The Open Medicinal Chemistry Journal




ISSN: 1874-1045 ― Volume 13, 2019
RESEARCH ARTICLE

Hexacyclododecylamines with Sigma-1 Receptor Affinity and Calcium Channel Modulating Ability



Jacques Joubert1, Natasha Strydom1, Werner J. Geldenhuys2, Yolande Greyling3, Sandra V. Dyk3, Sarel F. Malan1, *
1 Pharmaceutical Chemistry , School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville, South Africa
2 Department of Pharmaceutical Sciences, West Virginia University, School of Pharmacy, Morgantown WV, USA
3 Pharmaceutical Chemistry, School of Pharmacy, North-West University, Private Bag X6001, Potchefstroom, South Africa

Abstract

Introduction:

Recent research points to the Sigma Receptor (σR) as a possible neuromodulatory system with multi-functional action and σ1Rs have been suggested as a drug target for a number of CNS conditions. Hexacyclododecylamines have shown σ1R activity and provide an advantageous scaffold for drug design that can improve the blood-brain barrier permeability of privileged structures.

Methods and Materials:

A series of oxa- and aza- hexaxcyclododecylamines were synthesised and evaluated for sigma-1 receptor activity and voltage-gated calcium channel blocking ability to determine the effect of inclusion of amine containing heterocycles.

Results & Discussion:

The compounds had promising σ1R activities (Ki = 0.067 – 11.86 µM) with the aza-hexacyclododecylamines 12, 24 and 27 showing some of the highest affinities (Ki = 0.067 µM, 0.215 µM and 0.496 µM respectively). This confirms, as observed in previous studies, that the aza compounds are more favourable for σ1R binding than their oxa counterparts. The addition of the amine heterocycle showed affinities similar to that of related structures with only two lipophilic binding regions. This indicates that the inclusion of an amine heterocycle into these structures is a viable option in the design of new σ1R ligands. Significant voltage-gated calcium channel blocking ability was also observed for 12, 24 and 27, suggesting a link between σ1R activity and intracellular calcium levels.

Conclusion:

The σ1R activity and potential effect on other receptor classes and calcium channels could prove beneficial in pharmacological application.

Keywords: Hexacyclododecylamines, Sigma-1 receptor, Voltage-gated calcium channels, Neuromodulatory, Calcium levels, Pharmacological application.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 29
Last Page: 39
Publisher Id: TOMCJ-13-29
DOI: 10.2174/1874104501913010029

Article History:

Received Date: 13/11/2018
Revision Received Date: 28/01/2019
Acceptance Date: 06/02/2019
Electronic publication date: 28/02/2019
Collection year: 2019

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© 2019 Joubert et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Correspondence: Address correspondence to this author at the School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; Tel: +27 219593190; Fax: +27 219591588; E-mail: sfmalan@uwc.ac.za




1. INTRODUCTION

The sigma receptor, which was originally thought to be an opioid receptor, is divided into two subtypes; the Sigma-1 Receptor (σ1R) and the Sigma-2 Receptor (σ2R) which are now classified as distinct receptors [1Martin, W.R.; Eades, C.G.; Thompson, J.A.; Huppler, R.E.; Gilbert, P.E. The effects of morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther., 1976, 197(3), 517-532.[PMID: 945347] , 2Quirion, R.; Chicheportiche, R.; Contreras, P.; Johnson, K.; Lodge, D.; William Tam, S.; Woods, J.; Zukin, S. Classification and nomenclature of phencyclidine and sigma receptor sites. Trends Neurosci., 1987, 10, 444-446.[http://dx.doi.org/10.1016/0166-2236(87)90094-4] ] and do not share homology with any other known mammalian enzyme or receptor [3Hellewell, S.B.; Bowen, W.D. A sigma-like binding site in rat pheochromocytoma (PC12) cells: Decreased affinity for (+)-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain. Brain Res., 1990, 527(2), 244-253.[http://dx.doi.org/10.1016/0006-8993(90)91143-5] [PMID: 2174717] -5Hanner, F.; Moebius, A.; Flandorfer, H.; Knaus, J.; Striessnig, E. Kempner; Glossmann, H. Purification, molecular cloning, and expression of the mammalian sigma1-binding site. P. Natl. Acad. Sci., 1996, 93, 8072-8077.[http://dx.doi.org/10.1073/pnas.93.15.8072] ]. The σ1R has been cloned and is implicated in intracellular signalling, synaptic transmission, apoptosis and is able to mediate effects on calcium conductance through Voltage-Gated Calcium Channels (VGCC), N-methyl-D-Aspartate (NMDA) receptor activity, potassium channel activity, protein kinases and modulation of inositol phosphatases [6Aydar, E.; Palmer, C.P.; Klyachko, V.A.; Jackson, M.B. The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. Neuron, 2002, 34(3), 399-410.[http://dx.doi.org/10.1016/S0896-6273(02)00677-3] [PMID: 1198 8171] -8Jupp, B.; Lawrence, A.J. New horizons for therapeutics in drug and alcohol abuse. Pharmacol. Ther., 2010, 125(1), 138-168.[http://dx.doi.org/10.1016/j.pharmthera.2009.11.002] [PMID: 1991 7308] ]. The σ1R receptor is located on the endoplasmic reticulum and it is likely that the σ1R mediates its response via translocation from the endoplasmic reticulum to other cellular compartments [9Rousseaux, C.G.; Greene, S.F. Sigma receptors [σRs]: Biology in normal and diseased states. J. Recept. Sig. Transd., 2016, 4(36), 327-388.[http://dx.doi.org/10.3109/10799893.2015.1015737] , 10Su, T.P.; Hayashi, T.; Vaupel, D.B. When the endogenous hallucinogenic trace amine N,N-dimethyltryptamine meets the sigma-1 receptor. Sci. Signal., 2009, 2(61), pe12-pe12.[http://dx.doi.org/10.1126/scisignal.261pe12] [PMID: 19278957] ].

The role of sigma receptors in mediating neuroprotection or neurodegeneration has been studied in several in vitro models of Central Nervous System (CNS) injury [11DeCoster, M.A.; Klette, K.L.; Knight, E.S.; Tortella, F.C. Sigma receptor-mediated neuroprotection against glutamate toxicity in primary rat neuronal cultures. Brain Res., 1995, 671(1), 45-53.[http://dx.doi.org/10.1016/0006-8993(94)01294-R] [PMID: 7728532] -13Hayashi, T.; Maurice, T.; Su, T.P. Ca(2+) signaling via sigma(1)-receptors: novel regulatory mechanism affecting intracellular Ca(2+) concentration. J. Pharmacol. Exp. Ther., 2000, 293(3), 788-798.[PMID: 10869377] ]. A consistent positive correlation between sigma neuroprotective potency and σ1R binding site affinity has been demonstrated, suggesting that the functional neuroprotective effect of σR ligands may be mediated by binding at the σ1R site [11DeCoster, M.A.; Klette, K.L.; Knight, E.S.; Tortella, F.C. Sigma receptor-mediated neuroprotection against glutamate toxicity in primary rat neuronal cultures. Brain Res., 1995, 671(1), 45-53.[http://dx.doi.org/10.1016/0006-8993(94)01294-R] [PMID: 7728532] ]. Activation of σ1R receptors results in a complex, bipolar modulation of calcium homeostasis. It facilitates the mobilisation of inositol triphosphate receptor-gated intracellular calcium pools at the endoplasmic reticulum level and modulates extracellular calcium influx through VGCC at the plasma membrane level [12Bowen, W.D. Sigma receptors: recent advances and new clinical potentials. Pharm. Acta Helv., 2000, 74(2-3), 211-218.[http://dx.doi.org/10.1016/S0031-6865(99)00034-5] [PMID: 1081 2960] , 13Hayashi, T.; Maurice, T.; Su, T.P. Ca(2+) signaling via sigma(1)-receptors: novel regulatory mechanism affecting intracellular Ca(2+) concentration. J. Pharmacol. Exp. Ther., 2000, 293(3), 788-798.[PMID: 10869377] ]. Overstimulation of σ1 receptors may contribute to toxic intracellular calcium concentrations and sigma receptors are thus a target for neuroprotective drugs aimed at calcium modulation [12Bowen, W.D. Sigma receptors: recent advances and new clinical potentials. Pharm. Acta Helv., 2000, 74(2-3), 211-218.[http://dx.doi.org/10.1016/S0031-6865(99)00034-5] [PMID: 1081 2960] , 13Hayashi, T.; Maurice, T.; Su, T.P. Ca(2+) signaling via sigma(1)-receptors: novel regulatory mechanism affecting intracellular Ca(2+) concentration. J. Pharmacol. Exp. Ther., 2000, 293(3), 788-798.[PMID: 10869377] ]. Additionally, the σ1R receptors and the ER play an important functional role with the mitochondria, where the ER-to-mitochondria miscommunication has been implicated in calcium exchange and other cellular processes like mitophagy [14MacVicar, T.D.; Mannack, L.V.; Lees, R.M.; Lane, J.D. Targeted siRNA screens identify ER-to-mitochondrial calcium exchange in autophagy and mitophagy responses in RPE1 cells. Int. J. Mol. Sci., 2015, 16(6), 13356-13380.[http://dx.doi.org/10.3390/ijms160613356] [PMID: 26110381] ].

