Synthesis, Crystal Structure, DFT Studies, Docking Studies, and Fluorescent Properties of 2-(Adamantan-1-Yl)-2H-Isoindole-1-Carbonitrile

crystals

Article Synthesis, Crystal Structure, DFT Studies, Docking Studies, and Fluorescent Properties of 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile

Jacques Joubert Pharmaceutical Chemistry, School of Pharmacy, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa; [email protected]; Tel.: +27-21-959-2195

Received: 4 December 2018; Accepted: 15 December 2018; Published: 31 December 2018 Abstract: 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) has been identified as a neurobiological fluorescent ligand that may be used to develop receptor and enzyme binding affinity assays. Compound 1 was synthesized using an optimized microwave irradiation reaction, and crystallized from ethanol. Crystallization occurred in the orthorhombic space group P212121 with unit cell parameters: a = 6.4487(12) Å, b = 13.648(3) Å, c = 16.571(3) Å, V = 1458(5) Å3, Z = 4. Density functional theory (DFT) (B3LYP/6-311++G (d,p)) calculations of 1 were carried out. Results indicated that the optimized geometry was similar to the experimental results, with a root-mean-squared deviation of 0.143 Å. In this paper, frontier molecular orbital energies and net atomic charges are discussed with a focus on potential biological interactions. Docking experiments within the active site of the neuronal nitric oxide synthase (nNOS) protein crystal structure were carried out and analyzed. Important binding interactions between the DFT-optimized structure and amino acids within the nNOS active site were identified that explained the strong NOS binding affinity reported. Fluorescent properties of 1 were studied using aprotic solvents of different polarities. Compound 1 showed the highest fluorescence intensity in polar solvents, with excitation and emission maximum values of 336 nm and 380 nm, respectively.

Keywords: 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile; X-ray diffraction; density functional theory (DFT); molecular orbital calculations; ﬂuorescence; docking; neuronal nitric oxide synthase (nNOS); ﬂuorescent ligand

1. Introduction Radioligand binding techniques are extensively used to explore biological proteins involved in the pathophysiology of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease [1–3]. Neuroprotective targets, including the neuronal nitric oxide synthase (nNOS) enzyme [4], the N-methyl-D-aspartate (NMDA) receptor [3], and voltage gated calcium channels (VGCC) [5] have been widely studied using radioligands. Radioligand binding techniques have also afforded a high level of usefulness and sensitivity in the study of pharmacological proteins. However, the use of alternative approaches, such as fluorescent methods, to study enzyme-ligand and receptor-ligand binding interactions can provide information that may not be readily available using conventional radiopharmacology techniques, and can also avoid some of the disadvantages associated with radiopharmacology, such as high costs, health hazards, disposal, and possible methodological implications [6–9]. The development of small molecule fluorescent probes to study neurobiological events has thus become a research focus area that has received considerable attention [9,10]. Currently, there are no commercially available fluorescent ligands that are able to directly bind to the nNOS enzyme, NMDA receptor, and/or VGCC that can be used to develop binding affinity studies using fluorescent displacement assays.

