Blarcamesine is a promising investigational drug for the treatment of Alzheimer's disease. The international nonproprietary name blarcamesine refers to a racemic compound, although it seems likely that it will be marketed in an enantiopure form. A resolution process has been described in the literature, but the absolute configurations of the enantiomers have not yet been disclosed. In the present study, crystals of (R)-(-)- and (S)-(+)-mandelate salts of (+)- and (-)-blarcamesine and also that of (R)-(+)-blarcamesine itself, suitable for single-crystal X-ray diffraction measurement were prepared and the absolute configurations of (+)- and (-)-blarcamesine have been determined

Dancsó, András

Lozsi, Károly

Simig, Gyula

Szlávik, László

Szokol, Zsuzsanna

Virág, Attila

Volk, Balázs

English

Periodica Polytechnica (Budapest University of Technology and Economics)

536|https://doi.org/10.3311/PPch.20662Creative Commons Attribution bPeriodica Polytechnica Chemical Engineering, 66(4), pp. 536–540, 2022Cite this article as: Szokol, Z., Lozsi, K., Virág, A., Dancsó, A., Szlávik, L., Simig, G., Volk, B. "Assignment of Absolute Configuration to Enantiomers of Anti-Alzheimer Drug Candidate Blarcamesine", Periodica Polytechnica Chemical Engineering, 66(4), pp. 536–540, 2022. https://doi.org/10.3311/PPch.20662Assignment of Absolute Configuration to Enantiomers of  Anti-Alzheimer Drug Candidate BlarcamesineZsuzsanna Szokol1, Károly Lozsi1, Attila Virág2, András Dancsó1, László Szlávik1, Gyula Simig1, Balázs Volk1*1 Directorate of Drug Substance Development, Egis Pharmaceuticals Plc., H-1475 Budapest, P. O. B. 100, Hungary2 Directorate of Finished Product Development, Egis Pharmaceuticals Plc., H-1475 Budapest, P. O. B. 100, Hungary* Corresponding author, e-mail: volk.balazs@egis.huReceived: 16 June 2022, Accepted: 19 July 2022, Published online: 30 August 2022AbstractBlarcamesine is a promising investigational drug for the treatment of Alzheimer's disease. The international nonproprietary name blarcamesine refers to a racemic compound, although it seems likely that it will be marketed in an enantiopure form. A resolution process has been described in the literature, but the absolute configurations of the enantiomers have not yet been disclosed. In the present study, crystals of (R)-(-)- and (S)-(+)-mandelate salts of (+)- and (-)-blarcamesine and also that of (R)-(+)-blarcamesine itself, suitable for single-crystal X-ray diffraction measurement were prepared and the absolute configurations of (+)- and (-)-blarcamesine have been determined.Keywordsblarcamesine, resolution, crystallization, absolute configuration, single-crystal X-ray diffraction1 IntroductionBlarcamesine (ANAVEX2-73, 1, Fig. 1) is an experimen-tal drug of Anavex Life Sciences acting as a σ1 receptor agonist and muscarinic receptor modulator. It is in human Phase III clinical stage, under development for the treat-ment of Alzheimer's disease, and neuroprotective and neu-rodevelopmental disorders [1–14].Blarcamesine is a racemic compound. In a patent appli-cation [15] the originator disclosed the advantageous pharmacological properties of the (-)-enantiomer over the (+)-enantiomer therefore launch of an enantiopure drug can be anticipated. Several methods for the preparation of enantiomers of blarcamesine (1) are known from the liter-ature [16]. The resolution of compound 1 (Scheme 1) was carried out with mandelic acid enantiomers [(R)-(-)-2 and (S)-(+)-2] resulting in diastereomeric salts (+)- and (-)-3. The enantiomers of 1 were liberated from the diastereo-meric salts 3 and obtained as solids after evaporation of ethereal solutions to dryness.Fig. 1 Structure of blarcamesine Scheme 1 Resolution of blarcamesine (1) according to the literatureSzokol et al.Period. Polytech. Chem. Eng., 66(4), pp. 536–540, 2022 |537A method for the synthesis of blarcamesine enantio-mers via separation of covalent diastereomeric deriva-tives of a racemic intermediate was also described [16]. Racemic alcohol 4 (Scheme 2) was acylated with (S)-(-)-camphanic chloride [(S)-5] and diastereomeric ester (-)-6 was isolated. Hydrolysis of the ester afforded enantiomeri-cally pure (-)-4 which was transformed to (-)-1 by conven-tional methods. A similar procedure was carried out for the synthesis of (+)-1 using (R)-5 as the acylating agent. The enantiomeric purities were characterized by optical rotation measurements, ee values were not determined. For the sake of completeness, it has to be mentioned that an enantioselective chemoenzymatic desymmetrization of one of the early intermediates of blarcamesine (1) using Amano Lipase PS30 enzyme has also been described [16]. However, the yields and the information disclosed sug-gest that the procedure is inappropriate for an economical scaled up implementation.2 Results and discussionIn the course of our work on the process development of blarcamesine drug substance (1) it appeared to us that the absolute configuration of its enantiomers had not been dis-closed in the literature. Therefore, we aimed to improve the resolution process and to obtain crystals suitable for evaluation of the absolute configurations by single-crystal X-ray diffraction (SCXRD) measurements.The crude salt (-)-3 obtained by treatment of racemic blarcamesine (1) with 0.52 equivalent of (S)-(+)-mandelic acid [(S)-(+)-2] recrystallized from propan-2-ol resulted in salt (-)-3 with good yield (39%) and excellent enantiomeric purity (99.88% ee, Scheme 3). The absolute configuration of the chiral center in the furan ring, determined by SCXRD measurement of salt (-)-3, proved to be S (Fig. 2 (a)).This result was further confirmed by the preparation and SCXRD measurement of salt (+)-3. The mother liquor of salt (-)-3 was treated with (R)-(-)-mandelic acid [(R)-(-)-2] to give salt (+)-3 (99.92% ee). SCXRD measurement of the lat-ter supported the expected R absolute configuration of the chiral center in the furan ring (Fig. 2 (b)). The absolute con-figuration of blarcamesine in salts (-)-3 and (+)-3 was deter-mined relative to the known configuration of mandelic acid and supported also by anomalous dispersion, which latter was weak due to the lack of heavy atoms.Liberation of the bases from salts (-)-3 and (+)-3 with aqueous NaOH solution resulted in (S)-(-)-1 (99.84% ee) and (R)-(+)-1 (99.92% ee) enantiomers of blarcamesine (1), respectively, in nearly quantitative yields. In the latter case, our effort to obtain crystals appropriate for SCXRD struc-ture determination was crowned with success (Fig. 2 (c)). The synthetic evidence for the absolute configuration was further supported by the measured anomalous dispersion.The resolution of 1 was also carried