Since the discovery of σRs,much research was done in this field due to the implication of these receptors in various major (CNS) diseases [15Maurice, T.; Su, T.P. The pharmacology of sigma-1 receptors. Pharmacol. Ther., 2009, 124(2), 195-206.[http://dx.doi.org/10.1016/j.pharmthera.2009.07.001] [PMID: 1961 9582] , 16Hayashi, T.; Stahl, S. The sigma-1 (delta-1) receptor and its role in the treament of mood disorders. Drugs Future, 2009, 34, 137.[http://dx.doi.org/10.1358/dof.2009.034.02.1336115] ]. Some of the earliest σR ligands identified were diverse clinical antipsychotics that bind to σ1Rs with nanomolar affinity [17Tam, S.; Cook, L. Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[3H] SKF 10,047 and [3H]haloperidol binding in guinea pig brain membranes. P. Natl. Acad. Sci., 1984, 81, 5618-5621.[http://dx.doi.org/10.1073/pnas.81.17.5618] ] and several antidepressants from unrelated pharmacological classes that interact with σ1Rs with high affinity [18Itzhak, Y.; Kassim, C.O. Clorgyline displays high affinity for sigma binding sites in C57BL/6 mouse brain. Eur. J. Pharmacol., 1990, 176(1), 107-108.[http://dx.doi.org/10.1016/0014-2999(90)90139-W] [PMID: 2155796] , 19Narita, N.; Hashimoto, K.; Tomitaka, S.; Minabe, Y. Interactions of selective serotonin reuptake inhibitors with subtypes of sigma receptors in rat brain. Eur. J. Pharmacol., 1996, 307(1), 117-119.[http://dx.doi.org/10.1016/0014-2999(96)00254-3] [PMID: 8831113] ]. The role of σ1R in Alzheimer’s disease [20Maurice, T. Improving Alzheimer’s Disease-Related Cognitive Deficits with sigma1 Receptor Agonists. Drug News Perspect., 2002, 15(10), 617-625.[http://dx.doi.org/10.1358/dnp.2002.15.10.740241] [PMID: 12677246] ], Parkinson’s disease [20Maurice, T. Improving Alzheimer’s Disease-Related Cognitive Deficits with sigma1 Receptor Agonists. Drug News Perspect., 2002, 15(10), 617-625.[http://dx.doi.org/10.1358/dnp.2002.15.10.740241] [PMID: 12677246] ], anxiety disorders [21Kulkarni, S.K.; Dhir, A. sigma-1 receptors in major depression and anxiety. Expert Rev. Neurother., 2009, 9(7), 1021-1034.[http://dx.doi.org/10.1586/ern.09.40] [PMID: 19589051] ], depression [22J. Pharmacol. Sci., 2005, 97, 317-336.[http://dx.doi.org/10.1254/jphs.CRJ04005X] [PMID: 15750289] ] and drug addiction [23Maurice, T.; Martin-Fardon, R.; Romieu, P.; Matsumoto, R.R. Sigma(1) (sigma(1)) receptor antagonists represent a new strategy against cocaine addiction and toxicity. Neurosci. Biobehav. Rev., 2002, 26(4), 499-527.[http://dx.doi.org/10.1016/S0149-7634(02)00017-9] [PMID: 1220 4195] ] is well accepted.

Polycyclic amines, such as the hexacyclododecylamine (HCD; also described as pentacycloundecylamines in literature) and the adamantane amines, were shown to be valuable scaffolds in the development of CNS drugs [24Geldenhuys, W.J.; Malan, S.F.; Bloomquist, J.R.; Marchand, A.P.; Van der Schyf, C.J. Pharmacology and structure-activity relationships of bioactive polycyclic cage compounds: A focus on pentacycloundecane derivatives. Med. Res. Rev., 2005, 25(1), 21-48.[http://dx.doi.org/10.1002/med.20013] [PMID: 15389731] , 25Joubert, J.; Geldenhuys, W.J.; Van der Schyf, C.J.; Oliver, D.W.; Kruger, H.G.; Govender, T.; Malan, S.F. Polycyclic cage structures as lipophilic scaffolds for neuroactive drugs. ChemMedChem, 2012, 7(3), 375-384.[http://dx.doi.org/10.1002/cmdc.201100559] [PMID: 22307951] ]. These polycyclic compounds possess their own neuromodulatory activity on important receptor classes that have been implicated in CNS disease states such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, schizophrenia, stroke and disease states as intricate as drug addiction [24Geldenhuys, W.J.; Malan, S.F.; Bloomquist, J.R.; Marchand, A.P.; Van der Schyf, C.J. Pharmacology and structure-activity relationships of bioactive polycyclic cage compounds: A focus on pentacycloundecane derivatives. Med. Res. Rev., 2005, 25(1), 21-48.[http://dx.doi.org/10.1002/med.20013] [PMID: 15389731] -26Nguyen, V.H.; Kassiou, M.; Johnston, G.A.; Christie, M.J. Comparison of binding parameters of sigma 1 and sigma 2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes. Eur. J. Pharmacol., 1996, 311(2-3), 233-240.[http://dx.doi.org/10.1016/0014-2999(96)00395-0] [PMID: 8891604] ]. They act as NMDA receptor antagonists [27Geldenhuys, W.J.; Malan, S.F.; Bloomquist, J.R.; Van der Schyf, C.J. Structure-activity relationships of pentacycloundecylamines at the N-methyl-d-aspartate receptor. Bioorg. Med. Chem., 2007, 15(3), 1525-1532.[http://dx.doi.org/10.1016/j.bmc.2006.09.060] [PMID: 17157509] , 28Lemmer, H.J.R.; Joubert, J.; van Dyk, S.; van der Westhuizen, F.H.; Malan, S.F. S-nitrosylation and attenuation of excessive calcium flux by pentacycloundecane derivatives. Med. Chem., 2012, 8(3), 361-371.[http://dx.doi.org/10.2174/1573406411208030361] [PMID: 22530904] ], are able to spontaneously increase dopamine release [29Geldenhuys, W.J.; Bezuidenhout, L.M.; Dluzen, D.E. Effects of a novel dopamine uptake inhibitor upon extracellular dopamine from superfused murine striatal tissue. Eur. J. Pharmacol., 2009, 619(1-3), 38-43.[http://dx.doi.org/10.1016/j.ejphar.2009.08.012] [PMID: 19686731] ] and have been shown to have neuroprotective activity through modulation of VGCC flux [28Lemmer, H.J.R.; Joubert, J.; van Dyk, S.; van der Westhuizen, F.H.; Malan, S.F. S-nitrosylation and attenuation of excessive calcium flux by pentacycloundecane derivatives. Med. Chem., 2012, 8(3), 361-371.[http://dx.doi.org/10.2174/1573406411208030361] [PMID: 22530904] , 30Van der Schyf, C.J.; Squier, G.J.; Coetzee, W.A. Characterization of NGP 1-01, an aromatic polycyclic amine, as a calcium antagonist. Pharmacol. Res. Commun., 1986, 18(5), 407-417.[http://dx.doi.org/10.1016/0031-6989(86)90162-1] [PMID: 3737654] ]. Binding studies have confirmed that they also have an affinity for the sigma receptor and that the polycyclic amine group is important for binding interactions [31Kassiou, M.; Nguyen, V.; Knott, R.; Christie, M.; Hambley, T. Trishomocubanes, a new class of selective and high affinity ligands for the sigma binding site. Bioorg. Med. Chem. Lett., 1996, 6, 595-600.[http://dx.doi.org/10.1016/0960-894X(96)00067-4] -33Geldenhuys, W.J.; Novotny, N.; Malan, S.F.; Van der Schyf, C.J. 3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor. Bioorg. Med. Chem. Lett., 2013, 23(6), 1707-1711.[http://dx.doi.org/10.1016/j.bmcl.2013.01.069] [PMID: 23414839] ]. They further have the ability to greatly improve lipophilicity of their conjugates, which is helpful in increasing blood-brain barrier permeability leading to increased concentration of CNS acting drugs in the brain, decreasing dosage and ultimately minimising peripheral side effects [34Zah, J.; Terre’Blanche, G.; Erasmus, E.; Malan, S. Physicochemical prediction of a brain-blood distribution profile in polycyclic amines. Bioorg. Med. Chem., 2003, 11, 3569-3578.[http://dx.doi.org/10.1016/S0968-0896(03)00365-1] [PMID: 1290 1901] , 35Prins, L.H.; du Preez, J.L.; van Dyk, S.; Malan, S.F. Polycyclic cage structures as carrier molecules for neuroprotective non-steroidal anti-inflammatory drugs. Eur. J. Med. Chem., 2009, 44(6), 2577-2582.[http://dx.doi.org/10.1016/j.ejmech.2009.01.030] [PMID: 19233517] ]. Several studies have shown that both HCD containing compounds and the structurally related adamantane moiety may present with sigma receptor activity [31Kassiou, M.; Nguyen, V.; Knott, R.; Christie, M.; Hambley, T. Trishomocubanes, a new class of selective and high affinity ligands for the sigma binding site. Bioorg. Med. Chem. Lett., 1996, 6, 595-600.[http://dx.doi.org/10.1016/0960-894X(96)00067-4] -33Geldenhuys, W.J.; Novotny, N.; Malan, S.F.; Van der Schyf, C.J. 3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor. Bioorg. Med. Chem. Lett., 2013, 23(6), 1707-1711.[http://dx.doi.org/10.1016/j.bmcl.2013.01.069] [PMID: 23414839] ].