Crystals 2019, 9, 24; doi:10.3390/cryst9010024 www.mdpi.com/journal/crystals Crystals 2018, 8, x FOR PEER REVIEW 2 of 12 nNOS enzyme, NMDA receptor, and/or VGCC that can be used to develop binding affinity studies using fluorescent displacement assays. 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1, Scheme 1) has shown the ability to bind to and inhibit the nitric oxide synthase (NOS) enzyme [11] and antagonize the NMDA receptor [12] and block VGCC [12]. Compound 1 also has inherent fluorescent properties because of the cyanoisoindole fluorophore in its structure [11,13]. The ability of 1 to bind to these targets and show strong fluorescence may enable the design of a neurobiological fluorescent ligand that could be used to develop receptor and enzyme binding affinity assays. This study therefore set out to improve the synthesis of 1, study its crystal structure and geometry in both the solid and free-form state, analyze Crystalsthe frontier 2018, 8 molecular, x FOR PEER orbitals REVIEW and atomic net charges, perform docking experiments to elucidate2 of the 12 potential biological binding interactions, and further explore its fluorescent properties. These studies nNOS enzyme, NMDA receptor, and/or VGCC that can be used to develop binding affinity studies will provide valuable information that may be used to develop fluorescent displacement assays using fluorescent displacement assays. utilizing compound 1. Crystals2-(Adamantan-1-yl)-22019, 9, 24 H-isoindole-1-carbonitrile (1, Scheme 1) has shown the ability to bind2 of to 12 and2. Results inhibit and the Discussionnitric oxide synthase (NOS) enzyme [11] and antagonize the NMDA receptor [12] and block VGCC [12]. Compound 1 also has inherent fluorescent properties because of the cyanoisoindole 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1, Scheme1) has shown the ability to bind to fluorophore2.1. Chemistry in its structure [11,13]. The ability of 1 to bind to these targets and show strong andfluorescence inhibit the may nitric enable oxide the synthase design (NOS) of a neurobiolo enzyme [11gical] and fluorescent antagonize ligand the NMDA that could receptor be [used12] and to blockdevelop2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile VGCC receptor [12]. Compoundand enzyme1 bindingalso has inherentaffinity assa fluorescentys. (1 )This was propertiesstudy obtained therefore because through set of outthe the to cyanoisoindoleconjugation improve the of fluorophoresynthesisamantadine of in1HCl, itsstudy with structure its o-phthaldialdehyde crystal [11,13 structure]. The ability and (OPD) ofgeometry1 into the bind presencein to both these the of targets sodiumsolid andand cyanide, show free-form strong under state, fluorescence optimized analyze maythemicrowave frontier enable molecular the(MW) design irradiation orbitals of a neurobiological and(150 atomic W, 100 net fluorescent °C,char 150ges, psi)perform ligand conditions that docking could (Scheme experiments be used 1). to develop Bothto elucidate OPD receptor and the andpotentialamantadine enzyme biological HCl binding are binding affinitycommercially interactions, assays. available. This and study furtherThe therefore re actionexplore set proceeded outits fluorescent to improve efficiently, properties. the synthesis providing These of 1the studies, study title itswillcompound crystal provide structure in valuable an excellent and information geometry yield inof that both91% may theafter solid be only us anded 10 free-formto min develop of reaction state, fluorescent analyze time. the displacementThis frontier is a significant molecular assays orbitalsutilizingimprovement andcompound atomic in yield net1. charges,and reduction perform in docking reaction experiments time compared to elucidate to the the conventional potential biological method bindingpreviously interactions, described, and where further a yield explore of its66% fluorescent was obtained properties. with Thesea reaction studies time will of provide 24 h [12]. valuable The information2.molecular Results andstructure that Discussion may of be 1 usedwas toconfirmed develop fluorescentby 1H and displacement13C nuclear magnetic assays utilizing resonance compound (NMR),1 high-. resolution mass spectroscopy, and X-ray diffraction. 2.1. Chemistry 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) was obtained through the conjugation of amantadine HCl with o-phthaldialdehyde (OPD) in the presence of sodium cyanide, under optimized microwave (MW) irradiation (150 W, 100 °C, 150 psi) conditions (Scheme 1). Both OPD and amantadine HCl are commercially available. The reaction proceeded efficiently, providing the title compound in an excellent yield of 91% after only 10 min of reaction time. This is a significant Amantadine HCl OPD Compound 1 improvement in yield and reduction in reaction time compared to the conventional method previouslyScheme described, 1.1.Reagents Reagents where and and conditions aconditions yield for of the 66%for synthesis thewas syntheob oftained 2-(adamantan-1-yl)-2H-isoindole-1-carbonitrilesis ofwith 2-(adamantan-1-yl)-2H-isoindole-1- a reaction time of 24 h [12]. The ◦ molecular(carbonitrile1): (i) MeOH,structure (1 NaCN,): (i) of MeOH, 1 H was2O, MW,NaCN,confirmed 150 H W,2O, 100 byMW, 1C,H 150 and psi,W, 13 C100 10 nuclear min, °C, 91%150 magneticpsi, yield. 10 OPD:min, re o-phthaldialdehyde.sonance91% yield. (NMR), OPD: o- high- resolutionphthaldialdehyde. mass spectroscopy, and X-ray diffraction. 2. Results and Discussion 2.2. Crystal Structure and Geometry Optimization 2.1. Chemistry A crystalline material was grown from EtOH at room temperature for 24 h. The rod-like crystal 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) was obtained through the conjugation of crystallized in the orthorhombic space group, P212121. The asymmetric unit cell of compound 1 amantadine HCl with o-phthaldialdehyde (OPD) in the presence of sodium cyanide, under optimized contained four molecules. The structure of 1 is shown in Figure 1, and the molecular crystal packing microwave (MW) irradiation (150 W, 100 ◦C, 150 psi) conditions (Scheme1). Both OPD and amantadine image is shown in Figure 2a,b. Selected bond lengths and bond angles are listed in Table 1. HCl are commercially available. The reaction proceeded efficiently, providing the title compound in an Amantadine HCl OPD Compound 1 excellent yield of 91% after only 10 min of reaction time. This is a significant improvement in yield and reductionScheme in reaction1. Reagents time comparedand conditions to the conventionalfor the synthe methodsis of previously2-(adamantan-1-yl)-2H-isoindole-1- described, where a yield of 66% wascarbonitrile obtained (1): with (i) MeOH, a reaction NaCN, time H of2O, 24 MW, h [12 150]. TheW, 100 molecular °C, 150 structurepsi, 10 min, of 191%was yield. confirmed OPD: o- by 1H and 13phthaldialdehyde.C nuclear magnetic resonance (NMR), high-resolution mass spectroscopy, and X-ray diffraction.

2.2. Crystal Structure and Geometry Optimization

A crystalline material was grown from EtOH at room temperature for 24 h. The rod-like crystal Figure 1. The asymmetric unit of compound 1 showing the atomic numbering scheme and 30% crystallized in thethe orthorhombicorthorhombic spacespace group,group, P2P211221121.. The The asymmetric asymmetric unit unit cell cell of of compound 1 containedprobability four molecules.displacement The ellipsoids structure of non-H of 1 is atoms. shown in Figure1 1,, andand thethe molecularmolecular crystalcrystal packingpacking imageCrystals is2018 shown, 8, x; doi: in FOR FigureFigure PEER2 2a,b.a,b. REVIEW SelectedSelected bondbondlengths lengthsand andbond bond angles angles are are listed www.mdpi.com/joulisted in in Table Table1 .1.rnal/crystals

Figure 1. TheThe asymmetric asymmetric unit unit of of compound 11 showing the atomic numbering scheme and 30% probability displacement ellipsoidsellipsoids ofof non-Hnon-H atoms.atoms.

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(a) (b)

FigureFigure 2.2. ((aa)) TheThe molecularmolecular crystalcrystal packingpacking ofof compoundcompound 11 viewedviewed alongalong thethe b-axis.b-axis. ((bb)) PartialPartial crystalcrystal packingpacking showing the H-bond (magenta) (magenta) and and C-H C-H⋯···π (lightπ (light blue) blue) intermolecular intermolecular interactions interactions along along the thec-axis. c-axis.

