Novel Potent N-Methyl-d-aspartate (NMDA) Receptor Antagonists or

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Novel Potent N-Methyl-D-Aspartate (NMDA) Receptor Antagonists or #1 Receptor Ligands Based on Properly Substituted 1,4-Dioxane Ring Alessandro Bonifazi, Fabio Del Bello, Valerio Mammoli, Alessandro Piergentili, Riccardo Petrelli, Cristina Cimarelli, Maura Pellei, Dirk Schepmann, Bernhard Wünsch, Elisabetta Barocelli, Simona Bertoni, Lisa Flammini, Consuelo Amantini, Massimo Nabissi, Giorgio Santoni, Giulio Vistoli, and Wilma Quaglia J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01214 • Publication Date (Web): 02 Oct 2015 Downloaded from http://pubs.acs.org on October 7, 2015

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Journal of Medicinal Chemistry

Novel Potent N-Methyl-D-Aspartate (NMDA) Receptor Antagonists or σ1 Receptor Ligands Based on Properly Substituted 1,4-Dioxane Ring

Alessandro Bonifazi,†,§,┬ Fabio Del Bello,†,┬ Valerio Mammoli,† Alessandro Piergentili,† Riccardo Petrelli,† Cristina Cimarelli,‡ Maura Pellei,‡ Dirk Schepmann,§ Bernhard Wünsch,§ Elisabetta Barocelli,# Simona Bertoni,# Lisa Flammini,# Consuelo Amantini,ǁ Massimo Nabissi,⊥ Giorgio Santoni,⊥ Giulio Vistoli,^ and Wilma Quaglia*,†

†

Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via S.

Agostino 1, 62032 Camerino, Italy §

Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße

48, 48149 Münster, Germany ‡

Scuola di Scienze e Tecnologie, Università di Camerino, Via S. Agostino, 1, 62032 Camerino,

Italy #

Dipartimento di Farmacia, Università degli Studi di Parma, V.le delle Scienze 27/A, 43124

Parma, Italy ǁ

Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, via Madonna delle Carceri

9, 62032 Camerino, Italy. ⊥Scuola

di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Via Madonna

delle Carceri 9, 62032 Camerino, Italy ^

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25,

20133 Milano, Italy

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ABSTRACT Two series of 1,4-dioxanes (4-11 and 12-19) were rationally designed and prepared to interact either with the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA) receptor or with σ1 receptors, respectively. The biological profiles of the novel compounds were assessed using radioligand binding assays and the compounds with the highest affinities were investigated for their functional activity. The results were in line with the available pharmacophore models and highlighted that the 1,4-dioxane scaffold is compatible with potent antagonist activity at NMDA receptor or high affinity for σ1 receptors. The primary amines 6b and 7 bearing a cyclohexyl and a phenyl ring or two phenyl rings in position 6, respectively, were the most potent noncompetitive antagonists at the NMDA receptor with IC50 values similar to those of the dissociative anaesthetic (S)-(+)-ketamine. The 5,5-diphenyl substitution associated with a benzylaminomethyl moiety in position 2, as in 18, favoured the interaction with σ1 receptors.


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INTRODUCTION

The ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated cation channels (Na+, K+, Ca2+) activated by (S)-glutamate. This family can be subdivided into three types of receptors: AMPA, kainate, and N-methyl-D-aspartate (NMDA) receptors. NMDA receptors are cation channels with high calcium permeability involved in many aspects of the biology of higher organisms.1 They assemble as di- or tri-heteromers composed of different subunits, namely GluN1, GluN2A-D, GluN3A-B.2,3 The opening of the NMDA receptor-associated cation channel is controlled by various ligands interacting with different binding sites at the receptor, such as the ones for glutamate, glycine, polyamines, Zn2+, Mg2+, H+, as well as phencyclidine (PCP). This last binding site is located within the cation channel and compounds interacting with the PCP site behave as noncompetitive NMDA receptor antagonists by inhibiting the Ca2+-ions influx through the cation channel blockade.4 The activation of NMDA receptors plays an important role in the development of the central nervous system (CNS) and, thus, it is associated with physiological processes such as learning, memory, and neuroplasticity.5,6 Consequently, both NMDA receptor hypofunction and overstimulation are involved in several neurological disorders. Indeed, NMDA receptor hypofunction can be responsible for cognitive deficits, whereas permanent increased activation of NMDA receptors causes damage of the neuronal cells (excitotoxicity) and subsequent development of several CNS disorders, including stroke, ischemia, amyotrophic lateral sclerosis, Huntington’s, Parkinson’s and Alzheimer’s diseases, epilepsy, neuropathic pain, alcohol dependency, schizophrenia, and mood disorders.5,6 Therefore, NMDA receptor antagonists are useful for the treatment of such pathological conditions. Until today poor efficacy along with various undesirable CNS side effects of drugs targeting NMDA receptors, such as PCP, has limited their clinical success.1 The high affinity of PCP and its slow release from the binding site in the channel pore were considered responsible for its severe CNS side effects. The noncompetitive “dissociative anaesthetic” ketamine replaced PCP because of its ability to produce ACS Paragon Plus Environment


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profound analgesia and amnesia without slowing heart or breathing rate. It is used today in specialist anaesthesia, in pediatrics and, particularly, in veterinary medicine in all animal species.7 Recent findings suggest a complex relationship between NMDA and σ functions. Indeed, glutamatergic responses mediated by NMDA receptors can be modulated by σ receptor ligands, with σ receptor agonists enhancing NMDA neurotransmission8 or improving the NMDA antagonist PCP-induced cognitive deficits in mice.9 Hence, the NMDA antagonist ketamine has been reported to produce antidepressant effects in animal models and humans8 and, on the other hand, the combination between σ1 agonists and NMDA antagonists proved to be effective in animal models of depression.10 σ Receptors were initially classified as opioid receptor subtypes11 and subsequently it was postulated that they were identical to the PCP binding site at the NMDA receptor channel.12 Further studies demonstrated that they were distinct from both opioid receptors and PCP/NMDA receptor complex.13 At present, σ receptors are considered to be a unique receptor family comprising at least two pharmacologically distinct subtypes, namely σ1 and σ2 receptors,14,15 which are widely distributed in the CNS as well as in some peripheral tissues such as gastrointestinal tract, kidney, liver, lung, heart, and adrenal medulla.16,17 In addition, both σ receptor subtypes are overexpressed in many human and rodent tumour cell lines.18 Because of their widespread expression in many human tissues and their involvement in several pathophysiological processes, σ receptors have proved to be highly attractive pharmacological targets for the potential treatment of various pathologies, including neuropathic pain, depression, cocaine abuse, epilepsy, psychosis, as well as Alzheimer’s and Parkinson’s diseases.19 Moreover, σ1 antagonists and σ2 agonists may be useful as anticancer agents and radiolabeled ligands as selective tumour imaging agents.20 In 1966 the (dioxolan-4-yl)piperidine derivatives dexoxadrol and etoxadrol (Chart 1) were synthesized and evaluated as analgesic and anaesthetic agents. However, severe side effects stopped their clinical evaluation. Later on, they were demonstrated to bind with high affinity to ACS Paragon Plus Environment