Based on the above observations a series of hexacyclododecylamines were designed and synthesised to evaluate the effect of including an amine-containing heterocyclic group compared to previous (HCD) structures containing only two lipophilic regions (Fig. 1 and Table 1). The inclusion of an amine-containing ring has been shown to improve σ1R activity in an array of structurally distinct compounds [36Ablordeppey, S.Y.; Fischer, J.B.; Glennon, R.A. Is a nitrogen atom an important pharmacophoric element in sigma ligand binding? Bioorg. Med. Chem., 2000, 8(8), 2105-2111.[http://dx.doi.org/10.1016/S0968-0896(00)00148-6] [PMID: 1100 3156] , 37Ablordeppey, S.Y.; Fischer, J.B.; Law, H.; Glennon, R.A. Probing the proposed phenyl-A region of the sigma-1 receptor. Bioorg. Med. Chem., 2002, 10(8), 2759-2765.[http://dx.doi.org/10.1016/S0968-0896(02)00096-2] [PMID: 1205 7665] ]. This design would, therefore, serve as a preliminary study into the potential of including an amine-containing heterocyclic ring between the two lipophilic regions of these compounds. This may lead to better alignment of the compounds in the σ1R and/or additional binding interactions with the σ1R. In addition, σ1R antagonists have shown to regulate L-type VGCCs which may affect Ca2+ flux through these channels [12Bowen, W.D. Sigma receptors: recent advances and new clinical potentials. Pharm. Acta Helv., 2000, 74(2-3), 211-218.[http://dx.doi.org/10.1016/S0031-6865(99)00034-5] [PMID: 1081 2960] , 13Hayashi, T.; Maurice, T.; Su, T.P. Ca(2+) signaling via sigma(1)-receptors: novel regulatory mechanism affecting intracellular Ca(2+) concentration. J. Pharmacol. Exp. Ther., 2000, 293(3), 788-798.[PMID: 10869377] ]. The purpose of this study was thus to evaluate a series of HCD derivatives for modulating effect on the σ1R and intracellular calcium concentrations through VGCCs in an attempt to explore the neuromodulatory potential of this structural class of compounds.

Fig. (1)
Structures of polycyclic amines; amantadine, NGP1-01 and related hexacyclododecylamines 1-3, with affinity for the σ1R.


2. EXPERIMENTAL SECTION

2.1. Chemistry: General Procedures

Unless otherwise specified, materials were obtained from Sigma Aldrich® and Merck® and used without further purification. All reactions were monitored by thin-layer chromatography on 0.20 mm thick aluminium silica gel sheets (Alugram® SIL G/UV254, Kieselgel 60, Macherey-Nagel, Düren, Germany). Visualisation was achieved using a Chromato-vue® Cabinet under UV light (254 nm and 366 nm) or with iodine vapours. Mobile phases were prepared on a volume-to-volume basis. Infra-Red (IR) spectra were recorded on a Perkin Elmer Spectrum 400 spectrometer, fitted with a diamond Attenuated Total Reflectance (ATR) attachment. Mass Spectra (MS) were recorded on an analytical VG 70-70E mass spectrometer using Electron Spray Ionisation (ESI) at 70 eV. 1H and 13C NMR spectra were obtained using a Varian Gemini 200 spectrometer at a frequency of 200 MHz and 50 MHz, respectively or a Bruker Advanced 600 Spectrometer at frequencies of 600 MHz and 150 MHz respectively. Tetramethylsilane (TMS) was used as a point of reference in all NMR experiments. All chemical shifts are reported in parts per million (ppm) relative to the signal from TMS (δ = 0), added to an appropriate deuterated solvent. All chemical shifts are reported in parts per million (ppm), relative to the internal standard. The following abbreviations are used to indicate the multiplicities of the respective signals: s – singlet; d - doublet; dd - doublet of doublets; t - triplet; m - multiplet; and AB-q - AB quartet. The multiplicity of the identified carbons was confirmed with DEPT-spectra. Microwave synthesis was performed using a CEM DiscoverTM microwave synthesis system. Compounds 6 [38Joubert, J.; Sharma, R.; Onani, M.; Malan, S. Microwave-assisted methods for the synthesis of pentacyclo [5.4.0.0 2,6 .0 3,10 .0 5,9] undecylamines Tetrahedron Lett., 2013, 54, 6923-6927.[http://dx.doi.org/10.1016/j.tetlet.2013.10.047] ], 8 [39Banister, S.D.; Moussa, I.A.; Jordan, M.J.; Coster, M.J.; Kassiou, M. Oxo-bridged isomers of aza-trishomocubane sigma (sigma) receptor ligands: Synthesis, in vitro binding, and molecular modeling. Bioorg. Med. Chem. Lett., 2010, 20(1), 145-148.[http://dx.doi.org/10.1016/j.bmcl.2009.11.019] [PMID: 19954972] ], 9 [40Buschauer, A.; Postius, S.; Szelenyi, I.; Schunack, W. Isohistamine and homologs as components of H2-antagonists. 22. H2-antihistaminics. Arzneimittelforschung, 1985, 35(7), 1025-1029.[PMID: 2864932] ], 13 [41Matsumoto, R.R.; Bowen, W.D.; Tom, M.A.; Vo, V.N.; Truong, D.D.; De Costa, B.R. Characterization of two novel sigma receptor ligands: antidystonic effects in rats suggest sigma receptor antagonism. Eur. J. Pharmacol., 1995, 280(3), 301-310.[http://dx.doi.org/10.1016/0014-2999(95)00208-3] [PMID: 8566098] ], 15 [30Van der Schyf, C.J.; Squier, G.J.; Coetzee, W.A. Characterization of NGP 1-01, an aromatic polycyclic amine, as a calcium antagonist. Pharmacol. Res. Commun., 1986, 18(5), 407-417.[http://dx.doi.org/10.1016/0031-6989(86)90162-1] [PMID: 3737654] ], 21 [42Glennon, R.A.; Ablordeppey, S.Y.; Ismaiel, A.M.; el-Ashmawy, M.B.; Fischer, J.B.; Howie, K.B. Structural features important for sigma 1 receptor binding. J. Med. Chem., 1994, 37(8), 1214-1219.[http://dx.doi.org/10.1021/jm00034a020] [PMID: 8164264] ] and 22 [40Buschauer, A.; Postius, S.; Szelenyi, I.; Schunack, W. Isohistamine and homologs as components of H2-antagonists. 22. H2-antihistaminics. Arzneimittelforschung, 1985, 35(7), 1025-1029.[PMID: 2864932] ] were synthesised as previously described and all physical characteristics were similar to that reported in the literature.