Table 1. Bond lengths (Å), angles (◦), and theoretical calculations of compound 1. Table 1. Bond lengths (Å), angles (°), and theoretical calculations of compound 1. a ◦ a BondBond Lengths Lengths (Å) (Å)X-ray X-ray Crystal Crystal DFTDFTa BondBond Angles Angles (°) ( ) X-ray X-ray Crystal Crystal DFT DFTa N1-C14N1-C14 1.3521.352 1.3601.360 C14-N1-C11 C14-N1-C11 108.77 108.77 109.13 109.13 N1-C1N1-C1 1.4981.498 1.4991.499 C1-N1-C14 C1-N1-C14 126.57 126.57 125.31 125.31 N1-C11 1.389 1.395 C2-C3-C10 110.53 109.52 N2-C19N1-C11 1.1361.389 1.1611.395 C2-C3-C10 C10-C3-C4 110.53 110.10 109.52 109.74 C3-C10N2-C19 1.5201.136 1.5391.161 C10-C3-C4 N1-C1-C2 110.10 109.54 109.74 109.72 C1-C2C3-C10 1.5311.520 1.5481.539 N1-C1-C2 C2-C1-C6 109.54 110.85 109.72 110.21 C11-C12C1-C2 1.3971.531 1.4081.548 C2-C1-C6 C6-C1-C7 110.85 108.39 110.21 108.39 C12-C13 1.433 1.433 C11-C12-C13 106.98 106.78 C11-C12 1.397 1.408 C6-C1-C7 108.39 108.39 C13-C18 1.412 1.417 C11-C12-C15 132.27 132.92 C5-C6C12-C13 1.5261.433 1.5421.433 C11-C12-C13 C13-C12-C15 106.98 120.67 106.78 120.30 C15-C16C13-C18 1.3751.412 1.3751.417 C11-C12-C15 N1-C11-C12 132.27 107.88 132.92 107.88 C16-C17C5-C6 1.4081.526 1.4251.542 C13-C12-C15 N1-C11-C19 120.67 125.49 120.30 125.69 C15-C16 1.375 1.375 N1-C11-C12C13-C18-C17 107.88 118.66 107.88 118.44 N2-C19-C11 176.20 176.79 C16-C17 1.408 1.425 N1-C11-C19C12-C15-C16 125.49 117.31 125.69 118.21 C13-C18-C17C15-C16-C17 118.66 122.20 118.44 121.68 a Density functional theory (DFT) optimization was performedN2-C19-C11 at B3LYP/6-311++G(d,p) 176.20 level.176.79 C12-C15-C16 117.31 118.21 Bond lengths, angles, and torsion angles of the adamantaneC15-C16-C17 ring system were122.20 consistent 121.68 with other previouslya Density reported functional compounds theory (DFT) [14 optimizatio]. The geometryn was performed of the cyano at B3LYP/6-311++G(d,p) moiety was within level. the usual range for nitriles. Generally, the bond lengths and bond angles of the isoindole structure were comparable to thoseBond reported lengths, for N-methylisoindole angles, and torsion [15 angles], suggesting of the adamantane that substitutions ring system to the pyrrolidine were consistent ring at with the C11other position previously do not reported signiﬁcantly compounds alter the [14]. solid-state The geom geometry.etry of the The cyano orientation moiety ofwas the within cyanoisoindole the usual moietyrange for in relationnitriles. to Generally, the adamantane the bond moiety lengths is deﬁned and bybond the angles bond angles of the of isoindole C1-N1-C11 structure (124.63◦ )were and C1-N1-C14comparable (126.56 to those◦), asreported well as thefor torsionN-methylisoindol angles of C1-N1-C11-C12e [15], suggesting (-177.56 that◦) andsubstitutions C1-N1-C14-C13 to the (178.93pyrrolidine◦). The ring crystal at the C11 packing position is not do planarnot signific andantly has an alter inversion-related the solid-state geometry. molecular The arrangement. orientation Anof the intermolecular cyanoisoindole hydrogen moiety bond in relation is observed to the at ad C2-H2Aamantane··· N2. moiety The Dis··· defineA distanced by the is bond 2.61 Å angles and the of D-HC1-N1-C11··· A angle (124.63°) is 161 ◦and. The C1-N1-C14 formation of(126.56°), the self-assembled as well as crystalthe torsion structure angles can of also C1-N1-C11-C12 be attributed to(- a177.56°) number and of weakC1-N1-C14-C13 C-H··· π interactions (178.93°). The between crystal thepacking adamantane is not planar and theand isoindole has an inversion-related moieties of the ⋯ ⋯ moleculesmolecular constitutingarrangement. the An crystal intermolecular assembly (Figure hydrogen2b). Thus,bond theis observed crystal structure at C2-H2A is builtN2. up The through D A ⋯ adistance combination is 2.61 of Å hydrogenand the D-H bondA and angle C-H is··· 161°.π interactions. The formation of the self-assembled crystal structure ⋯ can alsoA density be attributed functional to theorya number (DFT) of geometry weak C-H optimizationπ interactions of 1 was between performed the adamantane with the Gaussian09 and the (Gaussianisoindole Inc.,moieties Wallingford, of the molecules UK, 2016) constituting program package the crystal [16] using assembly Becke’s (Figure three parameters2b). Thus, Lee–Yang–the crystal structure is built up through a combination of hydrogen bond and C-H⋯π interactions.