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PCP binding site of the NMDA receptor.21 Recently, it has been demonstrated that the piperidine ring was not necessary for high NMDA receptor affinity because its replacement with aminomethyl or methylaminomethyl chains led to potent NMDA receptor antagonists.21 Ring and side chain homologues of aminomethyl analogues of dexoxadrol and etoxadrol resulted in 1,3dioxane compounds which showed high NMDA or σ1 receptor affinity, depending on the substitution at the nitrogen atom and the distance between the heterocycle oxygen atoms and the amino function.22 Among these compounds, the 4-(2-aminoethyl)-1,3-dioxane derivative 1 behaved as the most potent NMDA antagonist with an affinity value similar to those of dexoxadrol, etoxadrol, and PCP, whereas the N-benzyl-4-(2-aminoethyl)-1,3-dioxane derivatives 2 and 3 did not interact with the NMDA receptor, but showed high affinity for σ1 receptors (Chart 1). Moreover, in an in vivo assay, 2 behaved as a σ1 antagonist.22 The primary amino compound 1 and the N-benzyl derivatives 2-3 fit the features of the pharmacophore models proposed for the binding of ligands at the PCP binding site of the NMDA receptor and σ1 receptor, respectively.21,23,24 Indeed, while for the interaction with NMDA receptors a phenyl ring and a small basic amino moiety are necessary, the pharmacophore of σ1 receptor ligands consists of a secondary or tertiary amine flanked by two hydrophobic structural elements located at appropriate distances. For dexoxadrol, etoxadrol, and their ring and side chain 1,3-dioxane homologues high NMDA receptor affinity is obtained with compounds bearing a) two phenyl residues or one phenyl ring and an aliphatic residue in position 2, b) one or two oxygen atoms, c) a piperidine ring or aminomethyl, 2-aminoethyl, and 2-(methylamino)ethyl moieties in position 4 with the nitrogen atom separated by two or three carbon atoms from the oxygen atom in position 3.21 On the basis of the above observations, two series of analogues of 1-3 were rationally designed to selectively interact either with the PCP binding site of the NMDA (compounds 4-11) or σ1 receptors (compounds 12-19) (Chart 2). Such compounds will allow us to extend the structureactivity relationship (SAR) study and get further information about the role played by the ACS Paragon Plus Environment


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heterocycle oxygen atoms and the distances between the fragments constituting the pharmacophores, to better characterize these receptor systems. All the novel compounds bear the 1,4-dioxane ring, which is more stable in acidic solution than the acetalic 1,3-dioxolane of dexoxadrol and ethoxadrol and 1,3-dioxane of 1-3. Moreover, 1,4-dioxane nucleus, differently and properly substituted in positions 2 and 5 or 6, has already proved to be a suitable scaffold for building ligands selectively targeting different receptors.25-32 The first series of compounds 4-11, rationally designed to interact with the PCP binding site of the NMDA receptor, is characterized by an aminomethyl or 2-aminoethyl side chain in position 2 and alkyl and/or aryl substituents in positions 5 or 6 of the 1,4-dioxane ring. The second series includes compounds 12-19, the N-benzyl analogues of 4-11, in which the N-benzyl substituent has been selected to interact with σ1 receptors. The preparation of the cis and trans diastereomers allowed us to evaluate the role played by the relative configuration on the affinity/activity. Moreover, the novel 1,3-dioxane derivatives 20 and 21 were also prepared to complete the previously published 1,3-dioxane series, thus allowing us to compare the pharmacological properties of the novel 1,4-dioxanes with those of the corresponding 1,3-dioxanes. Though the shifting of the oxygen atom from the 3 to the 4 position of the dioxane nucleus seems to be a small change in the structure, this modification causes differences in acid stability and might affect affinity/activity and/or selectivity because the electronic properties of the compounds are completely different when the electronegative oxygen atom is in a different position. Therefore, ligand-based computational studies were conducted with a view to better investigating and rationalizing the structure-affinity relationships of 1,3- and 1,4-dioxanes and possible analogies/differences between the two series. Analogously to the previously reported 1,3-dioxanes22 the biological profiles of the novel compounds 4-21 were assessed using radioligand binding assays at the PCP binding site of the NMDA receptor and at σ1 and σ2 receptors. To determine the agonist or antagonist activity of the compounds with the highest affinities at the PCP binding site of the NMDA receptor and at σ1 ACS Paragon Plus Environment

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receptors, functional assays were performed. The functional activity at the PCP binding site of the NMDA receptor was investigated on mouse fibroblast L(tk-)-cells, stably expressing the NMDA receptor subunits GluN1a and GluN2A, evaluating the inhibition of the excitotoxicity induced by activation of NMDA receptors with (S)-glutamate and glycine.33 Because σ1 antagonists are reported to potentiate opioid antinociception in rats and mice,34,35 the algesiometric hot plate test36 was used to evaluate the ability of the ligand 18, showing the highest σ1 affinity, to modulate morphine antinociception. RESULTS Chemistry. The novel compounds 4-19 were synthesized according to the methods reported in Scheme 1 and were obtained as racemates. The opening of 2-ethyl-2-phenyloxirane (22)37 and 2-cyclohexyl-2-phenyloxirane (23)38 with allyl alcohol in basic conditions afforded alkenes 24 and 25, respectively, which were subjected to oxymercuration-reduction reaction with mercury(II) acetate, followed by an aqueous solution of iodine and potassium iodide, to yield the mixtures of diastereomers 29a/29b and 30a/30b, respectively. The pure diastereomers were separated by column chromatography. Alcohols 26a, 26b, 27a, and 27b, prepared as previously reported in the literature,27 were treated with tosyl chloride in pyridine to afford 28a, 28b, 32a, and 32b, respectively. Iodo derivatives 31 and 33 were prepared as previously reported.27 The amination of the intermediate 28-33 with benzylamine afforded the corresponding N-benzyl compounds 12-15, 17, and 18. The treatment of 12a, 12b, 14a, 14b, 15, 17a, 17b, and 18 with 10% Pd/C in 4.4% HCOOH/CH3OH yielded 4a, 4b, 6a, 6b, 7, 9a, 9b, and 10, respectively. An attempt to obtain primary amines 5a and 5b from 13a and 13b under the same conditions failed. Therefore, 5a and 5b were synthesized by treating iodo derivatives 29a and 29b with potassium phthalimide salt followed by hydrazinolysis. The treatment of iodo derivatives 3127 and 3327 with potassium cyanide, affording 34 and 35, followed by reduction with LiAlH4, gave the primary amines 8 and 11, which were condensed with



