7.54 min. LC-MS (ESI): m/z calcd for C23H20F3N3O4S (M+H)+ 476.12 found 476.1.White solid. Yield: 79% (3.34 g); UPLC (purity: >99%) R)-1-(2-Oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (RC1-)-28. (tR = 7.44 min. LC-MS (ESI): m/z calcd for C23H20F3N3O3 (M+H)+ 444.15 found 444.1. Chiral HPLC > 99% ee (tR = 76.57 min).White solid. Yield: 74% (3.12 g); UPLC (purity: >99%) S)-1-(2-Oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (SC1-)-28. (tR = 7.42 min. LC-MS (ESI): m/z calcd for C23H20F3N3O3 (M+H)+ 444.15 found 444.1. Chiral HPLC > 99% ee (tR = 70.94 min).White solid. Yield: 82% (3.42 g); UPLC (purity: >99%) R)-1-(2-Oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (RC1-)-29. (tR = 7.52 min. LC-MS (ESI): m/z calcd for C23H20F3N3O4 (M+H)+ 460.14 found 460.2. Chiral HPLC > 99% ee (tR = 74.20 min).White solid. Yield: 78% (3.16 g); UPLC (purity: >99%) R)-1-(2-Oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)-1H-pyrrole-2,5-dione (RC1-)-30. (tR = 7.54 min. LC-MS (ESI): m/z calcd for C23H20F3N3O4S (M+H)+ 476.12 found 476.1. Chiral HPLC > 99% ee (tR = 78.82 min).White solid. Yield: 81% (3.45 g); UPLC (purity: >99%) 10–12, (RC1-)-28–(RC1-)-30, or (SC1-)-28  in dry benzene (50 mL). The reaction was continued with stirring for about 8 h, then concentrated under reduced pressure. The crude product was purified by column chromatography. The compound was then converted into the hydrochloride salt by treating the compound with a 2M methanolic hydrochloric acid solution. The compound was obtained as solid after washing with Et2O.The respective primary or secondary amine solution in THF  was added to a solution of 13). 3-(Methylamino)-1-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.42 (S3); UPLC (purity: >99%): tR = 5.49 min. LC-MS (ESI): m/z calcd for C24H25F3N4O3 (M+H)+ 475.48, found 475.3; 1H NMR  δ 2.47 , 2.55 , 2.69–2.77 , 2.95 , 3.04–3.17 , 3.23–3.39 , 3.68 , 4.00 , 6.10 , 6.94–6.98 , 7.01 , 7.09 , 7.30–7.38 , 7.40–7.44 ; 13C NMR  δ 29.8, 33.5, 42.4, 45.7, 48.5, 48.8, 56.8, 57.3, 112.8, 116.9, 119.3, 124.2 , 128.9, 129.2, 129.8, 129.9, 131.6 , 132.6, 150.9, 165.0, 174.4, 177.3. Anal. calcd for C24H26ClF3N4O3 (510.94): C: 56.42, H: 5.13, N: 10.97; Found C: 56.30, H: 5.27, N: 10.81.White solid. Yield: 87% (0.91 g); mp. 161.2–163.4 °C; TLC: R14). 3-(Dimethylamino)-1-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)-piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.49 (S3); UPLC (purity: >99%): tR = 5.59 min. LC-MS (ESI): m/z calcd for C25H27F3N4O3 (M+H)+ 489.20, found 489.3; 1H NMR  δ 2.41 , 2.61 , 2.68–2.74 , 2.84 , 3.04–3.08 , 3.12 , 3.24–3.36 , 3.66–3.71 , 3.74 , 3.97–4.04 , 6.13 , 6.96 , 7.01 , 7.08 , 7.29–7.38 , 7.40–7.43 ; 13C NMR  δ 31.1, 41.4, 42.4, 45.7, 48.5, 48.7, 56.7, 62.3, 112.8, 116.8, 119.3, 124.2 , 128.9, 129.1, 129.8, 131.6 , 132.8, 150.9, 165.0, 174.5, 175.7. Anal. calcd for C25H28ClF3N4O3 (524.97): C: 57.20, H: 5.38, N: 10.67; Found C: 57.30, H: 5.29, N: 10.84.White solid. Yield: 83% (0.90 g); mp. 157.8–159.2 °C; TLC: R15). 