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CrystalsA2019 density, 9, 24 functional theory (DFT) geometry optimization of 1 was performed with4 ofthe 12 Gaussian09 (Gaussian Inc., Wallingford, UK, 2016) program package [16] using Becke’s three Parrparameters exchange Lee–Yang–Parr correlation exchange functional correlation (B3LYP). Thisfunctional calculation (B3LYP). combines This calculation the hybrid combines exchange the functionalhybrid exchange of Becke functional [17] with of the Becke gradient-correlation [17] with the gradient-correlation functional of Lee, Yang,functional and Parrof Lee, [18 ]Yang, using and the 6-311G++(d,p)Parr [18] using basisthe 6-311G++(d,p) set. Previous basis studies set. have Previous shown studies that gas have phase shown calculations that gas correlatephase calculations well with crystalcorrelate structures; well with therefore, crystal struct no solventures; therefore, corrections no were solvent made corrections [19]. The X-ray were refined made [19]. data wereThe X-ray used torefined select data a crystal were unit used containing to select a the crystal starting unit geometries containing of the compound starting geometries1. The geometry of compound of compound 1. The1 wasgeometry optimized, of compound and the DFT-optimized 1 was optimized, molecule and the was DFT-optimized generated. Table molecule1 contains was the generated. data comparing Table 1 thecontains experimental the data andcomparing calculated the structureexperimental of 1. and calculated structure of 1. The C-C C-C bond bond lengths lengths within within the the isoindole isoindole stru structurecture was was in the in therange range of 1.41–1.42 of 1.41–1.42 Å and Å 1.42– and 1.42–1.431.43 Å for Åthe for crystal the crystal structure structure and at and the atcalculated the calculated B3LYP/6-311G++(d,p) B3LYP/6-311G++(d,p) level, respectively. level, respectively. This is Thisconsiderably is considerably shorter shorter than typical than typical C-C single C-C singlebonds bonds (1.54 Å) (1.54 [20]. Å) The [20]. experimental The experimental (1.36–1.40 (1.36–1.40 Å) and Å) andcalculated calculated (1.37–1.41 (1.37–1.41 Å) C-C Å) C-C bonds bonds of ofthe the adamantane adamantane structure structure were were within within the the reported characteristic range range [14]. [14]. The The C=C C=C bonds bonds were were found found to be to marginally be marginally longer longer than thanstandard standard C=C bond C=C bondlengths lengths (1.34 Å) (1.34 [20] Å) for [20 both] for the both experimental the experimental (1.52–1.54 (1.52–1.54 Å) and Å) calculated and calculated (1.53–1.55 (1.53–1.55 Å) results. Å) results. For ForN2-C19, N2-C19, the thecalculated calculated C≡ CN≡ (nitrile)N (nitrile) bond bond length length was was 1.16 1.16 Å, Å, which which is is longer longer than than the the experimental experimental value of 1.14 Å and is exactly the same as the bond length (1.16 Å) described for nitriles in chemistry literature. The The N1–C1, N1–C1, N1–C11, N1–C11, and and N1–C14 N1–C14 bond bond lengths lengths were were calculated calculated as as 1.50, 1.50, 1.40, 1.40, and and 1.36 1.36 Å at Å atthe the B3LYP/6-311G B3LYP/6-311G level, level, and and the the crystal crystal structure structure va valueslues were were 1.49, 1.49, 1.39, 1.39, and and 1.35 1.35 Å, Å, respectively. respectively. Normal C-N bond lengths were reported as 1.47 Å; however, thethe N1–C11 and N1–C14 bond lengths of the isoindole structure were considerablyconsiderably shorter, while N1–C1 was nearer to the normal length (Table(Table1 1).). TheThe shorter shorter bondbond lengthslengths occurredoccurred becausebecause theythey werewere partpart ofof the the unconjugated unconjugated isoindoleisoindole structure. These These bond bond lengths lengths were were consistent consistent wi withth similar isoindole structures reported in the literature [[15].15]. The The calculated bond bond angles correlated well with that of the crystal structure with a maximum deviation of 1.261.26°◦ for C1–N1–C14, which is calculated as 125.31125.31°◦, and the crystal structure value was 126.57126.57°.◦. The largest deviation between bond lengths was 0.025 Å for N2–C19.N2–C19. These deviations between bond lengths and bond angles, although relatively minor, occurred because the experimental results represented the molecule in its solid state and thethe calculationscalculations represented the molecule in its gaseousgaseous state.state. These differences in bond parameters can be ascribedascribed to the intermolecular interactions and crystal fieldfield that has linked the molecules together in its solid state, thus changing the physical and chemical parameters,parameters, compared to the molecule in its gaseous state. OverlayOverlay analysisanalysis was was performed performed using using YASARA YASARA version version 18.11.21 18.11.21 (YASARA (YASARA Biosciences Biosciences GmbH, Vienna,GmbH, Austria,Vienna, 2018)Austria, to globally 2018) to compare globally the comp structureare the obtained structure with obtained the theoretical with calculationthe theoretical with thecalculation experimental with the crystal experimental structure (Figurecrystal3 structure). A relatively (Figure low 3). root-mean-squared A relatively low root-mean-squared deviation (RMSD) valuedeviation of 0.143 (RMSD) Å was value obtained, of 0.143 confirming Å was obtained, that there confirming were minor that differences there were between minor the differences calculated andbetween experimental the calculated structures. and experimental structures.

Figure 3. 3. Atom-by-atomAtom-by-atom superimposition superimposition of DFT-optimized of DFT-optimized compound compound 1 (magenta)1 (magenta) calculated calculated using usingB3LYP/6-311++G B3LYP/6-311++G (d,p) on (d,p) the X-ray on the structure X-ray structure (blue) of (blue)1 (RMSD of 1= (RMSD0.143 Å). = DFT: 0.143 density Å). DFT: functional density functionaltheory. theory. 2.3. Frontier Molecular Orbitals and Atomic Net Charges 2.3. Frontier Molecular Orbitals and Atomic Net Charges The Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital The Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular (LUMO) are the most significant orbitals found in molecules. HOMO has electron-donating ability, and Orbital (LUMO) are the most significant orbitals found in molecules. HOMO has electron-donating LUMO is able to obtain an electron [21]. According to the frontier molecular orbital theory, HOMO and LUMO are significant factors that affect the biological activity, molecular reactivity, ionization, Crystals 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/crystals Crystals 2018, 8, x FOR PEER REVIEW 5 of 12 Crystals 2018, 8, x FOR PEER REVIEW 5 of 12 ability, and LUMO is able to obtain an electron [21]. According to the frontier molecular orbital Crystals 2019, 9, 24 5 of 12 theory,ability, andHOMO LUMO and is LUMO able to areobtain significant an electron factors [21]. that According affect the to biologicalthe frontier activity, molecular molecular orbital reactivity,theory, HOMO ionization, and andLUMO electron are significantaffinity of moleculesfactors that [22–27]. affect Thus, the biologicalfrontier orbital activity, energy molecular studies andcanreactivity, electronprovide ionization, affinityvaluable of and moleculesinsight electron into [ 22affinity –the27]. Thus,potentialof molecules frontier biological [22–27]. orbital energymechanismsThus, frontier studies of orbital can biologically provide energy valuable studiesactive insightcompounds.can provide into the Invaluable Figure potential 4, insight the biological distribution into mechanisms the andpotential ener ofgy biologically biologicallevels of HOMO mechanisms active and compounds. LUMO of biologically orbitals In Figure computed 4active, the distributionatcompounds. the B3LYP/6-311G and In Figure energy level 4, levels the for distribution ofcompound HOMO and and1 are LUMOener shown.gy orbitalslevels of computed HOMO and at the LUMO B3LYP/6-311G orbitals computed level for compoundat the B3LYP/6-311G1 are shown. level for compound 1 are shown.