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benzaldehyde, followed by reduction of the intermediate imines with NaBH4, to yield 16 and 19, respectively. The N-benzyl derivatives 20 and 21 were obtained by treating 3639 and 3740 with benzylamine (Scheme 2). The stereochemical relationship between the 2-CH2NH2 chain and the 6-substituents of the diastereomers 5a, 5b and 6a, 6b were assigned by 1D Nuclear Overhauser Effect (NOE) measurements (1H NMR and NOE spectra are reported in the Supporting Information) (Figures 1S-4S). In the 1H NMR spectra of the oxalate salts of diastereomers 5a and 6a the axially oriented proton in position 3 at 3.18 and 3.11 ppm, respectively, showed two large coupling constants (J = 11.5 Hz and J = 10.3 Hz for 5a; J = 11.6 Hz and J = 11.1 Hz for 6a), one with the geminal equatorially positioned proton and one with the axially located proton in position 2. Consequently, the CH2NH2 chain in position 2 adopts the equatorial position. Moreover, an evident NOE between the axially oriented proton in position 2 and the protons of the ethyl or cyclohexyl group in position 6 was observed. Therefore, in 5a and 6a the stereochemical relationship between the 2side chain and the 6-phenyl substituent is cis (Figure 1). Analogously, as highlighted by the 1H NMR spectra of the oxalate salts of diastereomers 5b and 6b, the CH2NH2 chain in position 2 is equatorial because the axially oriented proton in position 3 at 3.21 and 3.16 ppm, respectively, showed two large coupling constants (J = 11.2 Hz and J = 10.4 Hz for 5b; J = 11.3 Hz and J = 10.8 Hz for 6b), one with the geminal equatorially oriented proton and one with the axially positioned proton in position 2. Moreover, an evident NOE between the axially oriented proton in position 2 and two protons of the aromatic ring in position 6 was observed, indicating a trans stereochemical relationship between the 2-side chain and the 6-phenyl substituent (Figure 1). Binding assays. The affinities of the novel compounds 4-21 to the PCP binding site of the NMDA receptor were determined in competition experiments with the radioligand [3H]-(+)-MK-801, which interacts with the ion channel binding site with high affinity and selectivity. In the assay, membrane preparations from pig brain cortex were used as receptor material. Nonspecific binding ACS Paragon Plus Environment

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was determined by saturation of the specific binding sites with a large excess of nontritiated (+)MK-801.41-43 In the σ1 assay, homogenates of guinea pig brains were used as receptor material. The σ1 selective ligand [3H]-(+)-pentazocine was employed as radioligand, and the nonspecific binding was determined by saturation of the specific binding sites with a large excess of (+)pentazocine.44,45 The σ2 assay was performed with rat liver membrane preparations and the nonselective radioligand [3H]-di-o-tolylguanidine ([3H]-DTG). In order to gain σ2 selectivity an excess of the σ1 selective ligand (+)-pentazocine was added to occupy the σ1 receptors. A concentration of 10 µM of nontritiated DTG was used for the determination of nonspecific binding. The affinity values, expressed as Ki values, of compounds 4-21 are reported in Tables 1 and 2 along with those of the reference compounds (S)-(+)-ketamine, PCP, dexoxadrol, etoxadrol, (+)-pentazocine, and DTG. Functional Studies. The antagonist activities, expressed as IC50 values, of the compounds 6a, 6b, and 7 along with those of the reference compounds (S)-(+)-ketamine, PCP, and dexoxadrol were assessed by inhibition of the excitotoxicity induced by activation of NMDA receptors with (S)glutamate and glycine (Table 1). In the assays, mouse fibroblast L(tk-)-cells, stably transfected with a dexamethasone inducible eukaryotic expression vector containing the encoding NMDA receptor subunits GluN1a and GluN2A, were employed. The expression of the functional NMDA receptors was induced by addition of dexamethasone in the presence of ketamine to inhibit damage of the cells. During the assay (S)-glutamate, glycine and different concentrations of the test compounds were added to the cells. After an incubation period of 12 h, the cell death rate was assessed by measuring the amount of a formazane dye corresponding to the amount of lactate dehydrogenase (LDH) released from lysed cells.46 In Vivo Study. The selected σ1 compound 18 was evaluated for its ability to modulate morphine antinociception using the algesiometric hot plate test (Figure 2).36 DISCUSSION Affinity for the PCP binding site of the NMDA receptor ACS Paragon Plus Environment


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An analysis of the results reported in Table 1 reveals that, among the compounds belonging to the aminomethyl or 2-aminoethyl series with one or two substituents in positions 5 or 6, 6a, 6b and 7 bound to the PCP binding site of NMDA receptor, 6b and 7 at σ2 and 10 at σ1, showing micromolar or sub-micromolar affinity values. Two bulky substituents are necessary in position 6 of the 1,4-dioxane ring to interact efficiently with the PCP binding site of the NMDA receptor. Indeed, the 6-monophenyl derivatives 4a and 4b and the 6-ethyl,6-phenyl-disubstituted compounds 5a and 5b were not able to bind to the receptor, whereas the 6,6-diphenyl derivative 7 showed a Ki value higher than those of dexoxadrol, etoxadrol, but similar to that of (S)-(+)ketamine. To investigate the nature of the site interacting with such a fragment of the molecule, one phenyl group of 7 was replaced by a cyclohexyl moiety, affording diastereomers 6a and 6b, which showed Ki values similar to that of 7. This observation suggests that only the steric bulk is necessary to result in high affinity, phenyl and cyclohexyl substituents having similar steric bulk (Molar Refractivity = 2.54 and 2.67, respectively), independently from the type of interaction between the two groups and the corresponding receptor site. Indeed, while the phenyl group is capable of partially contributing to binding at the PCP binding site through the formation of a charge-transfer complex, for the cyclohexyl group the steric bulk remains the major effect. Comparing the results obtained for the previously reported 1,3-dioxane derivatives21,22,39,47 and those for their 1,4-dioxane regioisomers of the present paper, different SARs were obtained. The most potent compound within the 1,3-dioxane series (1) showed a Ki value of 24 ± 2.0 nM, which is lower than that of the most potent compound within the 1,4-dioxane series (6b, Ki = 413 ± 20 nM), indicating that the 1,3-dioxane ring is more favourable for the interaction with NMDA receptor. The substituents constituting the hydrophobic part of the ligands and their distance to the basic amino moiety affected the affinity differently within the 1,3-dioxane and 1,4-dioxane series. Indeed, in the 1,3-dioxane series39 the 2-ethyl,2-phenyl-substituted compound (Ki = 449 ± 8 nM) showed a Ki value for NMDA receptors lower than those of the 2,2-diphenyl- (Ki = 1450 ± 90 nM) and 2-monophenyl- (Ki >10000 nM) derivatives. In the 1,4-dioxane series the order of affinity was ACS Paragon Plus Environment

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6,6-diphenyl (7, Ki = 712 ± 99 nM) < 6-ethyl,6-phenyl- (5, Ki >10000 nM) = 6-monophenyl- (4, Ki >10000 nM) derivative. For etoxadrol analogues a significantly higher NMDA affinity of transconfigured derivatives than of cis-configured derivatives was found.21 In the series of the novel 1,4-dioxanes, 6b (Ki = 413 ± 20 nM) with trans-configuration showed an affinity value slightly lower than that of 6a (Ki = 893 ± 40 nM) with cis-configuration. Therefore, while the axial orientation of the phenyl moiety in position 2 of the 1,3-dioxanes and etoxadrol has been demonstrated to be responsible for the high NMDA receptor affinity,47 such a requirement does not seem to be crucial for the 1,4-dioxanes. The 6,6-diphenyl-1,4-dioxane 7 showed a Ki value not significantly different from that of its 1,3dioxane analogue (Ki = 712 ± 99 nM and Ki = 1450 ± 90 nM, respectively).39 The elongation of their side chain caused different effects. In the case of 1,3-dioxanes such a modification did not affect NMDA affinities: the 2,2-diphenyl,4-aminomethyl derivative (Ki = 1450 ± 90 nM) showed an affinity value similar to that of its higher 4-(2-aminoethyl) side chain homologue (Ki = 2350 ± 180 nM).22 In the 1,4-dioxane series homologation of the side chain was detrimental, as the aminoethyl derivative 8, homologue of the aminomethyl derivative 7, did not reveal any NMDA receptor affinity. This result indicates that the distance between the hydrophobic moiety and the amino function plays a crucial role for the interaction with the PCP binding site. The 1,4-dioxane ring offers the opportunity to have the additional position 5 where it is possible to insert substituents, yielding derivatives useful to study such a distance. Compounds 9-11, the 5substituted analogues of 4, 7 and 8, respectively, were not able to bind to NMDA receptors (Ki >10000 nM), indicating that the PCP binding site of the NMDA receptor tolerated only the substitution in position 6 of 1,4-dioxanes. Indeed, only in this case the distance between the hydrophobic moiety and the amino function was the same as in its 1,3-dioxane regioisomer. To evaluate whether preferred conformations adopted by the two dioxane rings and/or stereoelectronic properties and/or different mutual arrangements of the nitrogen atom and the phenyl were responsible for different SARs obtained for 1,3- and 1,4-dioxane series computational ACS Paragon Plus Environment