3-(Diethylamino)-1-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.52 (S3); UPLC (purity: >99%): tR = 5.79 min. LC-MS (ESI): m/z calcd for C27H31F3N4O3 (M+H)+ 517.23, found 517.3; 1H NMR  δ 1.07 , 2.55–2.63 , 2.69–2.76 , 2.86 , 3.06 , 3.10–3.15 , 3.26–3.35 , 3.67–3.71 , 3.98 , 4.00–4.07 , 6.13 , 6.96 , 7.01 , 7.09 , 7.30–7.37 , 7.40–7.43 .13C NMR  δ 13.8, 32.2, 42.4, 44.9, 45.7, 48.5, 48.8, 56.7, 58.8, 112.8, 116.8, 119.3, 124.2 , 128.8, 129.1, 129.8, 131.6, 132.8, 150.9, 165.1, 174.7, 176.7. Anal. calcd for C27H32ClF3N4O3 (553.02): C: 58.64, H: 5.83, N: 10.13; Found C: 58.78, H: 5.69, N: 10.38.White solid. Yield: 88% (1.00 g); mp. 142.2–143.1 °C; TLC: R16). 3-Morpholino-1-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.58 (S3); UPLC (purity: >99%): tR = 6.75 min. LC-MS (ESI): m/z calcd for C27H29F3N4O4 (M+H)+ 531.21, found 531.4; 1H NMR  δ 2.61–2.67 , 2.68–2.73 , 2.79 , 2.87 , 3.04–3.08 , 3.13 , 3.24–3.37 , 3.65–3.73 , 4.01 , 6.13 , 6.96 , 7.01 , 7.09 , 7.30–7.38 , 7.40–7.43 ; 13C NMR  δ 31.0, 45.7, 48.5, 48.8, 49.4, 56.8, 62.3, 67.0, 112.9, 116.9, 119.3, 124.2 , 128.9, 129.2, 129.8, 129.9, 131.6 , 132.6, 150.9, 165.0, 174.3, 175.1. Anal. calcd for C27H30ClF3N4O4 (567.01): C: 57.19, H: 5.33, N: 9.88; Found C: 57.31, H: 5.51, N: 10.08.White solid. Yield: 84% (0.99 g); mp. 146.2–147.5 °C; TLC: R17). 3-Dimethylamino-1-(2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.62 (S3); UPLC (purity: >99%): tR = 5.90 min. LC-MS (ESI): m/z calcd for C25H27F3N4O4 (M+H)+ 505.20, found 505.3 1H NMR  δ 2.43 , 2.63 , 2.67–2.71 , 2.85 , 3.00–3.06 , 3.07–3.14 , 3.24–3.34 , 3.68 , 3.76 , 3.99 , 6.13 , 6.61 , 6.69–6.73 , 7.21 , 7.33–7.38 , 7.40–7.42 ; 13C NMR  δ 31.1, 41.4, 42.3, 45.7, 48.4, 48.7, 56.7, 62.3, 108.9, 112.2, 114.3, 120.4 , 128.9, 129.1, 129.8, 130.3, 132.8, 150.3, 152.0, 165.0, 174.4, 175.6. Anal. calcd for C25H28ClF3N4O4 (540.97): C: 55.51, H: 5.22, N: 10.36; Found C: 55.78, H: 5.14, N: 10.49.White solid. Yield: 82% (0.96 g); mp. 152.3–153.5 °C; TLC: R18). 3-Dimethylamino-1-(2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (f = 0.65 (S3); UPLC (purity: >99%): tR = 6.08 min. LC-MS (ESI): m/z calcd for C25H27F3N4O3S (M+H)+ 521.18, found 521.1; 1H NMR  δ 2.42 , 2.61 , 2.67–2.72 , 2.84 , 3.01–3.07 , 3.09–3.15 , 3.25–3.35 , 3.65–3.72 , 3.74 , 3.97–4.03 , 6.13 , 6.91 , 7.05 , 7.12 , 7.24–7.28 , 7.33–7.38 , 7.40–7.42 ; 13C NMR  δ 31.1, 41.4, 42.4, 45.7, 48.4, 48.7, 56.7, 62.3, 118.5, 123.7, 127.8, 128.8 , 128.9, 129.1, 129.8, 130.1, 132.8, 151.3, 165.0, 174.5, 175.7. Anal. calcd for C25H28ClF3N4O3S (557.03): C: 53.91, H: 5.07, N: 10.06 Found: C: 53.82, H: 5.14, N: 10.32.White solid. Yield: 81% (0.97 g); mp. 170.1–171.5 °C; TLC: RR)-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)-piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (RC1-)-31. 3-Dimethylamino-1-((f = 0.49 (S3); UPLC (purity: >99%): tR = 5.73 min. LC-MS (ESI): m/z calcd for C25H27F3N4O3 (M+H)+ 489.20, found 489.4; 1H NMR  δ 2.42 , 2.61 , 2.67–2.74 , 2.84 , 3.03–3.09 , 3.13 , 3.24–3.36 , 3.65–3.72 , 3.74 , 3.97–4.04 , 6.13 , 6.96 , 7.01 , 7.09 , 7.30–7.37 , 7.40–7.43 ; 13C NMR  δ 31.1, 41.4, 42.4, 45.7, 48.5, 48.8, 56.7, 62.3, 112.8, 116.8, 119.3, 124.2 , 128.9, 129.1, 129.8, 131.6 , 132.8, 150.9, 165.0, 174.5, 175.8. Anal. calcd for C25H28ClF3N4O3 (524.97): C: 57.20, H: 5.38, N: 10.67; Found: C: 57.27, H: 5.42, N: 10.59.White solid. Yield: 85% (0.92 g); mp. 169.8–170.6 °C; TLC: RS)-oxo-1-phenyl-2-(4-(3-(trifluoromethyl)phenyl)-piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (SC1-)-31. 