(a) (b)

Figure 4. TheThe frontier frontier molecular molecular orbitals orbitals of of compound compound 1 1calculatedcalculated using using B3LYP/6-311++G B3LYP/6-311++G (d,p). (d,p). (a) (LUMOFigurea) LUMO 4.(− 1.550The (−1.550 frontier eV), eV), (b )molecular HOMO (b) HOMO (− orbitals5.625 (−5.625 eV). of eV).compound 1 calculated using B3LYP/6-311++G (d,p). (a) LUMO (−1.550 eV), (b) HOMO (−5.625 eV). As observedobserved inin FigureFigure4 ,4, both both the the LUMO LUMO and and HOMO HOMO is predominantly is predominantly localized localized at the at nitrile the nitrile and isoindoleand isoindoleAs observed moieties. moieties. in This Figure This indicates 4, indicate both that thes that theLUMO activitythe activity and associated HO associatedMO is withpredominantly wi thisth this molecule molecule localized could could generallyat thegenerally nitrile be attributedbeand attributed isoindole to the moieties.to cyanoisoindolethe cyanoisoindole This indicate structure,s thatstructure, withthe activity the with adamantane associatedthe adamantane moiety with this mostly moiety molecule providing mostly could providing structuralgenerally bulkstructuralbe attributed and/or bulk lipophilic to and/or the cyanoisoindole function.lipophilic This function. finding structure, This is in finding linewith with the is hypothesesadamantanein line with from hypothesesmoiety previous mostly from studies providing previous [11,12]. Itstudiesstructural is thus [11,12]. postulated, bulk Itand/or is thus based lipophilic postulated, on HOMO function. based and onThis LUMO HOMO finding findings, and is LUMOin that line the findings,with cyanoisoindole hypotheses that the cyanoisoindolefrom structure previous will formstructurestudies interactions [11,12]. will form It withis interactionsthus amino postulated, acids with lining basedamino the on acids active HOMO lining sites and ofthe the LUMO active NOS sitesfindings, enzyme, of the NMDAthat NOS the enzyme, receptor, cyanoisoindole NMDA and/or VGCC,receptor,structure and and/orwill in form so VGCC, doing, interactions giveand in rise so with to doing, its amino reported give acids rise biological liningto its reported the activity. active biological sites of the activity. NOS enzyme, NMDA receptor,The atomicand/or netVGCC, charges and ofin theso doing, DFT-optimized give rise to structure its reported areare shownshown biological inin FigureFigure activity.5 5.. TheThe twotwo N-atomsN-atoms have Thethe highestatomic highest netelectronegativity electronegativity charges of the andDFT-optimized and showed showed the the structurelowest lowest net netare atomic atomicshown charge in charge Figure values values 5. The(N1 (N1 two= −0.32 = N-atoms− 0.32e and e andN2have = N2 the−0.19 = highest− e)0.19 within e)electronegativity within the structure the structure and of 1 showed. Therefore, of 1. Therefore, the lowestit is expected it net is expectedatomic that charge the that N-atoms thevalues N-atoms (N1will = be − will0.32 the be emost and the mostfavoredN2 = favored−0.19 to interacte) towithin interact with the positively withstructure positively chargedof 1. chargedTherefore, amino amino ac itids is acidsexpectedwithin within the that enzyme the the enzyme N-atoms and receptor and will receptor beactive the active sites.most sites.However,favored However, to eveninteract eventhough with though positivelythe N1 the atom N1 charged atom showed showed amino a lower ac a lowerids withinnet net atomic atomic the enzyme charge charge andcompared compared receptor to to activeN2, N2, it it sites. will encounterHowever, someeven difficultythoughdifficulty the to N1 form atom interactions showed witha lower amino net acids atomic because charge of compared the structural to N2, hindrance it will imposedencounter byby some thethe bulky bulkydifficulty adamantane adamantane to form interactions group. group. Therefore, Therefore, with amino interactions interactions acids arebecause are expected expected of the to structural beto presentbe present hindrance at the at the N2 positionN2imposed position becauseby thebecause bulky the nitrilethe adamantane nitrile group group is group. protruding is protrudi Therefore, outwardsng interactionsoutwards from thefrom are isoindole expectedthe isoindole structure, to be structure,present and should at and the presentshouldN2 position present with anbecause with effective an the effective handle nitrile forhandlegroup interactions foris protrudi interactions to takeng outwards place.to take place. from the isoindole structure, and should present with an effective handle for interactions to take place.

Figure 5. The atomic net charges e of the DFT-optimized structure of 1. Hydrogens are not shown. Figure 5. The atomic net charges e of the DFT-optimized structure of 1. Hydrogens are not shown. 2.4. DockingFigure Studies5. The atomic net charges e of the DFT-optimized structure of 1. Hydrogens are not shown. 2.4. Docking Studies Molecular modelling studies using the NMDA receptor and VGCC were not carried out because 2.4. Docking Studies the fullMolecular protein modelling crystal structure studies of using these the targets NMDA have receptor not yet and been VGCC published. were not Therefore, carried out in orderbecause to furtherthe fullMolecular exploreprotein modellingcrystal the interaction structure studies proﬁle of using these and the targets biologicalNMDA have receptor potential/applicationnot yet and been VGCC published. were of not Therefore,1, carried molecular out in dockingorderbecause to experimentsthe full protein were crystal performed structure using of these the crystal targets structure have not (Protein yet been Data published. Bank I.D.: Therefore, 1OM5) of in rat order nNOS to Crystals 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/crystals Crystals 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/crystals Crystals 2018, 8, x FOR PEER REVIEW 6 of 12

further explore the interaction profile and biological potential/application of 1, molecular docking

Crystalsexperiments 2018, 8, x FORwere PEER performed REVIEW using the crystal structure (Protein Data Bank I.D.: 1OM5) of rat6 nNOS of 12 co-crystallised with the known nNOS inhibitor, 3-bromo-7-nitroindazole (3B7NI) [28] (Figures 6 and Crystals 2019, 9, 24 6 of 12 further7). 3B7NI explore crystallized the interaction in the active profile heme and (HEM750) biological site potential/application domain of nNOS. of 1, molecular docking experiments were performed using the crystal structure (Protein Data Bank I.D.: 1OM5) of rat nNOS co-crystallisedco-crystallised with with the the known known nNOS nNOS inhibitor, inhibitor, 3-bromo-7-nitroindazole 3-bromo-7-nitroindazole (3B7NI) (3B7NI) [28 [28]] (Figures (Figures6 and 6 and7). 3B7NI7). 3B7NI crystallized crystallized in the in the active active heme heme (HEM750) (HEM750) site site domain domain of nNOS. of nNOS.