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studies were conducted. Concerning the conformation adopted by the dioxane ring, it should be remembered that computational and spectroscopic studies revealed a similar behavior for 1,4- and 1,3-dioxane rings48,49 since the chair geometry is the preferred one for both rings followed by the twist forms while the boat conformations appear to be much less favored. With a view to analyzing the effects of the substituents in the examined compounds, density functional theory (DFT) calculations on some representative ligands were performed. Table 2S (supporting information) reports the so computed differences between the chair and twist conformations for the simulated ligands and shows a different behavior between 1,4- and 1,3-dioxane derivatives. Indeed the chair geometries of the 1,4-dioxane derivatives were always found to be more stable than the corresponding twist forms, with the energy difference which increases with the steric hindrance of the substituents, as seen in the diphenyl derivatives, and decreases in those derivatives whose chair conformation arranges the phenyl ring in axial position (as seen in 4b and 9a). In contrast, the 1,3-dioxane derivatives show less marked differences between chair and twist forms, with the twist geometry appearing to be the favored one in the diphenyl analogue. Such a different trend is already noticeable in the simple aminomethyl derivatives since the 1,3-dioxane analogue shows a difference between chair and twist forms which is about half that seen for 1,4dioxane analogue. The different behavior between 1,4- and 1,3-dioxanes is interpretable in terms of different intramolecular interactions between the ammonium head and the oxygen atoms, which in turn is reflected in different stereo-electronic properties between these rings. As exemplified by molecular electrostatic potential (MEP) surfaces reported in Figure 5S (supporting information), in both geometries of the 1,4-dioxane derivatives the ammonium head tightly interacts only with the oxygen atom in position 1, thus rendering accessible only the less electronegative oxygen atom in position 4. The same behavior is shown by the 1,3-dioxane derivatives in their chair form, while the ammonium head of the 1,3-dioxane derivatives in their twist form can contact both oxygen atoms which remain partly accessible and more electronegative. ACS Paragon Plus Environment

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This is confirmed by the computed Parr’s ω values50 (Table 2S in supporting information) which reveal that the twist conformation of the 1,3-dioxane derivatives shows the highest nucleophilicity and consequently the highest capacity to accept H-bonds. Remarkably, among the simulated 1,4dioxane derivatives, the only compound 7, which binds to NMDA, is characterized by the most nucleophilic chair conformation, thus suggesting that, besides the other examined key structural features, the capacity to elicit strong H-bonds can also play a key role. The analysis of the NMDA affinity values of the derivatives 4-11 was mainly based on a comparison of the best conformation as obtained by MonteCarlo simulations with that similarly computed for dexoxadrol. Considering the reduced flexibility of these derivatives, the study was focused on the lowest energy geometry. Thus, Table 3 reports the root-mean-square deviation (rmsd) values as computed superimposing each ligand with dexoxadrol and considering as matching atoms the three heteroatoms plus the centroid of the phenyl ring. Table 3 also compiles the key distances between the ammonium head and the centroid of the phenyl ring which should be around 3.6 Å as suggested by the pharmacophore model proposed by Carpy and co-workers.51 In fact, the pharmacophore also includes angle parameters which are indirectly taken into account by the computed superimpositions with dexoxadrol. Table 3 shows that the inactive compounds are characterized by an unsuitable arrangement of the key moieties which is reflected in both unsuitably high distance values between the nitrogen atom and the phenyl center (in most inactive compounds this distance is greater than 4.0 Å) and unsatisfactory superimpositions (as confirmed by rmsd values clearly greater than 1.0 Å). By contrast, compounds with higher affinities (including also 1) shows the key distance below 4.0 Å and the rmsd values around 1.0 Å, an acceptable value considering the unavoidable differences in the arrangement of the oxygen atoms when comparing dioxane and dioxolane derivatives. These differences are illustrated by Figure 3 which compares the superimposition of 7 and 10 with dexoxadrol and confirms that 7 (Figure 3A) achieves a better superimposition with dexoxadrol especially concerning the arrangement of the oxygen atoms. The only exception in these trends is represented by 5a which is inactive while showing the best ACS Paragon Plus Environment


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superimposition with dexoxadrol and an optimal distance between the nitrogen atom and the phenyl center (Table 3). This finding confirms that a second bulky substituent is here required for affinity, as highlighted by the SAR study. Affinity for σ1 and σ2 receptors As expected, the N-benzylamino-substituted 1,4-dioxanes did not show any affinity for the PCP binding site of the NMDA receptor (Table 2). All the 6-substituted derivatives 12-16 showed µM affinity values at σ1 and most of them also at σ2 receptors, with a preference for σ1 with respect to σ2 receptors: only 15 showed a slightly higher affinity for σ2 receptors. In particular, the derivative with the highest affinity at σ1 receptors was the cis diastereomer 12a with a Ki value slightly lower than that of its 1,3-dioxane regioisomer 20 and a significantly increased σ1/σ2 selectivity. This result indicates that the oxygen atom in position 1 of the 1,3-dioxane ring of 20 is important for the interaction with the σ2, but is not crucial for that with the σ1 receptor. The introduction of an additional phenyl ring in both 12a and 20, affording 15 and 21, respectively, produced an inversion of selectivity between the two σ receptor subtypes, with 21 being the most σ2 selective compound (σ1/σ2 = 27). The increase of the steric bulk, obtained by replacing the hydrogen atom in position 6 of the 1,4-dioxane ring of 12a and 12b with an ethyl, cyclohexyl or phenyl group, affording 13-15, respectively, caused a gradual decrease in σ1 receptor affinity. The stereochemistry significantly affected the interaction with the σ1 receptor subtype only when one phenyl group is in position 6, the cis-configured 12a showing a Ki value lower than that of the trans-configured 12b. The observation that the 1,3-dioxane 2 (Chart 1) showed an affinity for σ1 receptor higher than that of its lower side chain homologue 20 confirmed that the distance between the two lipophilic portions plays a crucial role in the interaction with the receptor site. More productive was the substitution in position 5 that led to an improvement in affinity and selectivity for σ1 receptors. Indeed, the 5-monophenyl-derivative 17b showed a Ki value lower than that of its 6-phenyl analogue 12b and an increased selectivity for σ1 over σ2 receptors (σ2/σ1 > 270). The stereochemical relationship between the phenyl ring in position 5 and the side chain in position 2 ACS Paragon Plus Environment