3-Dimethylamino-1-((f = 0.49 (S3); UPLC (purity: >99%): tR = 5.53 min. LC-MS (ESI): m/z calcd for C25H27F3N4O3 (M+H)+ 489.20, found 489.2; 1H NMR  δ 2.42 , 2.61 , 2.67–2.74 , 2.84 , 3.03–3.09 , 3.13 , 3.24–3.37 , 3.65–3.72 , 3.74 , 3.97–4.04 , 6.13 , 6.96 , 7.01 , 7.09 , 7.30–7.38 , 7.40–7.43 ; 13C NMR  δ 31.1, 41.4, 42.4, 45.7, 48.5, 48.8, 56.7, 62.3, 112.8, 116.8, 119.3, 124.2 , 128.9, 129.1, 129.8, 131.6 , 132.8, 150.9, 165.0, 174.5, 175.8. Anal. calcd for C25H28ClF3N4O3 (524.97): C: 57.20, H: 5.38, N: 10.67; Found: C: 57.31, H: 5.27, N: 10.72.White solid. Yield: 88% (0.95 g); mp. 169.2–170.8 °C; TLC: RR)-2-oxo-1-phenyl-2-(4-(3-(trifluoromethoxy)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (RC1-)-32. 3-Dimethylamino-1-((f = 0.62 (S3); UPLC (purity: >99%): tR = 5.91 min. LC-MS (ESI): m/z calcd for C25H27F3N4O4 (M+H)+ 505.20, found 505.3; 1H NMR  δ 2.42 , 2.62 , 2.67–2.72 , 2.85 , 3.01–3.05 , 3.09–3.13 , 3.24–3.34 , 3.65–3.70 , 3.75 , 3.96–4.02 , 6.13 , 6.61 , 6.68–6.73 , 7.21 , 7.33–7.38 , 7.40–7.42 ; 13C NMR  δ 31.1, 41.4, 42.3, 45.6, 48.3, 48.7, 56.7, 62.3, 108.9, 112.2, 114.3, 120.5 , 128.9, 129.1, 129.8, 130.3, 132.8, 150.3, 152.0, 165.0, 174.5, 175.7. Anal. calcd for C25H28ClF3N4O4 (540.97): C: 55.51, H: 5.22, N: 10.36; Found: C: 55.62, H: 5.18, N: 10.28.White solid. Yield: 79% (0.92 g); mp. 164.8–165.7 °C; TLC: RR)-2-oxo-1-phenyl-2-(4-(3-((trifluoromethyl)thio)phenyl)piperazin-1-yl)ethyl)pyrrolidine-2,5-dione hydrochloride (RC1-)-33. 3-Dimethylamino-1-((f = 0.65 (S3); UPLC (purity: >99%): tR = 6.08 min. LC-MS (ESI): m/z calcd for C25H27F3N4O3S (M+H)+ 521.18, found 521.1; 1H NMR  δ 2.42 , 2.61 , 2.68–2.73 , 2.84 , 3.01–3.07 , 3.11 , 3.24–3.36 , 3.66–3.71 , 3.75 , 3.96–4.03 , 6.13 , 6.89–6.93 , 7.05 , 7.12 , 7.24–7.27 , 7.31–7.38 , 7.40–7.42 ; 13C NMR  δ 31.1, 41.4, 42.4, 45.7, 48.4, 48.7, 56.7, 62.3, 118.5, 123.7, 127.8, 128.9 , 129.1, 129.8, 130.1, 132.8, 151.4, 165.0, 174.5, 175.8. Anal. calcd for C25H28ClF3N4O3S (557.03): C: 53.91, H: 5.07, N: 10.06; Found: C: 53.82, H: 5.14, N: 10.32.White solid. Yield: 79% (0.94 g); mp. 173.2–174.7 °C; TLC: R50 or TD50 values, 3–4 groups of six animals were injected with various doses of tested compounds. All procedures involving animals and their care were performed in accordance with the current European Union Directive of 22 September 2010 (2010/63/EU) and Polish legislation on animal experimentation. The studies were carried out under experimental protocols approved by the I Local Ethical Committee for Experiments on Animals of the Jagiellonian University in Krakow, Poland . All substances were suspended in Tween 80 (1% aqueous solution) and administered i.p. as a single injection at a dose of 10 mL/kg body weight. On each day of experimentation, fresh solutions were prepared. In this study, we used adult male CD-1 mice  that weighed between 22 and 26 g. They were housed under standardized housing conditions in colony cages and had free access to food, as well as tap water. Four mice per group