Figure 6. The putative binding modes and orientation of 3-bromo-7-nitroindazole (3B7NI) (green) and compound 1 (red) within the rat neuronal nitric oxide synthase (nNOS) heme domain active site. The heme cofactor (HEM750) is shown in magenta, and the surrounding amino acids are shown in FigureFiguregrey. 6.6.The The putative putative binding binding modes modes and and orientation orientation of 3-bromo-7-nitroindazoleof 3-bromo-7-nitroindazole (3B7NI) (3B7NI) (green (green) and) compoundand compound1 (red 1) ( withinred) within the rat the neuronal rat neuronal nitric nitric oxide oxide synthase synthase (nNOS) (nNOS) heme heme domain domain active active site. Thesite. hemeThe heme cofactor cofactor (HEM750) (HEM750) is shown is shown in magenta, in magenta, and the and surrounding the surrounding amino amino acids are acids shown are shown in grey. in grey.

(a) (b)

Figure 7. Binding interactions and binding affinity (kcal/mol) of compound 1 and the co-crystallized ligand,Figure 3B7NI, 7. Binding within interactions the active site and of thebinding nNOS affinity heme domain. (kcal/mol) (a) Bindingof compound affinity 1( 1and) = −the5.722 co-crystallized kcal/mol, (b)ligand, Binding 3B7NI, affinity within( (3B7NI)a) the = −active6.000 site kcal/mol. of the nNOS heme domain. (a) (Bindingb) affinity (1) = −5.722 kcal/mol, (b) Binding affinity (3B7NI) = −6.000 kcal/mol. Inspection of the binding mode of 3B7NI indicated that two hydrogen bond contacts were formed Figure 7. Binding interactions and binding affinity (kcal/mol) of compound 1 and the co-crystallized through the interaction of the 7-NO moiety with residues Tyr588 and Met589 (Figure7). A π-π ligand,Inspection 3B7NI, of within the bindingthe active mode site2 of of the 3B7NI nNOS indicated heme domain. that (twoa) Binding hydrogen affinity bond (1) contacts= −5.722 were interaction was also observed between the pyrazole ring of the indazole structure and HEM750. formedkcal/mol, through (b) Binding the interaction affinity (3B7NI) of the = 7-NO −6.0002 moietykcal/mol. with residues Tyr588 and Met589 (Figure 7). A Theseπ-π interactionsinteraction was within also the observed active sitebetween could the be responsiblepyrazole ring for of the the potent indazole nNOS structure inhibitory and activityHEM750. µ reportedTheseInspection interactions for 3B7NI of the within (rat binding nNOS the active mode IC50 =site of 0.17 could3B7NIM) be indicated [responsible28]. The that binding for two the hydrogen potent afﬁnity nNOS of bond 3B7NI inhibitory contacts was found activitywere to be −6.000 kcal/mol. The DFT-optimized (B3LYP/6-311++G (d,p)) structure of 1 was used for formedreported through for 3B7NI the interaction (rat nNOS of IC the50 = 7-NO 0.17 µM)2 moiety [28]. with The residuesbinding Tyr588affinity andof 3B7NI Met589 was (Figure found 7). to A be furtherπ-−π6.000 interaction docking kcal/mol. was studies The also DFT-optimized (Figure observed7). Compoundbetween (B3LYP/6-311++G the pyrazo1 was dockedle ring (d,p)) of using the structure indazole the protocol of structure 1 was as described used and HEM750.for infurther the experimentalThese interactions section within and was the ableactive to site access could and be bind responsible to the active for the heme potent site domainnNOS inhibitory of nNOS, activity similar toCrystals 3B7NI. 2018 Two, 8, x; H- doi:π interactions FOR PEER REVIEW were observed between the adamantane moietywww.mdpi.com/jou and HEM750,rnal/crystals and reported for 3B7NI (rat nNOS IC50 = 0.17 µM) [28]. The binding affinity of 3B7NI was found to be between−6.000 kcal/mol. Gln478 andThe theDFT-optimized pyrrolidine ring(B3LYP/6-311++G of the isoindole (d,p)) structure. structure Compound of 1 was 1usedalso for showed further a hydrogen bond interaction between the electronegative nitrile moiety and Gln478. This result was as Crystals 2018, 8, x; doi: FOR PEER REVIEW www.mdpi.com/journal/crystals Crystals 2018, 8, x FOR PEER REVIEW 7 of 12

docking studies (Figure 7). Compound 1 was docked using the protocol as described in the experimental section and was able to access and bind to the active heme site domain of nNOS, similar to 3B7NI. Two H-π interactions were observed between the adamantane moiety and HEM750, and Crystals 2019, 9, 24 7 of 12 between Gln478 and the pyrrolidine ring of the isoindole structure. Compound 1 also showed a hydrogen bond interaction between the electronegative nitrile moiety and Gln478. This result was as expected,expected, based based on on the the frontier frontier molecular molecular orbital orbita andl netand atomic net atomic charges charges findings. findings. The binding The binding affinity ofaffinity1 was of found 1 was to found be − 5.722to be − kcal/mol.5.722 kcal/mol. The bindingThe binding interactions interactions and and binding binding affinity affinity of 1 of, which 1, which is similaris similar to 3B7NI,to 3B7NI, may may be thebe reasonthe reason for thefor significantthe significant NOS NOS inhibitory inhibitory activity activity reported reported (rat NOS (rat ICNOS50 =IC 0.2950 = 0.29µM) µM) [12]. [12]. A virtual A virtual derivative derivative of 1 ofdevoid 1 devoid of of the the nitrile nitrile group group was was also also docked, docked, and and showed showed nono bindingbinding interactions interactions within within the the active active site. site. ThisThis findingfinding demonstratesdemonstrates the the importanceimportance of of thethe nitrilenitrile moietymoiety in in this this structure structure for nNOSfor nNOS inhibitory inhibitory activity. activity. In addition, In addition, no covalent no intermolecularcovalent intermolecular biological interactionsbiological interactions were identified, were suggesting identified, thatsuggesting this molecule that this will molecule be suited will for fluorescentbe suited for displacement fluorescent studiesdisplacement to determine studies the to bindingdetermine affinity the binding of test compounds.affinity of test compounds. ItIt isis importantimportant to note that that 11 hashas only only been been evaluated evaluated on on a cru a crudede NOS NOS extract extract [12] [12 containing] containing the theinducible-, inducible-, endothelial-, endothelial-, and and neuronal neuronal NOS NOS isoforms isoforms,, and and further further biological biological studies studies are are necessary necessary in inorder order to to determine determine the the NOS NOS isoform isoform selectivity selectivity profile profile of this compound.compound. However,However, thisthis dockingdocking studystudy indicates indicates that that1 1should should show show significant significant nNOS nNOS inhibitory inhibitory activity activity which which is is vital vital in in order order to to treattreat neurodegenerativeneurodegenerative disorders disorders or or to to develop develop molecular molecular probes probes for for neurobiological neurobiological studies. studies.