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affected both affinity and selectivity for σ1 over σ2 receptors, trans diastereomer 17b showing affinity for σ1 and selectivity over σ2 higher than those of the cis diastereomer 17a. The steric bulk in position 5 caused an effect contrary to that obtained in position 6, the 5,5-diphenyl analogue 18 being the most promising compound with the highest affinity (Ki = 7.37 ± 0.3 nM), similar to that of the reference compound (+)-pentazocine, and good selectivity for σ1 over σ2 receptors (σ2/σ1 = 136). The elongation of the side chain of 18, affording 19, maintained the affinity for σ2 but was detrimental for the interaction with σ1 receptors. Taken together all the results confirm that the distance between the two hydrophobic portions in 1,4-dioxanes is crucial for a high affinity for σ1 and selectivity over σ2 receptors and is optimal in compounds bearing a benzylaminomethyl side chain in position 2 and one phenyl group with trans-configuration or two phenyl groups in position 5 of the 1,4-dioxane ring (17b and 18, respectively). With regard to σ1 affinity of the derivatives 12-21, Table 4 compiles the key intramolecular distances which define the Glennon’s pharmacophore model.24 Compounds 2-3 are also included in Table 4 for extended comparison. The model includes a central basic group with two lateral hydrophobic regions located at different distances. As previously proposed by Gilligan and recently by Zampieri,52,53 the model can be completed by a hydrogen bonding acceptor group located at a well-defined distance from the central basic nitrogen atom. Hence, Table 4 includes four key distances: (1) distance between the nitrogen atom and the proximal hydrophobic region (dN-Ph1, optimal value = 3 ± 1 Å), (2) distance between the nitrogen atom and the distal hydrophobic region (dN-Ph2, optimal value = 6 ± 2 Å), (3) distance between the two hydrophobic regions (dPh1-Ph2, optimal value = 10 ± 2 Å), (4) distance between the nitrogen atom and the Hbonding group (dN-HB, optimal value = 3 ± 1 Å). Moreover and considering the flexibility of the simulated compounds, such an analysis is performed on all the non-redundant conformers as derived by MonteCarlo simulations and, thus, Table 4 reports for each monitored distance the mean and range values. The compiled values clearly show that all simulated ligands are able to assume suitable values concerning the distances between the nitrogen atom and the proximal ACS Paragon Plus Environment


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hydrophobic region as well as between the nitrogen atom and the H-bonding group with the ligand’s flexibility having marginal effects on these distances. In contrast, the other two distances show a noteworthy variability and in some cases fail to remain within the allowed ranges. When considering the determined affinity, one may note that the compounds with higher affinities show a rather large dN-Ph2 mean value combined with very narrow range values, thus suggesting that they are characterized by stably extended conformations. Conversely, the ligands with lower affinity values are endowed with more folded geometries and, despite being more flexible (as parameterized by the corresponding range values), they show unsuitably low dN-Ph2 mean values. The same trend is noticeable in the distance between the two hydrophobic regions even though the overall flexibility renders the differences between folded and extended geometries less evident. This trend is confirmed by the N-benzyl-4-(2-aminoethyl)-1,3-dioxane derivatives 2 and 3, which, while possessing a marked flexibility, show satisfactorily high dN-Ph2 mean values which are clearly ascribable to their longer aminoethyl linker and can rationalize their higher affinity for σ1 receptors compared to the derivative 20. The comparison of the three considered subsets (2,6substituted-1,4-dioxanes, 2,5-substituted-1,4-dioxanes, and 1,3-dioxane analogues, as exemplified by 15, 18 and 21, respectively) reveals that the suitably extended conformations are assumed by 2,5-substituted-1,4-dioxane ligands probably because the reciprocal arrangement of the substituents as well as the steric hindrance imposed by the dioxane ring prevents the intramolecular charge transfer interaction, thus favoring more extended conformations. In contrast, in the other derivatives (i.e. 2,6-substituted-1,4-dioxanes, and 1,3-dioxanes) the arrangement of the substituents permits their reciprocal approaching, thus favoring more folded conformations. The discussed differences between the three subsets are further clarified by Figure 4 which reports the superimposition of 15, 18 and 21 with haloperidol as computed by considering as matching atoms the heteroatoms plus the centroids of the phenyl rings. One may observe that only 18 assumes an extended conformation (Figure 4B) which permits a satisfactory superimposition with haloperidol, while the folded conformations of 15 and 21 cannot be conveniently superimposed with ACS Paragon Plus Environment

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haloperidol (Figures 4A and 4C, respectively) especially concerning the arrangement of the corresponding phenyl rings. Functional activities at NMDA and σ1 receptors Several NMDA receptor antagonists have been reported to require agonist binding and channel opening to get access to the binding site within the cation channel. The channel closure and the subsequent agonist dissociation can occur while the blocker is bound, trapping the antagonist within the receptor channel. Ketamine, binding to a very deep binding site in the ion channel, has been classiﬁed as a ‘full-trapping’ channel blocker.54 This peculiarity justifies the strong antagonist potency of ketamine observed in the excitotoxicity assay despite a not very high affinity in the binding studies. The observation that compounds 6a, 6b, and 7, though structurally unrelated to (S)-(+)-ketamine, showed similar Ki affinity values prompted us to evaluate their functional activity at NMDA receptor. Their IC50 are reported in Table 1 along with those of (S)(+)-ketamine, PCP, and dexoxadrol as reference compounds. An analysis of the data showed that the 1,4-dioxanes 6a, 6b, and 7 behaved as potent NMDA receptor antagonists. In particular, compound 6b, endowed with the highest affinity, was also the most potent antagonist with cytoprotective potential not significantly different from those of the reference channel blockers. As mentioned above, the noncompetitive NMDA antagonist ketamine is clinically used in specialist anaesthesia and in veterinary medicine and it is also effective at low doses for the treatment of neuropathic pain which is notoriously difficult to treat.7 Compounds 6b and 7, for which a full-trapping binding profile might be hypothesized, showed low binding affinities and high antagonist activities which were superimposable to that of (S)-(+)-ketamine. Therefore, due to the therapeutic use of (S)-(+)-ketamine, these compounds would deserve a deeper investigation and might be considered as novel lead compounds for the design of NMDA receptor antagonists with a promising therapeutic potential and favourable side effect profile. Previous studies have demonstrated the involvement of σ1 receptors in opioid antinociception. In particular, it has been reported that σ1 antagonists are able to enhance the antinociception due to ACS Paragon Plus Environment