were randomly assigned to each experimental group  with each mouse being used only once. To evaluate the EDscPTZ) [The detailed in vivo procedures were described elsewhere; maximal electroshock seizure test (MES) , subcutascPTZ) , the 6 HscPTZ) , the rotscPTZ) .50 and TD50 values with 95% confidence limits were calculated by probit analysis [50 value, as determined in the rotarod test, by the respective ED50 value, as determined in the MES, scPTZ, or 6 Hz (32 mA or 44 mA) tests. The protective index was considered as an index of the margin of safety and tolerability between anticonvulsant doses and doses of the compounds exerting acute adverse effects such as sedation, motor coordination impairment, ataxia, or other neurotoxic manifestations.The EDanalysis . The pro14 on neuromuscular strength, body temperature, and seizure threshold, compound 14 was suspended in a 1% solution of Tween 80 and administered i.p., 30 min before the tests. In this study, we used adult male Swiss albino mice  license no. 106/2019 . In studies assessing the acute effect of compound ivPTZ test was employed to evaluate the acute effect of compound 14 on the seizure thresholds for (1) the first myoclonic twitch, (2) generalized clonic seizure with loss of righting reflex, and (3) forelimb tonus. The experimental procedure was described in detail elsewhere [The timed lsewhere . The cha14 or vehicle and 30 min later. The difference between the pre- and post-injection temperature value was then calculated (Δ °C). The probe remained in the rectum for several second, allowing for temperature stabilization. The temperature was recorded just before injection of compound 14 on neuromuscular strength was quantified using the grip-strength apparatus  according to the method described elsewhere [The acute effect of compound lsewhere .The experiments were conducted on adult male Albino Swiss mice  and male Wistar rats (Krf:(WI) WU), 180–250 g) as described previously . The minp < 0.05. The log-probit method was applied to statistically determine the ED50 values with 95% confidence limitsData were presented as means ± standard error of the mean (S.E.M). The Graph Pad Prism 8.0.1 was used to analyze the vast majority of data. Statistically significant differences between groups were calculated using one-way analysis of variance (ANOVA) and the post hoc Dunnett’s multiple comparison test or two-way analysis of variance (ANOVA) and the post hoc Tukey’s comparison when appropriate. The criterion for significance was set at i.p. at a dose of 40 mg/kg. The mice were sacrificed by decapitation under isoflurane anesthesia. Blood samples were collected at 5, 15, 30, 60, 120, 240, and 480 min after dosing and brains were harvested at the same time points. Blood was allowed to clot at room temperature for 20 min and serum was separated by centrifugation at 3000 rpm for 10 min . The samples were stored at −80 °C until analysis. All animal procedures were approved by the First Ethical Committee on Animal Experimentation in Krakow, Poland (no. 270/2019). Male CD-1 mice weighting 28–33 g housed in conditions of the constant temperature with a 12:12 h light–dark cycle with free access