2.2. Synthesis of Compounds

2.2.1. 5-[2-(4-Benzylpiperazin-1-yl)ethyl]-5-azahexacyclo[5.4. 1.02,6.03,10.04,8.09,12]dodecan4-ol (12)

Pentacyclo[5.4.0.02,6.03,10.05,9]undecane-8,11-dione-ethylene acetal (9, 200 mg, 0.917 mmol) and compound 8 (200 mg, 0.913 mmol) were dissolved in 5 ml EtOH and reacted under microwave conditions at a maximum temperature of 100°C, power setting of 150 W and pressure of 150 psi for 30 minutes. The reaction mixture was allowed to cool and directly used in the next step. The cooled solution of crude compound 10 was dissolved in 5 ml EtOH and NaBH4 (159 mg, 4.20 mmol, 1.4 equiv.) was added. The mixture was stirred at room temperature for 8 hours where after EtOH was evaporated under reduced pressure, water (10 ml) was added and the mixture was extracted with DCM (3 × 10 ml). The combined organic extracts were washed with brine (10 ml), dried (Na2SO4), and concentrated in vacuo. To this crude material (11), acetone (25 ml) and 4 M aq. HCl (15 ml) were added. After stirring at room temperature for 6 h, the mixture was diluted with H2O (200 ml), basified to pH 14 with 1 M aq. NaOH, and extracted with DCM (3 × 15 ml). The combined organic extracts were dried (Na2SO4) and concentrated in vacuo. The crude product was purified by recrystallization from EtOH to yield the desired compound (12) as colourless crystals (Yield = 183 mg, 53%) [38Joubert, J.; Sharma, R.; Onani, M.; Malan, S. Microwave-assisted methods for the synthesis of pentacyclo [5.4.0.0 2,6 .0 3,10 .0 5,9] undecylamines Tetrahedron Lett., 2013, 54, 6923-6927.[http://dx.doi.org/10.1016/j.tetlet.2013.10.047] ]. Physical data: 1H NMR (200 MHz, CDCl3) δH: 7.26 – 7.16 (m, 5H), 3.43 (s, 2H), 3.32 (t, 1H, J = 4.9 Hz), 2.96 – 2.30 (m, 20H), 1.79-1.41 (AB-q, 2H, J = 10.6 Hz). 13C NMR (50 MHz, CDCl3) δc: 137.9, 129.1, 128.2, 127.0, 70.8, 62.7, 58.6, 56.9, 52.7, 53.4, 51.5, 46.6, 45.7, 45.4, 43.3, 43.2, 42.1, 41.9, 41.7. IR (υmax): 3242.86, 2949.29, 2812.87, 1319.78, 1284.36 cm-1. HR-ESI [M+H]+: calcd. 378.2540, found. 378.2546.

2.2.2. N-[2-(4-benzylpiperidin-1-yl)ethyl]-5-oxahexacyclo[5.4. 1.02,6.03,10.04,8.09,12]dodecan-4-amine (18)

Methanesulfonyl chloride (261 mg, 2.28 mmol) dissolved in 10 ml of a 50:40:10 diethyl ether:DCM:triethylamine solvent system was added dropwise to a stirred solution of compound 15 (500 mg, 2.28 mmol, 1.00 equiv.) in 20 ml of the same solvent system under nitrogen gas on an external ice bath consisting of ice, acetone and sodium chloride which cooled the reaction mixture to approximately -8 °C. At the moment of addition, an exothermal reaction took place, gas was expelled and the reaction vessel’s temperature increased despite the external ice bath. The reaction was allowed to stir overnight at room temperature. The solvents were evaporated in vacuo and the mixture was dissolved in 10 ml of DCM and washed with 2 x 10 ml of brine. The combined organic fractions were dried over MgSO4, filtered and evaporated in vacuo to produce a dark yellow oil. The product ((16, 2-({5-oxahexacyclo [5.4.1.02,6.03,10.04,8.09,12]dodecan-4-yl}amino)ethyl methanesulfonate)) was confirmed to be pure enough for subsequent reactions (Yield = 193 mg, 20%). Physical data: 1H NMR (200 MHz, CDCl3): δH: 4.68 - 4.53 (t, 1H, J = 5.6 Hz), 3.57 (t, 2H, J = 5.8 Hz), 3.05 (t, 2H, J = 5.8 Hz), 2.90 – 2.32 (m, 9H), 1.95 – 1.46 (AB-q, 2H, J = 10.5). Next, 16 (193 mg, 0.65 mmol) was reacted with 4-benzylpiperidine (17, 114 mg, 0.65 mmol, 1.00 equiv.). The reactants were dissolved in 7 ml of acetonitrile and a spatula point (150 – 200 mg) of potassium carbonate was added. It was then reacted in a microwave reactor for 10 min at a maximum temperature of 120 °C, a pressure which fluctuated around 90 psi and power output between 50 W and 100 W. The reaction mixture was then dissolved in 10 ml of DCM and washed with 2 x 5 ml of water. Further purification by column chromatography was done starting with hexane as mobile phase and gradually incorporating ethyl acetate into the mobile phase at increments of 10%. The product eluded at 50% ethyl acetate:hexane, but still contained impurities. The eluded crude compound was dissolved in 10 ml of DCM and extracted with 3 x 10 ml 0.01 M HCl solution. The acidic phases were collected and made basic with a 0.1 M NaOH solution. The compound was then extracted with 3 x 30 ml of DCM. The organic fractions were collected, dried with MgSO4 overnight, filtered and dried in vacuo to yield the desired compound (18) as a viscous yellow-orange oil (Yield = 23 mg, 9%). Physical data: 1H NMR (200 MHz, CDCl3): δH: 7.38 – 7.19 (m, 5H), 4.62 (t, 1H, J = 5.6 Hz), 3.17 – 3.13 (d, 2H, J = 8.2 Hz), 2.79 – 2.40 (m, 21H), 1.90-1.48 (AB-q, 2H, J = 10.5). 13C NMR (50 MHz, CDCl3): δc: 136.8, 128.5, 127.4, 126.3, 82.6, 60.9, 55.0, 53.2, 51.9, 47.9, 47.6, 44.7, 44.5, 43.6, 43.4, 41.9, 41.7, 41.6, 41.3, 29.6. IR (υmax): 2961.45, 2861.88, 1739.92, 1341.79, 1008.16 cm-1. HR-ESI [M+H]2+: calcd. 378.2587, found. 378.2574.