2.5.2.5. FluorescentFluorescent PropertiesProperties TheThe excitationexcitation andand emissionemission spectraspectra ofof compoundcompound 11 recordedrecorded inin aa dimethyldimethyl sulfoxidesulfoxide (DMSO)(DMSO) solutionsolution (1 ×× 1010−5 −mol/L)5 mol/L) is shown is shown in Figure in Figure 8a. A 8BioTeka. A BioTekSynergy Synergy (BioTek (BioTekInstruments, Instruments, Inc., Winooski, Inc., Winooski,VT, USA, 2013) VT, USA, fluorescent 2013) fluorescentplate reader plate was used reader for was all fluorescence used for all measurements. fluorescence measurements. The excitation Theabsorption excitation maximum absorption and maximum the emission and maximum the emission were maximum found at were 336 foundnm and at 380 336 nm, nm andrespectively. 380 nm, respectively.The strong emission The strong can emission be explained can be explainedby compar bying comparing the fluorescent the fluorescent properties properties of the 1-nitrile of the 1-nitrilesubstituted substituted compound compound 1 with1 withthat thatof a of reported a reported unsubsti unsubstitutedtuted N-methylisoindole N-methylisoindole compoundcompound (emission(emission == 345345 nm) [15]. [15]. When When the the electron-withdrawi electron-withdrawingng nitrile nitrile group group is ispresent, present, a red-shift a red-shift in the in theemission emission maximum maximum and andincrease increase in fluorescence in fluorescence intensity intensity is observed. is observed. This is This because is because the charge the chargeseparation separation of the cyanoisoindole of the cyanoisoindole moiety is moiety increased is increased due to an due internal to an Stark internal effect Stark [29–31]. effect Therefore, [29–31]. Therefore,the nitrile themoiety nitrile is of moiety significant is of significantimportance importance in order for in compound order for compound1 to show strong1 to show fluorescent strong fluorescentproperties. properties.

FigureFigure 8.8.( a(a)) ExcitationExcitation andand emissionemission spectraspectra ofof compoundcompound1 1.(. (bb)) EmissionEmission spectraspectra ofof compoundcompound 11 showingshowing the the effect effect of of different different aprotic aprotic solvents solvents of of varying varying polarities. polarities.

FigureFigure8 b8b shows shows the the influence influence of of different different aprotic aprotic solvents solvents of of varying varying polarities polarities on on the the emission emission −5 spectraspectra ofof compoundcompound1 1(1 (1× ×10 10−5 mol/L). No No noticeable noticeable shift in the emissionemission maximummaximum ofof 11 waswas observedobserved inin thethe differentdifferent solvents.solvents. TheThe results,results, however,however, indicateindicate thatthat thethe solventsolvent polaritypolarity hadhad aa profoundprofound effecteffect onon thethe emission intensity of of 11.. The The fluorescence fluorescence intensity intensity significantly significantly decreased decreased as asthe the polarity polarity of of the the solvents solvents decreased decreased (Figure (Figure 88b).b). This initial studystudy indicatesindicates thatthat 11 exhibitsexhibits aa solvatochromic effect in the different solvents with varying polarity, most probably due to an internal chargeCrystals transfer2018, 8, x; phenomenon.doi: FOR PEER REVIEW www.mdpi.com/journal/crystals Fluorescent properties of the solid crystalline state of 1 were also determined. Interestingly, a definite red shift was observed for both the excitation (336 to 346 nm) and emission (380 to 400 nm) Crystals 2019, 9, 24 8 of 12 for the solid state of 1. In the solid state, there were intermolecular interactions present between the molecules in the crystal field (see Figure2b). These interactions altered the physical and chemical properties, which led to a change in the photochemical properties of 1 compared to when the compound was dissolved in organic solvents and intermolecular bonds were broken.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich or Merck. All the chemicals were of analytical grade and used without further purification. Compound 1 was characterized using nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. NMRs were obtained using a Bruker 400 MHz instrument. The chemical shifts are reported as δ values in ppm downfield from tetramethylsilane as an internal standard. Deuterated chloroform (CDCl3) was used as NMR solvent. The multiplicities of NMR signals are expressed as: s, singlet or m, multiplet. HRMS was recorded on a Waters API Q-Tof Ultima (Waters Corporation, Milford, MA, USA, 2011) electro-spray ionization mass spectrometer at 70 eV and 100 ◦C. The melting point was determined using a Stuart SMP-10 (Cole-Palmer, Staffordshire, UK, 2010) melting-point apparatus. The melting point is uncorrected. Microwave synthesis was performed using a CEM DiscoverTM (CEM Corporation, Matthews, NC, USA, 2014) focused closed vessel (maximum capacity = 30 ml) microwave synthesis system.

3.1.2. Synthesis of 2-(adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) Amantadine hydrochloride (0.600 g, 3.301 mmol) and sodium cyanide (NaCN, 0.132 g, 3.301 mmol) was dissolved in 15 ml methanol. Distilled water (1 mL) and o-phthaldialdehyde (0.442 g, 3.301 mmol) were added. The reaction mixture was placed in the microwave reactor and irradiated at 150 W, 100 ◦C, and 150 psi for 10 min. Upon cooling in an ice bath, a white precipitate formed and was ﬁltered and washed twice with cold methanol (10 ml) and twice with distilled water (10 ml) to yield the pure product (1) as a white amorphous solid in high yield (0.828 g, 2.997 mmol, 91%). Further crystallization by slow diffusion of a solution in EtOH was carried out to provide a rod-like single crystal suitable for X-ray diffraction analysis. The structure elucidation data is similar to that previously reported [12]. ◦ 1 Mp: 160 C; H NMR (400 MHz, CDCl3) δH: 7.69–7.64 (m, 2H), 7.51 (s, 1H), 7.26–7.19 (m, 1H), 7.12–7.04 13 (m, 1H), 2.45 (m, 6H), 2.32 (s, 3H), 1.85-1.80 (m, 6H); C NMR (100 MHz, CDCl3) δC: 150.6, 133.9, 125.3, 122.7, 122.4, 120.9, 117.8, 116.6, 115.9, 60.4, 43.00, 35.9, 30.0; HRMS m/z: Calculated for C19H20N2 (M + H); 276.16366, found 276.16265.