to food and water were used in this study. The investigated compound was dissolved in sterile water for injection  and administered m/z 489 to 132 (CE = 65 eV) and m/z 489 to 231 (CE = 29 eV) for compound 14  and m/z 436 to 235 (CE = 42 eV) for an internal standard . The mass spectrometric conditions were optimized by continuous infusion of the standard solution at the rate of 7 μL/min using a Harvard infusion pump. The ion source temperature was maintained at 450 °C and the ion spray voltage was set at 5500 V. The curtain gas (CUR) was set at 40 psi and the collision gas (CAD) at medium. Data acquisition and processing were accomplished using the Analyst version 1.7 software. The calibration curves were constructed by plotting the ratio of the peak area of the studied compound to internal standard (IS) versus drug concentration and generated by weighted (1/x·x) linear regression analysis. The validated quantitation ranges for this method were within the expected concentration ranges, namely, from 0.1 to 20 µg/mL of serum and from 0.05 to 10 µg/g of brain tissue with accuracy from 93.73 to 104.29% and from 88.33 to 107% for serum and brain, respectively. No significant matrix effect was observed and there were no stability-related problems during the routine analysis of the samples. Concentrations of compound 14 in mouse brains and serum were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) method using SCIEX QTRAP 4500 triple quadrupole mass spectrometer coupled to Excion LC AC HPLC system . Chromatographic separation was carried out on the Hypersil GoldTM C18 analytical column  with the oven temperature set at 40 °C. The mobile phase containing 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B) was delivered at the flow rate of 0.4 µL/min in the gradient mode. The initial mobile phase composition was 95% B for the first 2 min with a linear gradient to 5% B in the next 2 min, then isocratic mode for 2 min with the following rapid change back to 95% B in 0.1 min. The remaining time of elution was set at 95% B. The whole HPLC operation lasted 10 min. Electrospray ionization (ESI) in the positive ion mode was used for ion production. The tandem mass spectrometer was operated at unit resolution in the selected reaction monitoring mode (SRM), monitoring the transition of the protonated molecular ions 14 was prepared in methanol at the concentration of 1 mg/mL. Working standard solutions were prepared in the same solvent, by the serial dilution of the stock solution, at the following concentrations: 1, 2.5, 5, 10, 50, 100, and 200 µg/mL for serum analysis and 0.1, 0.25, 0.5, 1, 5, 10, and 20 µg/mL for analysis of brain homogenates. To prepare samples for calibration curve, 45 μL of matrix (plasma or brain homogenate) was spiked with 5 μL of standards working solution at appropriate concentration level and vortexed for 10 s.The stock solution of compound w/v) with a tissue homogenizer LabGEN 125 . Brain homogenates or serum samples (50 µL) were deproteinized with 150 μL of 0.1% formic acid in acetonitrile with an addition of internal standard, shaken for 10 min  and then centrifuged for 5 min at the speed of 8000× g . Serum