2.2.3. 5-(3-{3-[(Piperidin-1-yl)methyl]phenoxy}propyl)-5-aza- hexacyclo[5.4.1.02,6.03,10.04,8.09,12]dodecan-4-ol (24)

Pentacyclo[5.4.0.02,6.03.10.05,9]undecane-8,11-dione (13, 1.4 g, 8.04 mmol) was reacted with 1.7 g (8.04 mmol) of N-[3-(3-piperidin-1-ylmethylphenoxy)propyl]amine (22) in 10 ml dry THF at -10 °C. The carbinolamine that formed after 10 min was filtered off and refluxed under Dean-Stark conditions for 1 hour in benzene. The benzene was evaporated under vacuum and the residue was dissolved in 6 ml dry methanol and 30 ml dry THF. To this, 0.3 g NaBH3CN was added as a reducing agent. The mixture was stirred overnight at room temperature. The methanol and THF were evaporated in vacuo and the residue was extracted with 4 x 25 ml DCM and the organic fraction was dried with MgSO4, filtered and evaporated in vacuo. The desired product (24) was obtained as a yellow oily substance after column chromatography using DCM: EtO Ac:PE, 1:1:1 (Rf = 0.42), as mobile phase (Yield: 0.43 g, 1.09 mmol, 13%). Physical data: 1H NMR (300 MHz, CDCl3) δH: 7.26 (t, 1H, J = 5.8 Hz), 6.95-6.88 (m, 1H), 6.80-6.74 (m, 2H), 4.10 (t, 2H, J = 5.1 Hz), 3.63 (t, 1H, J = 5.0 Hz), 3.52 (s, 2H) 3.13 (t, 2H, J = 5.2 Hz), 3.09-2.55 (m, 14H), 2.10-1.94 (m, 4H), 1.85-1.55 (AB-q, 2H, J = 11.3 Hz), 1.53-1.45 (m, 2H). 13C NMR (75 MHz, CDCl3) δc: 158.0, 140.2, 129.1, 122.0, 115.2, 113.0, 66.9, 64.8, 63.6, 54.8, 54.3, 54.3, 53.6, 46.3, 44.6, 43.9, 43.8, 43.1, 41.9, 41.6, 41.2 40.9, 38.9, 26.9, 26.0, 24.2 IR (υmax): 3342.8, 2969.1, 2865.4, 1731.0, 1337.7, 1274.1, 1098.0, 1557.9 cm-1. HR-ESI [M+H]: calcd. 407.2693, found. 407. 2697.

2.2.4. N-(3-{3-[(Piperidin-1-yl)methyl]phenoxy}propyl)-5-ox- ahexacyclo[5.4.1.02,6.03,10.04,8.09,12]dodecan-4-amine (25)

Pentacyclo[5.4.0.02,6.03.10.05,9]undecane-8,11-dione (13, 1.4 g, 8.04 mmol) was reacted with 1.7 g (8.04 mmol) of N-[3-(3-piperidin-1-ylmethylphenoxy)propyl]amine (22) in 10 ml dry THF at -10 °C. The carbinolamine that formed after 10 min was filtered off and refluxed under Dean-Stark conditions for 1 hour in benzene. The benzene was evaporated under vacuum and the residue was dissolved in 6 ml dry methanol and 30 ml dry THF. To this 0.30 g NaBH4 was added as reducing agent. The mixture was stirred overnight at room temperature. The methanol and THF were evaporated under vacuum and the residue was extracted with 4 x 25 ml DCM and the organic fraction was dried with magnesium sulphate, filtered and evaporated. The desired product (25) was obtained as an light yellow oily substance after column chromatography using DCM:EtOAc:PE, 1:1:1 (Rf = 0.43), as mobile phase. (Yield: 1.31 g, 35%). Physical data: 1H NMR (300 MHz, CDCI3) δH: 7.20 (t, 1H, J = 8.2 Hz), 6.93-6.85 (m, 2H), 6.78-6.74 (m, 1H), 4.7 (t, 1H, J = 5.4 Hz), 4.02 (t, 2H, J = 5.2 Hz), 3.43 (s, 2H), 3.17 (t, 2 H, J = 5.3 Hz), 3.10-3.01 (m, 4H), 2.96-2.65 (m, 10H), 2.15-2.09 (m, 4H), 2.05-1.87 (AB-q, 2H, J = 11.5), 1.60-1.53 (m, 2H); 13C NMR (75 MHz, CDCI3) δc: 159, 140.0, 128.1, 122.0, 115.4, 112.6, 110.0, 82.3, 65.2, 64.9, 64.0, 54.9, 54.8, 54.6, 54.0, 46.0, 44.5, 43.9, 43.5, 43.0, 41.5, 41.2 40.5 38.8, 26.8, 26.2, 24.2. IR (υmax): 3357.2, 2964.7, 2863.9, 1706.0, 1211.2, 1131.8 cm-1; HR-ESI [M+H]: calcd. 407.2693, found. 407.2696.

2.2.5. 5- [2- (Piperidin-1-yl) ethyl]-5- azahexacyclo [5.4.1.0 2,6. 03,10.04,8.09,12]dodecan-4-ol (27)

Pentacyclo[5.4.0.02,6.03.10.05,9]undecane-8,11-dione (13, 1 g, 8.04 mmol) was reacted with 1 ml of 1-(2-aminoethyl) piperidine (26, 5.74 mmol) in 10 ml dry THF at -10 °C. The carbinolamine that formed was filtered off and refluxed under Dean-Stark conditions for 1 hour in benzene. The benzene was evaporated in vacuo and the residue was dissolved in 6 ml dry methanol, 30 ml dry THF and 0.3 g NaBH3CN was added as reducing agent and the mixture was stirred overnight at room temperature. The methanol and THF were evaporated in vacuo and the residue was extracted with 4 x 25 ml DCM, dried with MgSO4, filtered and evaporated. The desired product (27) was obtained as a yellow oily substance after column chromatography using DCM:EtOAc:PE, 1:1:1 (Rf = 0.31), as mobile phase. (Yield: 0.70 g, 2.33 mmol, 41%). Physical data: 1H NMR (300 MHz, CDCl3) δH: 3.80 (t, 1H, J = 5.3 Hz), 2.80-2.10 (m, 16H), 1.90 (d, 1H, J = 10.40 Hz), 1.75-1.40 (m, 7H); 13C NMR (75 MHz, CDCI3) δc: 82.4, 71.9, 59.1, 58.1, 54.9, 54.8, 45.0, 44.8, 43.0, 42.6, 40.0, 35.2, 34.4, 30.5, 26.0, 24.6; IR (υmax): 3000, 2938.1, 2860.3, 2324.8, 2168.4 cm-1; HR-ESI [M+H]: calcd. 287.2118, found. 287.2121.

2.3. Sigma-1 Receptor Binding Affinity Studies

Binding affinities for the σ1R were determined as described previously [41Matsumoto, R.R.; Bowen, W.D.; Tom, M.A.; Vo, V.N.; Truong, D.D.; De Costa, B.R. Characterization of two novel sigma receptor ligands: antidystonic effects in rats suggest sigma receptor antagonism. Eur. J. Pharmacol., 1995, 280(3), 301-310.[http://dx.doi.org/10.1016/0014-2999(95)00208-3] [PMID: 8566098] , 42Glennon, R.A.; Ablordeppey, S.Y.; Ismaiel, A.M.; el-Ashmawy, M.B.; Fischer, J.B.; Howie, K.B. Structural features important for sigma 1 receptor binding. J. Med. Chem., 1994, 37(8), 1214-1219.[http://dx.doi.org/10.1021/jm00034a020] [PMID: 8164264] ]. Briefly, rat liver lysates were incubated with 3 nM [3H](+)-pentazocine (Perkin Elmer USA), 100 ug/mL tissue lysate and compounds in 50 mM Tris pH 8.0. After 1 hour incubation, the reaction was terminated with the addition of 1 ml cold 50 mM Tris pH 8.0 buffer and filtered through Wathman B filter paper using a vacuum manifold. The filters were pre-soaked in 0.3% polyethylene imine for at least 30 min before the experiment was terminated. The filters were additionally washed twice with 1 ml cold buffer. The remaining radioactivity remaining on the filter paper was counted using scintillation counting. All data analysis, calculation and graphs were done using Prism 6.0® (GraphPhad, La Jolla, CA). Experiments were repeated three times on different tissue preparations with three determinations in each replicate. The final results were expressed as Ki and IC50 values.