3.2. X-Ray Crystallography Single-crystal X-ray intensity data were collected on a Bruker 3-circle Apex II DUO X-ray diffractometer equipped with an INCOATEC IµS HB microfocus sealed tube (MoKα radiation λ = 0.71073 Å) fitted with a multilayer monochromator. Data were captured with a charge-coupled device (CCD) area detector. Data collection was carried out at 100 K using an Oxford Cryosystems cryostat (700 series Cryostream Plus) attached to the diffractometer. Data collection and reduction were carried out using the Bruker software package, APEX3 [32], using standard procedures. All structures were solved and refined using SHELX-2016 [33] employed within the X-Seed [34,35] environment. Hydrogen atoms were placed in calculated positions using riding models. Table2 contains the crystal data and refinement parameters. Crystals 2019, 9, 24 9 of 12

Table 2. Crystal data and reﬁnement parameters of the tile compound 1.

Crystal Data 1

Chemical formula C19H20N2 Mr 276.37 Crystal system, space group Orthorhombic, P212121 Temperature (K) 100 a, b, c (Å) 6.4487 (12), 13.648 (3), 16.571 (3) V (Å3) 1458.4 (5) Z 4 Radiation type Mo Kα µ (mm−1) 0.07 Crystal size (mm) 0.59 × 0.05 × 0.05 Diffractometer Bruker APEX II DUO CCD Tmin, Tmax 0.931, 1.000 No. of measured, independent, and observed [I > 2σ(I)] reﬂections 14040, 3372, 2528 Rint 0.072 −1 (sin θ/λ)max (Å ) 0.650 R[F2 > 2σ(F2)], wR(F2), S 0.074, 0.199, 1.04 No. of reﬂections 3372 No. of parameters 190 −3 ∆ρmax, ∆ρmin (e Å ) 0.69, −0.28

A CIF ﬁle containing complete information of the studied structure was deposited with CCDC, deposition number 1881043, and is freely available upon request from the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-1223-336033; e-mail: [email protected]) or from the following website: www.ccdc.cam.ac.uk/data_request/cif.

3.3. Theoretical Calculations A crystal unit within the crystal structure of 1 was selected as the starting structure for the DFT calculations. DFT-B3LYP/6-311G++(d,p) methods in Gaussian09 [16] was used for structural optimization. Solvent corrections were not made for these calculations. Vibration analysis showed no negative eigenvalues, indicating that the optimized structure represents a minimum on the potential energy surface. For the optimized structure, the HOMO and LUMO were drawn using Avogadro 1.2.0 [36,37]. Atomic net charges were also calculated using Avogadro 1.2.0.

3.4. Docking Studies The docking studies were performed using the rat nNOS heme domain crystal structure (Protein Data Bank I.D.: 1OM5). The docking method employed is similar to that reported by our group for studies on a protease enzyme [38] The Molecular Operating Environment (MOE) 2018 software suite [39] was used for docking studies with the following protocol. (1) The enzyme protein structure was checked for missing atoms, bonds, and contacts. (2) Removal of water molecules, 3D protonation, and energy minimization was carried out with parameters, force field: MMFF94X+solvation, gradient: 0.05, chiral constraint and current geometry. This minimized structure was used as the enzyme for docking analysis. (3) The DFT B3LYP/6-311++G (d,p) optimized structure of 1 was saved as a pdb file and imported into the MOE database. (4) Compound 1 was subsequently docked within the nNOS heme domain active site using the MOE Dock application. The active site was selected based on the proximity of the co-crystallized ligand, 3B7NI, with the help of the MOE Site Finder tool. The docking algorithm, which was chosen for these experiments, was based on induced fit docking to allow for flexible interactions of the test ligand with the protein. (5) The best binding pose of compound 1 was visually inspected, and the interactions with the binding pocket residues were analyzed. The selected parameters that were used to calculate the score and interaction of the ligand molecule with the nNOS enzyme were as follows: Rescoring function, London dG; Placement, Triangle matcher; Retain, 30; Crystals 2019, 9, 24 10 of 12

Refinement, Force field; Rescoring 2, London dG. The build-in scoring function of MOE, S-score, was used to predict the binding affinity (kcal/mol) of the ligand with the enzyme protein active site after docking.

4. Conclusions 2-(Adamantan-1-yl)-2H-isoindole-1-carbonitrile (1) was synthesized using an optimized microwave irradiation synthesis method in high yield. The three-dimensional structure of 1 was confirmed by single crystal X-ray diffraction. Using DFT calculations at the B3LYP level of theory and the 6-311G++(d,p) basis set, the molecular structure and electronic properties of 1 were deduced. Molecular docking studies indicated important interactions of 1 with the active site of the nNOS enzyme and showed that the nitrile moiety is imperative for nNOS inhibitory activity. The fluorescent properties of 1 were studied, and strong fluorescence was shown at 380 nm in polar aprotic solvents. This study has therefore provided valuable information of 1 that may be used to develop a pharmacological tool to investigate enzyme–ligand and/or receptor–ligand interactions utilizing modern fluorescent imaging techniques.

Funding: This research was funded by the National Research Foundation (NRF, South Africa, Grant Number: 11181) and the University of the Western Cape. Acknowledgments: E. Antunes from the Chemistry Department of the University of the Western Cape is acknowledged for NMR data collection. L. Loots from the Chemistry and Polymer Science Department of Stellenbosch University is acknowledged for the X-ray diffraction data collection and analysis. The Centre for High Performance Computing at the Council for Scientific and Industrial Research (South Africa) is acknowledged for granting access to Gaussian09 software. Conflicts of Interest: The authors declare no conflict of interest.

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