out by changing the sequence of addition of the resolving agents with a smaller batch size (starting from 5.30 g, 18.7 mmol blarcames-ine). In this case we first prepared mandelate (+)-3 (38%, 99.72% ee) and then, from the mother liquor the diastereo-meric salt (-)-3 (38%, 99.74% ee).Scheme 2 Resolution of blarcamesine intermediate (4) via covalent diastereomeric derivatives Scheme 3 Improved process for the resolution of blarcamesine (1)538|Szokol et al.Period. Polytech. Chem. Eng., 66(4), pp. 536–540, 20223 ConclusionThe resolution procedure of blarcamesine with (R)- and (S)-mandelic acid has been improved by recrystalliza-tion of the corresponding salts from propan-2-ol result-ing in excellent ee values and yields. Crystals suitable for SCXRD measurement were grown from (-)-blarcamesine (S)-(+)-mandelate, (+)-blarcamesine (R)-(-)-mandelate, and also from (R)-(+)-blarcamesine, and the absolute con-figuration of blarcamesine enantiomers was determined.4 Supporting informationThe "Supporting Information" contains the ORTEP dia-grams, cif, and checkcif files, and structure report files of (-)-3, (+)-3 and (R)-(+)-1 as a zip file and is available on the journal website.5 Experimental sectionAll melting points were determined on a Büchi Melting Point B-540 melting point apparatus and are uncorrected. IR spectra were obtained on a Bruker ALPHA FT-IR spec-trometer operating in transmission mode, in KBr pellets. NMR spectra were recorded at 295 K on a Bruker Avance III HD 600 (600 and 150 MHz for 1H and 13C NMR spec-tra, respectively) spectrometer equipped with a Prodigy cryo-probehead. 1H assignments were accomplished using general knowledge of chemical shift dispersion with the aid of the 1H-1H coupling pattern. CDCl3 was used as the solvent and tetramethylsilane (TMS) as the internal standard. Chemical shifts (δ) and coupling constants (J) are given in ppm and in Hz, respectively. All reagents were purchased from commercial sources and were used without further purification. Reactions were followed by HPLC-MS on a Shimadzu LC-30 HPLC equipment (Kyoto, Japan) equipped with a quaternary pump, degas-ser, autosampler, column oven, diode array detector and an LCMS-2020 quadrupole mass spectrometer. Elemental analyses were performed on an Elementar Vario MICRO cube CHNS elemental analyzer. Single-crystal X-ray dif-fraction (SCXRD) measurements were carried out on a Rigaku R-Axis Spider diffractometer with imaging plate area detector using graphite monochromatic Cu-Kα radia-tion. Single-crystal X-ray structures were deposited at the Cambridge Crystallographic Data Centre under the fol-lowing numbers: CCDC 2177899 [(-)-3], CCDC 2177900 [(+)-3], CCDC 2177901 [(R)-(+)-1].Salt of {[(3S)-2,2-diphenyloxolan-3-yl]methyl}dime-thylamine formed with (S)-(+)-mandelic acid [(-)-3]: Blarcamesine (1, 60.0 g, 213 mmol) was dissolved in tert-butyl methyl ether (MTBE, 180 mL) and (S)-(+)-mandelic acid [(S)-(+)-2, 16.9 g, 110.9 mmol] dissolved in warm propan-2-ol (IPA, 50 mL) was added. The mixture was stirred at room temperature for 2 h. The crude crystalline product separated was filtered, washed with MTBE, dried to give (-)-3 (40.5 g, 44%, 85.12% ee). Recrystallization from IPA resulted in pure (-)-3 (35.6 g, 39%, 99.88% ee). Mp 139–141 °C (IPA). IR (KBr, cm−1): 3410, 2893, 2419, 1626, 1347, 1325, 1050, 1020, 730, 694. 