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opioid agonists in mice and rats.34,35,55 In this study, we provide evidence that compound 18, selected on the basis of its high σ1 affinity and selectivity over the σ2 receptor, was able to increase significantly morphine-induced antinociception. In fact, as shown in Figure 2, 18 had no antinociceptive effect when given alone up to 50 mg/kg, but significantly improved the antinociceptive response induced by morphine, as evidenced by the increased reaction latencies expressed as %MPE in the hot plate test. Indeed, while pre-treatment with compound 18 at 30 mg/kg did not modify the antinociceptive effect produced by a sub-threshold dose of morphine (4 mg/kg), at the highest tested dose (50 mg/kg) 18 significantly increased the antinociception produced by the opiate agent until 60 minutes after its injection. Therefore, according to the literature, our in vivo data provided circumstantial evidence for the σ1 antagonist profile of compound 18.22,55 CONCLUSION The present study highlights that the 1,4-dioxane scaffold is compatible with potent antagonist activity at NMDA receptor or high affinity for σ1 receptors and that the SARs obtained for the 1,4dioxanes are different from those of their 1,3-dioxane regioisomers. With respect to 1,3-dioxane series it was possible to obtain both cis and trans configured 1,4-dioxane diastereomers, which allowed us to value the role of stereochemistry on the affinity/activity. The selective interaction with the PCP binding site of the NMDA or σ1 receptors depends on the substituents in positions 5 or 6 and the amino function. Indeed, 6b and 7 bearing a cyclohexyl and a phenyl ring or two phenyl rings in position 6, respectively, and a primary aminomethyl moiety in position 2 showed the highest affinities and were potent noncompetitive antagonists at the NMDA receptor with Ki and IC50 values similar to those of the dissociative anaesthetic (S)-(+)-ketamine, while the 5,5diphenyl substitution and a benzylaminomethyl moiety in position 2, affording the σ1 ligand 18, favoured the interaction with the σ1 receptor subtype. Computational studies showed that the role of these key moieties and of their mutual arrangements can be conveniently rationalized by exploiting well-known pharmacophore models as well as by considering the stereo-electronic ACS Paragon Plus Environment

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differences due to the chair and twist conformations of the dioxane rings. Therefore, 6b and 7 as well as 18 might be considered novel starting points for the design of antagonists for NMDA or σ1 receptor ligands, respectively, with promising therapeutic potential. Given the complex relationship between NMDA and σ1 functions, such compounds may be useful tools to characterize these receptor systems and to better understand the physiological role in which they are involved. The synthesis of the enantiomers of these compounds is already being planned to evaluate the role of chirality on affinity/activity at NMDA and σ1 receptors. EXPERIMENTAL SECTION Chemistry. Melting points were taken in glass capillary tubes on a Büchi SMP-20 apparatus and are uncorrected. IR and NMR and NOE spectra were recorded on Perkin-Elmer 297 and Varian Mercury AS400 instruments, respectively. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), d (doublet), dd (double doublet), t (triplet), or m (multiplet). IR spectral data (not shown because of the lack of unusual features) were obtained for all compounds reported and are consistent with the assigned structures. The microanalyses were recorded on FLASH 2000 instrument (ThermoFisher Scientific). The elemental composition of the compounds agreed to within ±0.4% of the calculated value. When the elemental analysis was not included, crude compounds were used in the next step without further purification. Chromatographic separations were performed on silica gel columns (Kieselgel 40, 0.040-0.063 mm, Merck) by flash chromatography. The term “dried” refers to the use of anhydrous sodium sulfate. Chemical names were generated according to IUPAC Nomenclature of Organic Chemistry (http://www.acdlabs.com/iupac/nomenclature). The purity of the novel compounds was determined by combustion analysis and was ≥95%. (±)-cis-(6-Phenyl-1,4-dioxan-2-yl)methanamine (4a). A solution of 12a (0.70 g, 2.47 mmol) in 4.4% HCOOH/MeOH (35 mL) was added dropwise to a mixture of 10% Pd/C (1.80 g) in 4.4% HCOOH/MeOH (70 mL). The mixture was stirred overnight at room temperature, under nitrogen atmosphere. After the catalyst was filtered off over celite and washed with MeOH, the solvent was ACS Paragon Plus Environment


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evaporated and the residue was taken up with 2N NaOH and extracted with CHCl3. The organic phase was dried over Na2SO4. After evaporation of the solvent the residue was purified by column chromatography eluting with EtOAc/MeOH (95:5) to obtain an oil (0.15 g; 31% yield). 1H NMR (CDCl3): δ 1.67 (br s, 2H, NH2, exchangeable with D2O), 2.80 (d, J = 5.5 Hz, 2H, CH2N), 3.40 (m, 2H, 3-CH2), 3.82 (m, 3H, 2-CH, 5-CH2), 4.69 (dd, J = 10.3, 2.7 Hz, 1H, 6-CH), 7.33-7.40 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from MeOH: mp 187-189 °C. Anal. (C11H15NO2.H2C2O4) C, H, N. (±)-trans-(6-Phenyl-1,4-dioxan-2-yl)methanamine (4b). This compound was prepared starting from 12b (1.0 g; 3.53 mmol) following the procedure described for 4a: an oil was obtained (0.29 g; 43% yield). 1H NMR (CDCl3): δ 1.58 (br s, 2H, NH2, exchangeable with D2O), 2.79 (dd, J = 13.3, 8.7 Hz, 1H, HA CH2N), 3.22 (dd, J = 13.3, 5.0 Hz, 1H, HB CH2N), 3.62-3.95 (m, 5H, 2-CH, 3-CH2, 5-CH2), 4.84 (dd, J = 7.6, 3.1 Hz, 1H, 6-CH), 7.25-7.43 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from MeOH: mp 201-202 °C. Anal. (C11H15NO2.H2C2O4) C, H, N. (±)-cis-(6-Ethyl-6-phenyl-1,4-dioxan-2-yl)methanamine (5a) A mixture of 29a (1.5 g, 4.52 mmol), potassium pthalimide salt (1.06 g, 5.72 mmol) and K2CO3 (0.90 g, 6.51 mmol) in DMF (25 mL) was heated to 80 °C for 3 h. The reaction mixture was filtered off over celite and the filtrate was diluted with EtOAc. The organic phase was washed with water, brine and dried over Na2SO4. After the evaporation of the solvent, the crude cis-2-((6-ethyl-6-phenyl-1,4-dioxan-2yl)methyl)isoindoline-1,3-dione was dissolved in THF (40 mL) and a solution of hydrazine hydrate (1.28 g, 25.57 mmol) in THF (30 mL) was added dropwise. After the reaction was refluxed for 2 h, the suspension was filtered off over celite and washed with THF. After evaporation of the filtrate, the residue was purified by column chromatography eluting with EtOAc/MeOH (9:1) to obtain an oil (0.56 g; 56% yield). 1H NMR (CDCl3): δ 0.69 (t, J = 7.3 Hz, 3H, CH3), 1.62 (m, 1H, HA CH2), 2.11 (br s, 2H, NH2, exchangeable with D2O), 2.62 (m, 1H, HB CH2), 2.73 (m, 2H, CH2N), 3.21 (m, 2H, 3-CH2), 3.75 (m, 2H, 2-CH, HA 5-CH2), 3.85 (m, 1H, HB ACS Paragon Plus Environment