2.4. Sigma-1 Receptor Docking Studies

The docking studies were performed using the recently published σ1R crystal structure (PDB: 5HK2) [43Schmidt, H.R.; Zheng, S.; Gurpinar, E.; Koehl, A.; Manglik, A.; Kruse, A.C. Crystal structure of the human σ1 receptor. Nature, 2016, 532(7600), 527-530.[http://dx.doi.org/10.1038/nature17391] [PMID: 27042935] ]. The Molecular Operating Environment (MOE) 2015 software suite was used for the docking studies with the following protocol. (1) The receptor protein structure was checked for missing atoms, bonds and contacts. (2) Hydrogens and partial charges were added using the protonate 3D application in MOE. Protein structure preparation was done by setting the pH of the protein at 7.4. (3) The ligands were constructed using the builder module and were energy minimized using CHARMm. (4) Ligands were docked within the σ1R active sites using MOEDock application. The active site was selected based on the position of the co-crystallised ligand, 4-IBP. The docking algorithm which was chosen for these experiments was based on induced fit docking to allow for flexible interactions of the test compounds with the receptor. (5) The top binding best pose of each compound was visually inspected and the interactions with binding pocket residues were analyzed. To determine the accuracy of this docking protocol, the co-crystallised ligand, 4-IBP (PDB ID: 5HK2), was redocked into the σ1R active site. The best ranked solution of 4-IBP exhibited an RMSD values of less than 0.56 Å from the position of the co-crystallised ligand. In general, RMSD values smaller than 2.0 Å, indicate that the docking protocol is capable of accurately predicting the binding orientation of the co-crystallised ligand [49Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson, D.E.; Mattevi, A. Insights into the mode of inhibition of human mitochondrial monoamine oxidase B from high-resolution crystal structures. Proc. Natl. Acad. Sci. USA, 2003, 100(17), 9750-9755.[http://dx.doi.org/10.1073/pnas.1633804100] [PMID: 12913124] , 50Boström, J.; Greenwood, J.R.; Gottfries, J. Assessing the performance of OMEGA with respect to retrieving bioactive conformations. J. Mol. Graph. Model., 2003, 21(5), 449-462.[http://dx.doi.org/10.1016/S1093-3263(02)00204-8] [PMID: 1254 3140] ]. This protocol was thus deemed to be suitable for the docking of the test compounds.

2.5. Voltage Gated Calcium Channel Assay

The fluorescent ratiometric indicator, Mag-Fura-2/AM, and a Bio-Tek® fluorescent plate reader were used to evaluate the influence of the test compounds on calcium homeostasis via the VGCC utilizing murine synaptoneurosomes at 37 ºC. The preparation of synaptoneurosomes, solutions and experimental techniques was similar to those of published studies [44Zindo, F.T.; Barber, Q.R.; Joubert, J.; Bergh, J.J.; Petzer, J.P.; Malan, S.F. Polycyclic propargylamine and acetylene derivatives as multifunctional neuroprotective agents. Eur. J. Med. Chem., 2014, 80, 122-134.[http://dx.doi.org/10.1016/j.ejmech.2014.04.039] [PMID: 24769350] , 45Hollingsworth, E.B.; McNeal, E.T.; Burton, J.L.; Williams, R.J.; Daly, J.W.; Creveling, C.R. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes. J. Neurosci., 1985, 5(8), 2240-2253.[http://dx.doi.org/10.1523/JNEUROSCI.05-08-02240.1985] [PMID: 2991484] ]. All data analysis, calculation and graphs were conducted using Prism 6.0® (GraphPhad, La Jolla, CA). Data analysis was carried out using the Student Newman Keuls multiple range test and the level of significance was accepted at p < 0.05. Experiments were repeated three times on different tissue preparations with three determinations in each replicate

A 1990 µl suspension of synaptoneurosomes was allowed to reach room temperature, thereafter 10 µl of Fura-2 AM (1 mM in DMSO) was added to produce a final concentration of 5 µM. Synaptoneurosomes were then incubated at 37 ºC for 30 min after which the suspension was centrifuged on a desktop centrifuge at 7000 g for 5 min and the supernatant decanted to remove all extracellular Fura-2 AM. The resulting pellet was resuspended in 2 mM CaCl2 containing buffer to obtain a final protein concentration of 0.6 mg/ml.

For the screening test, 10 mM stock solutions of the compounds in DMSO were prepared, with the control containing 1% DMSO and no test compound. The individual stock solutions were added in 2 µl portions to a 96 well plate in triplicate followed by the addition of 200 µl of synaptoneurosomal-Fura-2 AM solution prepared above. This gave rise to a final concentration of 100 µM of the compounds. The 96 well plate was shaken and incubated for 30 min at 37 ºC and used immediately after incubation. The measurement was then performed at 37 ºC in a 96 well plate using dual wavelength excitation at 340 nm and 380 nm. The resting fluorescence was measured at 510 nm after which the changes in fluorescence intensity following the addition of 10 µl KCl (140 mM) depolarization solution using auto-injectors were recorded over a period of 5 min. The changes in fluorescence indicated the effect of the test compound on calcium flux. The data obtained from the fluorescent readings of each well were expressed in the form of a ratio (340 nm reading / 380 nm reading). This ratio indicated the net movement of Ca2+ ions across the membrane as it represents the ratio between unbound-Ca2+ and Ca2+-bound to Fura-2 AM. The average ratio over a 10 second interval before stimulation was subtracted from the average ratio 10 second after stimulation to give the net change in Ca2+ movement (Nc) across the membrane. This calculation was performed for each individual well and the averages of wells containing the same test compound were calculated (NcAve). These averages of three independent experiments were used to calculate the percentage inhibition of the test compounds relative to the control by the use of the following equation:

% inhibition = [NcAve (Control) – NcAve (Test compound)] / NcAve (Control) x 100%

3. RESULTS & DISCUSSION

3.1. Chemistry

To obtain the required novel oxa- and aza-hexacyclododecylamine compounds (12, 18, 24, 25 and 27), bimolecular nucleophilic substitution, nucleophilic addition and selective reduction methods were utilized. The synthesis of the aza-HCD compound 12 commenced from a series of microwave-assisted SN2 reactions to produce the intermediate 2-(4-benzylpiperazin-1-yl)ethanamine (8, Scheme 1), as previously described [38Joubert, J.; Sharma, R.; Onani, M.; Malan, S. Microwave-assisted methods for the synthesis of pentacyclo [5.4.0.0 2,6 .0 3,10 .0 5,9] undecylamines Tetrahedron Lett., 2013, 54, 6923-6927.[http://dx.doi.org/10.1016/j.tetlet.2013.10.047] ]. Compound 8 was conjugated to the pentacycloundecane (PCU) ketal (9) [39Banister, S.D.; Moussa, I.A.; Jordan, M.J.; Coster, M.J.; Kassiou, M. Oxo-bridged isomers of aza-trishomocubane sigma (sigma) receptor ligands: Synthesis, in vitro binding, and molecular modeling. Bioorg. Med. Chem. Lett., 2010, 20(1), 145-148.[http://dx.doi.org/10.1016/j.bmcl.2009.11.019] [PMID: 19954972] ] through microwave irradiation at 100 °C, 150 W and 150 psi for 30 minutes to produce the PCU-imine (10). The PCU-imine was reduced using NaBH4 to produce the endo-amine (11) and the final aza-HCD (12) was obtained through hydrolysis and subsequent transannular cyclization using 3 M HCl/acetone.