1H NMR (CDCl3, 600 MHz): δ 7.49 (m, 2H), 7.45 (m, 2H), 7.32 (m, 2H), 7.29 (m, 2H), 7.26 (m, 2H), 7.24 (m, 2H), 7.24 (m, 1H), 7.20 (m, 1H), 7.17 (m, 1H), 4.97 (s, 1H), 4.03 (m, 1H), (a)Fig. 2 Single-crystal X-ray structures of (-)-3 (a), (+)-3 (b) and (R)-(+)-1 (c)(b) (c)Szokol et al.Period. Polytech. Chem. Eng., 66(4), pp. 536–540, 2022 |539References[1] Vamvakides, A., Colocouris, N., Foscolos, G., Fytas, G., Papadopoulou-Daifoti, Z., Pouli, N. "Tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanemethanamine, its enantiomers, and their pharmaceutically acceptable acid addition salts", WO 97030983A1, 1997.[2] Foscolos, G. B., Kolocouris, N., Fytas, G., Marakos, P., Pouli, N., Vamvakides, A. "Synthesis and pharmacological study of some new beta-(dialkylaminomethyl)-gamma-butyrolactones and their tetrahydrofuran analogues", Farmaco, 51(1), pp. 19–26, 1996.[3] Vamvakides, A. "New sigma(σ)-receptor ligands with anti-apop-totic and/or pro-apoptotic properties over cellular biochemical mechanisms, with neuroprotective, anticancer, anti-metastatic and anti-(chronic) inflammatory action", WO 2008087458A2, 2008.[4] Villard, V., Espallergues, J., Keller, E., Vamvakides, A., Maurice, T. "Anti-amnesic and neuroprotective potentials of the mixed musca-rinic receptor/sigma1 (σ1) ligand ANAVEX2-73, a novel aminotet-rahydrofuran derivative", Journal of Psychopharmacology, 25(8), pp. 1101–1117, 2011. https://doi.org/10.1177/0269881110379286[5] Lahmy, V., Meunier, J., Malmström, S., Naert, G., Givalois, L., Kim, S. H., Villard, V., Vamvakides, A., Maurice, T. "Blockade of Tau Hyperphosphorylation and Aβ1-42 Generation by the Aminotetrahydrofuran Derivative ANAVEX2-73, a Mixed Muscarinic and σ1 Receptor Agonist, in a Nontransgenic Mouse Model of Alzheimer's Disease", Neuropsychopharmacology, 38(9), pp. 1706–1723, 2013.  https://doi.org/10.1038/npp.2013.703.71 (m, 1H), 3.28 (m, 1H), 2.57 (m, 1H), 2.56 (s, 6H), 2.39 (dd, J1 = 2.8 Hz, J2 = 13.0 Hz, 1H), 2.04 (m, 1H), 1.93 (m, 1H) ppm. 13C NMR (CDCl3, 150 MHz): 178.25, 144.51, 142.29, 141.98, 128.54, 128.34, 127.96, 127.30, 127.04, 126.37, 125.92, 125.50, 89.31, 74.01, 64.85, 59.58, 43.56, 41.91, 29.28 ppm. Elemental analysis calculated for C27H31NO4 (433.55): C 74.80, H 7.21, N 3.23%, found C 74.79, H 7.47, N 3.19%. [α]25D = −128° (c = 1, MeOH).Salt of {[(3R)-2,2-diphenyloxolan-3-yl]methyl}dime-thylamine formed with (R)-(-)-mandelic acid [(+)-3]: (R)-(-)-Mandelic acid [(R)-(-)-2, 16.9 g, 111 mmol] was added to the mother liquor of the crude product (-)-3, and the mixture was stirred at room temperature for 2 h. The crystalline product separated was filtered, washed with MTBE and dried to give (+)-3 (42.8 g, 46%, 91.68% ee). Recrystallization from IPA resulted in pure (+)-3 (38.0 g, 41%, 99.92% ee). Mp 139–141 °C (IPA). IR (KBr, cm−1): 3410, 2893, 2419, 1626, 1347, 1326, 1050, 1020, 731, 694. 1H NMR (CDCl3, 600 MHz): δ 7.49 (m, 2H), 7.45 (m, 2H), 7.32 (m, 2H), 7.29 (m, 2H), 7.26 (m, 2H), 7.24 (m, 2H), 7.24 (m, 1H), 7.20 (m, 1H), 7.17 (m, 1H), 4.97 (s, 1H), 4.03 (m, 1H), 3.71 (m, 1H), 3.28 (m, 1H), 2.57 (m, 1H), 2.56 (s, 6H), 2.39 (dd, J1 = 2.6 Hz, J2 = 12.9 Hz, 1H), 2.05 (m, 1H), 1.94 (m, 1H) ppm. 13C NMR (CDCl3, 150 MHz): 178.27, 144.52, 142.31, 141.97, 128.56, 128.35, 127.97, 127.31, 127.04, 126.38, 125.93, 125.52, 89.32, 74.01, 64.86, 59.61, 43.58, 41.93, 29.30 ppm. Elemental analysis calculated for C27H31NO4 (433.55): C 74.80, H 7.21, N 3.23%, found C 74.78, H 7.52, N 3.18%. [α]25D = +128° (c = 1, MeOH).