supernatant was additionally diluted 10 times with the deproteinization solution. Then, brain or serum supernatants were transferred into the autosampler vials. The autosampler temperature was maintained at 15 °C and a sample volume of 2 μL was injected into the LC-MS/MS system.The brains were homogenized in distilled water at the ratio of 1:4  and Eurofins Panlabs Discovery Services Taiwan, Ltd.  using testing procedures reported previously. The general information is listed in 2 (2), and Na2ATP (4) at pH 7,4. The composition of extracellular solution was (in mM) NaCl (30), choline chloride (90), TEA-Cl (30), CaCl2 (2), MgCl2 (2), glucose (15), HEPES (10), LaCl3 (0.001), and CdCl2 (0.4) at pH 7.4. Compound 14 was tested at concentration of 1 µM.The methodology of slice preparation, slice preincubation, and patch-clamp technique was the same as in our previous study . In this14’s toxicity were described previously. The luminescent CYP3A4 P450-Glo™ and CYP2D6 P450-Glo™ assays and protocols were provided by Promega® . Compound 14 was tested in triplicate at the final concentrations from 0.1 to 25 μM. Hepatoma HepG2 (ATCC® HB-8065™) and neuroblastoma SH-SY5Y (ATCC® CRL-2266™) cell lines were obtained directly from ATCC® . The CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS) was purchased from Promega . Compound 14 was incubated with cells in four repetitions in the concentration range 0.1–100 μM. The additional test was performed with SH-SY5Y cells in the concentration range 0.001–1 µM. During the toxicity examination, the luminescent signal and the absorbance (490 nm) were measured by using a microplate reader EnSpire PerkinElmer .All applied in vitro methods for assessment of compound scPTZ seizure tests. The most beneficial anticonvulsant properties and safety profile revealed compound 14. In addition, compound 14 showed potent effectiveness by decreasing pain responses in the formalin-induced tonic pain, the capsaicin-induced neurogenic pain, as well as in the OXPT-induced neuropathic pain in mice. Notably, compound 14 also displayed anti-inflammatory activity in the model of carrageenan-induced aseptic inflammation. The in vitro ADME-Tox studies revealed that compound 14 inhibited CYP3A4 activity, but only at the highest used concentration, showed negligible influence on CYP2D6, had a very low hepatotoxic potential, and notably increased the viability of SH-SY5Y cells. Overall, the obtained results indicated that compound 14 seems to be an interesting candidate for further and more advanced preclinical development in epilepsy and, potentially, neuropathic pain.In the current study, we generated a series of water-soluble hydrochlorides of the pyrrolidine-2,5-dione derivatives targeted as potential anticonvulsants with additional antinociceptive properties. The preclinical data in mice revealed that these compounds exhibited potent protection and showed broad-spectrum activity in different animal models of seizures, such as the MES, 6 Hz (32/44 mA), and/or The authors declare no competing financial interest. The results are subject of the international WIPO patent application, no. WO 2020/145831 (publication date 16 July 2020).