To synthesize the oxa-HCD derivative (18), the obtained 2-aminoethanol-HCD structure (15, Scheme 2) [28Lemmer, H.J.R.; Joubert, J.; van Dyk, S.; van der Westhuizen, F.H.; Malan, S.F. S-nitrosylation and attenuation of excessive calcium flux by pentacycloundecane derivatives. Med. Chem., 2012, 8(3), 361-371.[http://dx.doi.org/10.2174/1573406411208030361] [PMID: 22530904] ] was reacted with methanesulfonyl chloride to provide a leaving group for reaction with the N-benzylpiperazine. Mesylate compounds generally show selectivity for amine groups over hydroxyl groups, but the NMR of compound 16 and the NMR, IR and MS data of 18 confirmed that the mesylate substitution of 16 took place on the hydroxyl moiety. This is probably due to steric hindrance from the hexacyclododecane structure at the amine. The benzyl piperidine (17) was then capable of displacing the methane-sulfonyl group and the final compound (18) was obtained using microwave-irradiation conditions (Scheme 2).

Compounds 21 and 22 were synthesised according to the methods proposed by Buschauer et al., (1985) [40Buschauer, A.; Postius, S.; Szelenyi, I.; Schunack, W. Isohistamine and homologs as components of H2-antagonists. 22. H2-antihistaminics. Arzneimittelforschung, 1985, 35(7), 1025-1029.[PMID: 2864932] ]. Intermediate 22 served as the functional moiety that was conjugated to 13 in order to obtain imine-PCU compound 23. Further reduction using NaCNBH3 and NaBH4 produced the desired aza-HCD (24) and oxa-HCD (25) respectively (Scheme 3). Compound 27 was synthesised by conjugating 13 and 26 followed by Dean-Stark dehydration and subsequent reductive amination using NaCNBH3 (Scheme 4).

Scheme 1
Reagents and conditions: (i) CH3CN, MW, 100 °C, 150 W, 100 psi, 5 min, 94%; (ii) CH3CN, MW, 100 °C, 150 W, 100 psi, 10 min, 92% yield from 5; (iii) EtOH, MW, 100 °C, 150 W, 150 psi, 30 min, quantitative yield; (iv) EtOH, NaBH4, rt, 8 h; (v) acetone, 3 M HCl(aq), 6 h, 53% [38Joubert, J.; Sharma, R.; Onani, M.; Malan, S. Microwave-assisted methods for the synthesis of pentacyclo [5.4.0.0 2,6 .0 3,10 .0 5,9] undecylamines Tetrahedron Lett., 2013, 54, 6923-6927.[http://dx.doi.org/10.1016/j.tetlet.2013.10.047] ].


Scheme 2
Reagents and conditions: (i) THF, MeOH, NaBH4, rt, 4 h, 32%; (ii) Mesyl chloride, DCM, Et3N, Et2O, -8 °C, overnight, 20%; (iii) CH3CN, K2CO3, EtOH, MW, 120 °C, 100 W, 90 psi, 10 min, 9.3%.


Scheme 3
Reagents and conditions: (i) Formic acid, 110 °C for 2 h, 74%; (ii) DMF, 3-chloropropylamine, NaOH, 80-90 °C, 2 h, 69%; (iii) THF, -10 °C, 10 min; (iv) benzene, Dean-Stark, 1 h; (v) MeOH/AcOH, NaCNBH3, rt, 2 h, 13%; (vi) THF/MeOH, NaBH4, rt, 8 h 35%;


Scheme 4
(i) THF, -10 °C, 10 min; (ii) benzene, Dean-Stark, 1 h (iii) MeOH/AcOH, NaCNBH3, rt, 2 h, 17%.


3.1.1. Sigma-1 Receptor Binding Affinity

σ1R binding affinity of the test compounds (Table 1) was evaluated according to the method by Matsumoto et al. In the experiments, rat liver membranes were used as the source of σ1Rs. [3H](+)-pentazocine was used as a radioligand and all stock solutions and procedures including data processing were adhered to as previously described [41Matsumoto, R.R.; Bowen, W.D.; Tom, M.A.; Vo, V.N.; Truong, D.D.; De Costa, B.R. Characterization of two novel sigma receptor ligands: antidystonic effects in rats suggest sigma receptor antagonism. Eur. J. Pharmacol., 1995, 280(3), 301-310.[http://dx.doi.org/10.1016/0014-2999(95)00208-3] [PMID: 8566098] ]. The final results were expressed as Ki and IC50 values and further compared to known σ1R ligands. The novel HCD compounds (12, 18, 24, 25 and 27) showed σ1R affinities in the same range as that described for similar HCD structures (NGP1-01 and 1-3, Table 1) with 5-[2-(4-benzylpiperazin-1-yl)ethyl]-5- azahexacyclo [5.4.1.02,6 .03,10 .04,8.09,12] dodecan-4-ol (12) showing the best affinity (67 nM). Amongst the tested compounds, the aza structures generally are more favourable for σ1R binding than their oxa counterparts (cf. 12 and 18; 24 and 25). With the inclusion of the amine heterocycle between the cage and the aromatic ring (12 and 18) σ1R affinity was retained (cf. 12 with 2 and 3) or even improved (cf. 18 with NGP1-01 and 1) (Table 1). The addition of the HCD scaffold showed a definite increase in σ1R binding affinity from the respective intermediate counterparts without the HCD scaffold (cf. 8, 12 and 18; 22 and 24). Compounds 24 and 25 that consisted of the HCD connected to a phenyl ether with a terminal piperidine moiety, when compared to compounds 12 and 18, showed a reduced affinity and suggested that the HCD linked through an amine-containing heterocycle connected to a benzyl moiety is preferable for σ1R affinity and supports the pharmacophore model suggested by Glennon et al. [42Glennon, R.A.; Ablordeppey, S.Y.; Ismaiel, A.M.; el-Ashmawy, M.B.; Fischer, J.B.; Howie, K.B. Structural features important for sigma 1 receptor binding. J. Med. Chem., 1994, 37(8), 1214-1219.[http://dx.doi.org/10.1021/jm00034a020] [PMID: 8164264] ] The aza compound 24 (Ki = 0.215 µM) compared to the oxa compound 25 (Ki = 11.855 µM) showed a significant increase in σ1R binding affinity which strongly suggests that the aza compound is preferable for the σ1R affinity. This preference for the aza-structure is further evident in the binding affinities observed for the aza-HCDs 2, 3, 12, 24 and 27. This is also in agreement with the previously published σ1R affinities for HCD derivatives [33Geldenhuys, W.J.; Novotny, N.; Malan, S.F.; Van der Schyf, C.J. 3D-QSAR and docking studies of pentacycloundecylamines at the sigma-1 (σ1) receptor. Bioorg. Med. Chem. Lett., 2013, 23(6), 1707-1711.[http://dx.doi.org/10.1016/j.bmcl.2013.01.069] [PMID: 23414839] , 39Banister, S.D.; Moussa, I.A.; Jordan, M.J.; Coster, M.J.; Kassiou, M. Oxo-bridged isomers of aza-trishomocubane sigma (sigma) receptor ligands: Synthesis, in vitro binding, and molecular modeling. Bioorg. Med. Chem. Lett., 2010, 20(1), 145-148.[http://dx.doi.org/10.1016/j.bmcl.2009.11.019] [PMID: 19954972] ]. Intermediate compound 21 has good affinity (Ki = 0.349 µM) as it possesses the necessary features of the pharmacophore model including the basic amine and the adjacent lipophilic region which support this result. It is also important to note that intermediate compound 22 (Ki = 0.251 µM), despite the lack of a second lipophilic binding area, still showed significant σ1R binding. Finally, the comparison of compound 12 (0.067 µM) with compound 27 (Ki = 0.469 µM) indicates that the addition of N-benzyl group is favoured for σ1R affinity. When the activities of 12 and 27 are compared to 2 and 3, it is clear that the inclusion of the amine heterocycle leads to σ1R binding affinity in the same range. This suggests that the additional amine heterocycles retained the optimal alignment of compound 12 and 27 for σ1R binding affinity as suggested by the pharmacophore model proposed by Glennon et al. [42Glennon, R.A.; Ablordeppey, S.Y.; Ismaiel, A.M.; el-Ashmawy, M.B.; Fischer, J.B.; Howie, K.B. Structural features important for sigma 1 receptor binding. J. Med. Chem., 1994, 37(8), 1214-1219.[http://dx.doi.org/10.1021/jm00034a020] [PMID: 8164264] ].