{[(3S)-2,2-Diphenyloxolan-3-yl]methyl}dimethylamine [(S)-(-)-1]: (S)-Mandelate salt (-)-3 (32.0 g, 73.8 mmol, 99.88% ee) was dissolved in water (220 mL), then aque-ous NaOH solution (10%, 120 mL) and EtOAc (240 mL) were added, and the mixture was intensively stirred for 20 min. The phases were separated, the aqueous phase was extracted with EtOAc (80 mL). The combined organic phases were extracted with water and brine, dried (MgSO4) and evaporated to give (S)-(-)-1 (20.6 g, 99%, 99.84% ee). Mp 73–74 °C. IR (KBr, cm−1): 2966, 2761, 1490, 1445, 1050, 748, 706. 1H NMR (CDCl3, 600 MHz): δ 7.53 (m, 2H), 7.32 (m, 2H), 7.29 (m, 2H), 7.24 (m, 2H), 7.23 (m, 1H), 7.16 (m, 1H), 4.18 (m, 1H), 3.83 (m, 1H), 3.10 (m, 1H), 2.19 (s, 6H), 2.09 (m, 1H), 2.03 (m, 1H), 1.93 (m, 2H) ppm. 13C NMR (CDCl3, 150 MHz): δ 146.43, 143.72, 128.20, 127.68, 126.83, 126.50, 126.38, 126.14, 89.15, 65.61, 61.11, 45.92, 44.43, 29.93 ppm. Elemental analysis calculated for C19H23NO (281.39): C 81.10, H 8.24, N 4.98%, found C 81.24, H 8.31, N 5.00%. [α]25D = −243° (c = 1, MeOH).{[(3R)-2,2-Diphenyloxolan-3-yl]methyl}dimethylamine [(R)-(+)-1]: Starting from (R)-mandelate salt (+)-3 (32.0 g, 73.8 mmol, 99.92% ee) and by following the same proce-dure as for (-)-3 above, (R)-(+)-1 (20.2 g, 97%, 99.92% ee) was prepared. Mp 73–74 °C. IR (KBr, cm−1): 2969, 2766, 1491, 1446, 1047, 753, 700. 1H NMR (CDCl3, 600 MHz): δ 7.53 (m, 2H), 7.32 (m, 2H), 7.29 (m, 2H), 7.24 (m, 2H), 7.23 (m, 1H), 7.16 (m, 1H), 4.18 (m, 1H), 3.83 (m, 1H), 3.10 (m, 1H), 2.19 (s, 6H), 2.09 (m, 1H), 2.03 (m, 1H), 1.93 (m, 2H) ppm. 13C NMR (CDCl3, 150 MHz): 146.43, 143.71, 128.20, 127.67, 126.83, 126.50, 126.37, 126.14, 89.15, 65.61, 61.11, 45.92, 44.43, 29.92 ppm. Elemental analysis calculated for C19H23NO (281.39): C 81.10, H 8.24, N 4.98%, found C 81.12, H 7.92, N 4.96%. [α]25D = +243° (c = 1, MeOH).540|Szokol et al.Period. Polytech. Chem. Eng., 66(4), pp. 536–540, 2022[6] Vamvakides, A. 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P., Nedelkoska, L., Benjamins, J. A. "Sigma-1 receptor agonists as potential protective therapies in multiple sclerosis", Journal of Neuroimmunology, 342, 577188, 2020. https://doi.org/10.1016/j.jneuroim.2020.577188[13] Missling, C. U., Selvey, A. "Anavex2-73 for the treatment of genetic neurodevelopmental disorders", WO 2021016314A1, 2021.[14] Reyes, S. T., Deacon, R. M. J., Guo, S. G., Altimiras, F. J., Castillo, J. B., van der Wildt, B., Morales, A. P., Park, J. H., Klamer, D., Rosenberg, J., Oberman, L. M., Rebowe, N., Sprouse, J., Missling, C. U., McCurdy, C. R., Cogram, P., Kaufmann, W. E., Chin, F. T. "Effects of the sigma-1 receptor agonist blarcamesine in a murine model of fragile X syndrome: neurobehavioral phenotypes and receptor occupancy", Scientific Reports, 11(1), 17150, 2021. https://doi.org/10.1038/s41598-021-94079-7[15] Neliat, G. 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Assignment of Absolute Configuration to Enantiomers of Anti-Alzheimer Drug Candidate Blarcamesine

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Assignment of Absolute Configuration to Enantiomers of Anti-Alzheimer Drug Candidate Blarcamesine

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