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5-CH2), 7.19-7.35 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 153-155 °C. 1H NMR (DMSO): δ 0.54 (t, J = 7.3 Hz, 3H, CH3), 1.68 (m, 1H, HA CH2), 2.47 (m, 1H, HB CH2), 2.82 (dd, J = 13.1, 8.2 Hz, 1H, HA CH2N), 2.96 (dd, J = 13.1, 3.4 Hz, 1H, HB CH2N), 3.18 (dd, J = 11.5, 10.3 Hz, 1H, HA 3-CH2), 3.21 (d, J = 11.3 Hz, 1H, HA 5-CH2), 3.82 (dd, J = 11.5, 2.7 Hz, 1H, HB 3-CH2), 3.92 (d, J = 11.3 Hz, 1H, HB 5-CH2), 4.11 (m, 1H, 2-CH), 6.25 (br s, 2H, NH2, exchangeable with D2O), 7.20-7.57 (m, 5H, ArH). Anal. (C13H19NO2.H2C2O4) C, H, N. (±)-trans-(6-Ethyl-6-phenyl-1,4-dioxan-2-yl)methanamine (5b) This compound was prepared starting from 29b (4.30 g, 12.94 mmol) following the procedure described for 5a: an oil was obtained (1.40 g; 49% yield). 1H NMR (CDCl3): δ 0.63 (t, J = 7.4 Hz, 3H, CH3), 1.70 (m, 4H, CH2, NH2, exchangeable with D2O), 2.63 (m, 2H, CH2N), 3.33 (dd, J = 11.4, 10.9 Hz, 1H, HA 3CH2), 3.55 (m, 2H, 2-CH, HA 5-CH2), 3.64 (dd, J = 11.4, 2.8 Hz, 1H, HB 3-CH2), 4.51 (d, J = 12.1 Hz, 1H, HB 5-CH2), 7.20-7.44 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 172-174 °C. 1H NMR (DMSO): δ 0.58 (t, J = 7.5 Hz, 3H, CH3), 1.58 (m, 2H, CH2), 2.78 (m, 2H, CH2N), 3.21 (dd, J = 11.2, 10.4 Hz, 1H, HA 3-CH2), 3.41 (d, J = 12.1 Hz, 1H, HA 5-CH2), 3.62 (m, 2H, 2-CH, HB 3-CH2), 4.52 (d, J = 12.1 Hz, 1H, HB 5CH2), 7.08 (br s, 2H, NH2, exchangeable with D2O), 7.18-7.48 (m, 7H, ArH). Anal. (C13H19NO2.H2C2O4) C, H, N. (±)-cis-(6-Cyclohexyl-6-phenyl-1,4-dioxan-2-yl)methanamine

(6a).

This

compound

was

prepared starting from 14a (0.90 g, 2.46 mmol) following the procedure described for 4a. an oil was obtained (0.61 g; 90% yield). 1H NMR (CDCl3): δ 0.62-2.55 (m, 11H, cyclohexyl), 2.77 (m, 2H, CH2N), 3.24 (dd, J = 11.1, 10.9 Hz, 1H, HA 3-CH2), 3.43 (d, J = 11.5 Hz, 1H, HA 5-CH2), 3.87 (dd, J = 11.1, 2.9 Hz, 1H, HB 3-CH2), 4.05 (m, 1H, 2-CH), 4.49 (d, J = 11.5 Hz, 1H, HB 5CH2), 4.67 (br s, 2H, NH2, exchangeable with D2O), 7.20-7.37 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 222-224 °C. 1H-NMR (DMSO): δ 0.41-2.37 (m, 11H, cyclohexyl), 2.74 (dd, J = 13.0, 8.7 Hz, 1H, HA CH2N), 2.95 (dd, J ACS Paragon Plus Environment


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= 13.0, 2.4 Hz, 1H, HB CH2N), 3.11 (dd, J = 11.6, 11.1 Hz, 1H, HA 3-CH2), 3.21 (d, 1H, J = 11.7 Hz, HA 5-CH2), 3.85 (dd, J = 11.1, 2.5 Hz, 1H, HB 3-CH2), 4.14 (m, 1H, 2-CH2), 4.57 (d, 1H, J = 11.7 Hz, HB 5-CH2), 7.21-7.40 (m, 5H, ArH), 7.82 (br s, 2H, NH2, exchangeable with D2O). Anal. (C17H25NO2.H2C2O4.0.5H2O) C, H, N. (±)-trans-(6-Cyclohexyl-6-phenyl-1,4-dioxan-2-yl)methanamine (6b) This compound was prepared starting from 14b (1.50 g, 4.10 mmol) following the procedure described for 4a: an oil was obtained (0.85 g; yield 75%). 1H-NMR (CDCl3): δ 0.60-1.85 (m, 13H, cyclohexyl, NH2, exchangeable with D2O), 2.60 (m, 2H, CH2N), 3.24 (dd, J = 10.9, 10.4 Hz, 1H, HA 3-CH2), 3.55 (m, 3H, 2-CH, HB 3-CH2, HA 5-CH2), 4.47 (d, J = 12.0 Hz, 1H, HB 5-CH2), 7.18-7.35 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 173-175 °C. 1H-NMR (DMSO): δ 0.50-1.82 (m, 11H, cyclohexyl), 2.72-2.92 (m, 2H, CH2N), 3.16 (dd, J = 11.3, 10.8 Hz, 1H, HA 3-CH2), 3.40-3.69 (m, 3H, 2-CH, HB 3-CH2, HA 5-CH2), 4.61 (d, J = 12.1 Hz, 1H, HB 5-CH2), 4.90 (br s, 2H, NH2, exchangeable with D2O), 7.20-7.46 (m, 5H, ArH). Anal. (C17H25NO2.H2C2O4.1.5H2O) C, H, N. (±)-(6,6-Diphenyl-1,4-dioxan-2-yl)methanamine (7). This compound was prepared starting from 15 (1.08 g, 3.0 mmol) following the procedure described for 4a: an oil was obtained (0.34 g; 42% yield). 1H NMR (CDCl3): δ 1.96 (br s, 2H, NH2, exchangeable with D2O), 2.78 (m, 2H, CH2N), 3.40-3.80 (m, 4H, 2-CH, 3-CH2, HA 5-CH2), 4.60 (d, J = 12.1 Hz, 1H, HB 5-CH2), 7.18-7.57 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 211-212 °C. Anal. (C17H19NO2.H2C2O4) C, H, N. (±)-2-(6,6-Diphenyl-1,4-dioxan-2-yl)ethanamine (8). A solution of 34 (0.25 g, 0.89 mmol) in THF (7 mL) was added dropwise, at 0 °C, to a suspension of LiAlH4 (0.36 g, 9.49 mmol) in THF (8 mL). The reaction was stirred at room temperature for 2 h and then refluxed for 24 h. The reaction was quenched with saturated Na2SO4 solution and filtered. After evaporation of the filtrate, the residue was purified by column chromatography eluting with CHCl3/MeOH (95:5) to obtain an oil (0.08 g; 32% yield). 1H NMR (CDCl3): δ 1.58 (m, 2H, CH2), 1.95 (br s, 2H, NH2, ACS Paragon Plus Environment