3.1.2. Sigma-1 Receptor Docking Studies

To gain insight into the mode of interaction between the compounds, σ1R, docking studies were performed using the Molecular Operating Environment (MOE) 2016 software package and the recently published crystal structure of the σ1R (PDB:5HK2) [43Schmidt, H.R.; Zheng, S.; Gurpinar, E.; Koehl, A.; Manglik, A.; Kruse, A.C. Crystal structure of the human σ1 receptor. Nature, 2016, 532(7600), 527-530.[http://dx.doi.org/10.1038/nature17391] [PMID: 27042935] ]. The docking results indicate that the HCU amine moiety seemed to play an important role in orienting the compounds for favorable hydrogen bonding interactions with several amino acids, specifically HIS154 and GLU172. For instance, compound 2, 3, 12 and 24 were found to form lipophilic interactions between the HCD carbons and HIS154, and the HC hydroxyl moiety with GLU172 (Fig. 2) through a hydrogen bond. Compound 18, an oxa-HCD showed an interaction with HIS154 but lacked the interaction with GLU172 as observed for the aza-HCDs 2, 3 and 12. This may explain why better σ1R affinity was observed for the aza-HCDs. Compound 8 without the HCD moiety did not show interactions with these two amino acids, which may account for the loss of affinity as compared to compounds 12 and 18 for instance. Compound 27, that lacks the benzyl group observed in 12 and 18, adopted an orientation where a hydrogen bond is observed between the hydroxyl moieties of the aza-HCD and TYR120. The Ki value of 27 was significantly higher compared to 12 and 18. This indicates that the benzyl moiety of 12 and 18 is important to enable optimal alignment of both the amine heterocycle and aza-HCD of these molecules within the σ1R binding pocket, enabling important interactions with HIS154 and GLU172.

3.2. Voltage-gated Calcium Channel Activity

All HCD amine compounds (Table 1) were screened at 100 µM for their potential inhibitory activity on the VGCC as previously described [44Zindo, F.T.; Barber, Q.R.; Joubert, J.; Bergh, J.J.; Petzer, J.P.; Malan, S.F. Polycyclic propargylamine and acetylene derivatives as multifunctional neuroprotective agents. Eur. J. Med. Chem., 2014, 80, 122-134.[http://dx.doi.org/10.1016/j.ejmech.2014.04.039] [PMID: 24769350] ] using the fluorescent ratiometric indicator Fura-2 AM. Fresh synaptoneurosomes were prepared from rat brain homogenate [45Hollingsworth, E.B.; McNeal, E.T.; Burton, J.L.; Williams, R.J.; Daly, J.W.; Creveling, C.R. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate-generating systems, receptors, and enzymes. J. Neurosci., 1985, 5(8), 2240-2253.[http://dx.doi.org/10.1523/JNEUROSCI.05-08-02240.1985] [PMID: 2991484] ] and incubated with Fura-2 AM. Thereafter the test compounds were incubated for 30 minutes and 140 mM KCl solution was added to depolarize the cell membranes to stimulate calcium influx. Calcium influx was then monitored based on the fluorescence intensity relative to that of a blank control (without inhibiting compound) over a 5 minute period. Two positive controls were included in the VGCC assay; nimodipine, a commercially available dihydropyridine calcium channel blocker and NGP1-01, the prototype HCD compound. Amantadine, a polycyclic NMDA receptor channel inhibitor, was also included as a negative control. Dose-response curves were plotted for selected compounds which showed a high degree of VGCC inhibition at 100 µM. The novel aza derivatives 24 and 27 exhibited comparable activities (IC50 < 10 µM) in the KCl initiated calcium flux assays to NGP1-01 (IC50 = 86 µM) and related HCD’s 1-3 (IC50 = 8 – 51 µM). The aza-HCD compound (12) showed moderate calcium flux inhibition (IC50 < 100 µM) and the two oxa-HCD derivatives 18 and 25 exhibited the weakest calcium flux inhibition (26% and 25%) at 100 µM (Table 1). From the VGCC results compounds 12, 24 and 27 can be considered as lead compounds to develop more potent calcium channel inhibitors.

Table 1
Summary of effects of the hexacyclododecylamine derivatives and related compounds on affinity for sigma-1 receptors and voltage gated calcium channel modulation.


Fig. (2)
Docking studies of compounds in σ1R. Compounds A) 2, B) 3, C) 8, D) 12, E) 18 and F) 27. The three amino acids that were found to play a role in binding of the HCD amines were HIS154, GLU172 and TYR120. The aza-HCD moiety provides for the optimal orientation to allow for hydrogen bonding with HIS154 and GLU172.


CONCLUSION

The synthesised HCD derivatives had inhibitory effects on calcium flux and thus decreased intracellular calcium concentration. In many cases, this effect might be linked to the compounds’ interaction with sigma receptors present in the synaptoneurosomal preparation and implicates σ1R antagonism. This is corroborated by the correlation observed between the VGCC inhibitory activity and the σ1R affinity of NGP1-01, 1, 2, 3 and between 12 and 18, and 24 and 25 – as the σ1R affinity for these compounds increased, the VGCC inhibition activity also increased. The decrease in intracellular calcium detected in the VGCC assays could thus in part be linked to the interaction of these compounds with σ1R in this assay. The compounds where this correlation is not observed will also be useful in identifying structural features to manipulate the specific or dual activities of future structures in this series. The observations and conclusions obtained from this study do not exclude interaction with other receptors such as the NMDA and AMPA/KA receptors and further investigation to characterise the calcium modulating effects of these compounds will follow.

Test compounds 12, 24 and 27 show potential to act as dual σ1R antagonists and VGCC blockers. Compound 12 exhibited VGCC inhibition in the same range as NGP1-01 with 24 and 27 showing activity comparable to the most potent aza-HCD compound 3 (Table 1). All three of these compounds showed nanomolar σ1R binding affinity (Ki = 0.067 – 0.469 µM). The dual and possible multifunctional activity of these compounds justifies further biological evaluation to establish their potential as multifunctional neurotherapeutic agents. A further important consideration when comparing the HCDs affinity to other σ1R ligands is their ability to cross the blood-brain barrier [34Zah, J.; Terre’Blanche, G.; Erasmus, E.; Malan, S. Physicochemical prediction of a brain-blood distribution profile in polycyclic amines. Bioorg. Med. Chem., 2003, 11, 3569-3578.[http://dx.doi.org/10.1016/S0968-0896(03)00365-1] [PMID: 1290 1901] , 35Prins, L.H.; du Preez, J.L.; van Dyk, S.; Malan, S.F. Polycyclic cage structures as carrier molecules for neuroprotective non-steroidal anti-inflammatory drugs. Eur. J. Med. Chem., 2009, 44(6), 2577-2582.[http://dx.doi.org/10.1016/j.ejmech.2009.01.030] [PMID: 19233517] ] and other relevant neuroprotective /neurological activities, providing a multitarget-directed drug approach. This study has also shown that the inclusion of an amine heterocycle into these structures could be a viable option in the design of new σ1R ligands with VGCC activity. The reported compounds could prove to be effective lead structures in the development of therapies for the prevention and treatment of neurodegenerative and neurological disorders.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No animals/humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors would like to thank the National Research Foundation and Medical Research Council of South Africa for research funding. The project described was supported by the National Institute Of General Medical Sciences (NIH: NIG MS), U54GM104942. The content is solely the responsibility of the authors and does not represent the official views of the funding bodies.

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Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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