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exchangeable with D2O), 2.85 (m, 2H, CH2N), 3.42 (dd, J = 11.2, 10.6 Hz, 1H, HA 3-CH2), 3.55 (d, J = 12.3 Hz, 1H, HA 5-CH2), 3.68 (m, 2H, 2-CH, HB 3-CH2), 4.60 (d, J = 12.3 Hz, 1H, HB 5CH2), 7.19-7.52 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 183-184 °C. Anal. (C18H21NO2.0.5H2C2O4.H2O) C, H, N. (±)-cis-(5-Phenyl-1,4-dioxan-2-yl)methanamine (9a). This compound was prepared starting from 17a (0.70 g, 2.47 mmol) following the procedure described for 4a: an oil was obtained (0.47 g; 98% yield). 1H NMR (CDCl3): δ 1.44 (br s, 2H, NH2, exchangeable with D2O), 2.80 (dd, J = 13.3, 4.8 Hz, 1H, HA CH2N), 3.26 (dd, J = 13.3, 9.0 Hz, 1H, HB CH2N), 3.63 (m, 1H, 2-CH), 3.79 (m, 2H, 3-CH2), 3.86 (m, 2H, 6-CH2), 4.64 (dd, J = 7.8, 3.3 Hz, 1H, 5-CH), 7.22-7.41 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from MeOH: mp 211-213 °C. Anal. (C11H15NO2.H2C2O4) C, H, N. (±)-trans-(5-Phenyl-1,4-dioxan-2-yl)methanamine (9b). This compound was prepared starting from 17b (1.0 g, 3.53 mmol) following the procedure described for 4a: an oil was obtained (0.66 g; 97% yield). 1H NMR (CDCl3): δ 2.84 (m, 2H, CH2N), 3.58 (m, 2H, 3-CH2), 3.72 (m, 1H, 2CH), 3.92 (m, 2H, 6-CH2), 4.57 (dd, J = 8.7, 2.1 Hz, 1H, 5-CH), 4.98 (br s, 2H, NH2, exchangeable with D2O), 7.21-7.40 (m, 5H, ArH). The free base was transformed into the oxalate salt that was crystallized from MeOH: mp 200-202 °C. Anal. (C11H15NO2.H2C2O4) C, H, N. (±)-(5,5-Diphenyl-1,4-dioxan-2-yl)methanamine (10). This compound was prepared starting from 18 (0.52 g, 1.45 mmol) following the procedure described for 4a: an oil was obtained (0.09 g; 23% yield). 1H NMR (CDCl3): δ 2.17 (br s, 2H, NH2, exchangeable with D2O), 2.62 (m, 2H, CH2N), 3.39 (dd, J = 11.2, 10.9 Hz, 1H, HA 3-CH2), 3.65-3.80 (m, 3H, 2-CH, HB 3-CH2, HA 6CH2), 4.61 (d, J = 12.4 Hz, 1H, HB 6-CH2), 7.18-7.51 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 207-208 °C. Anal. (C17H19NO2.H2C2O4.H2O) C, H, N. (±)-2-(5,5-Diphenyl-1,4-dioxan-2-yl)ethanamine (11). This compound was prepared starting from 35 (0.80 g, 2.86 mmol) following the procedure described for 8: an oil was obtained (0.25 g; ACS Paragon Plus Environment


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31% yield). 1H NMR (CDCl3): δ 1.60 (m, 2H, CH2), 2.97 (t, J = 6.4 Hz, 2H, CH2N), 3.22 (br s, 2H, NH2, exchangeable with D2O), 3.35 (dd, J = 11.3, 11.0 Hz, 1H, HA 3-CH2), 3.60 (dd, J = 11.3, 2.3 Hz, 1H, HB 3-CH2), 3.72 (d, J = 12.5 Hz, 1H, HA 6-CH2), 3.85 (m, 1H, 2-CH), 4.62 (d, J = 12.5 Hz, 1H, HB 6-CH2), 7.18-7.59 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 189-190 °C. Anal. (C18H21NO2.H2C2O4.0.5H2O) C, H, N. (±)-cis-N-Benzyl-1-(6-phenyl-1,4-dioxan-2-yl)methanamine (12a). Benzylamine (1.0 g, 9.33 mmol) was added to a solution of 28a (1.5 g, 4.31 mmol) in 2-methoxyethanol (30 mL). The reaction was heated to 150 °C and left for 4 h under stirring. After distillation of the solvent under vacuum, the residue was treated with 2N NaOH and the solution was extracted with CHCl3. The organic layer was dried over Na2SO4. After evaporation of the solvent the residue was purified by column chromatography eluting with EtOAc/MeOH (95:5) to obtain an oil (0.70 g; 57% yield). 1H NMR (CDCl3): δ 1.42 (br s, 1H, NH, exchangeable with D2O), 2.56 (m, 2H, CH2N), 3.31-3.45 (m, 2H, 3-CH2), 3.83 (m, 3H, 2-CH, CH2Ar), 4.08 (m, 2H, 5-CH2), 4.67 (dd, J = 10.4, 2.8 Hz, 1H, 6CH), 7.21-7.38 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 116-117 °C. Anal. (C18H21NO2.HCl.2H2O) C, H, N. (±)-trans-N-Benzyl-1-(6-phenyl-1,4-dioxan-2-yl)methanamine (12b). This compound was prepared starting from 28b (1.7 g, 4.88 mmol) following the procedure described for 12a: an oil was obtained (0.97 g; 70% yield). 1H NMR (CDCl3): δ 1.80 (br s, 1, NH, exchangeable with D2O), 2.78 (dd, J = 12.3, 4.9 Hz, 1H, HA CH2N), 3.20 (dd, J = 12.3, 8.7 Hz, 1H, HB CH2N), 3.51-4.07 (m, 5H, 2-CH, 3-CH2, CH2Ar), 4.33 (dd, J = 10.3, 6.3 Hz, 1H, HA 5-CH2), 4.42 (dd, J = 10.3, 6.9 Hz, 1H, HB 5-CH2), 4.79 (dd, J = 7.6, 3.1 Hz, 1H, 6-CH), 7.22-7.41 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 145-147 °C. Anal. (C18H21NO2.HCl.H2O) C, H, N. (±)-cis-N-Benzyl-1-(6-ethyl-6-phenyl-1,4-dioxan-2-yl)methanamine (13a) This compound was prepared starting from 29a (0.66 g, 1.99 mmol) following the procedure described for 12a: an oil was obtained (0.25 g; 40% yield). 1H NMR (CDCl3): δ 0.68 (t, J = 7.1 Hz, 3H, CH3), 1.70 (m, 1H, ACS Paragon Plus Environment

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HA CH2), 1.95 (br s, 1H, NH, exchangeable with D2O), 2.66-2.79 (m, 3H, HB CH2, CH2N), 3.193.38 (m, 3H, HA 3-CH2, CH2Ar), 3.80-3.97 (m, 3H, HB 3-CH2, 5-CH2), 4.19 (m, 1H, 2-CH), 7.227.40 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 198-199 °C. Anal. (C20H25NO2.HCl.0.5H2O) C, H, N. (±)-trans-N-Benzyl-1-(6-ethyl-6-phenyl-1,4-dioxan-2-yl)methanamine (13b) This compound was prepared starting from 29b (0.70 g, 2.11 mmol) following the procedure described for 12a: an oil was obtained (0.32 g; 49% yield). 1H NMR (CDCl3): δ 0.62 (t, J = 7.0 Hz, 3H, CH3), 1.66 (m, 2H, CH2), 2.09 (br s, 1H, NH, exchangeable with D2O), 2.60 (m, 2H, CH2N), 3.27-3.40 (m, 2H, CH2Ar), 3.55-3.66 (m, 2H, 3-CH2), 3.75-3.84 (m, 2H, 2-CH, HA 5-CH2), 4.50 (d, J = 12.3 Hz, 1H, HB 5-CH2), 7.30-7.50 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 186-187 °C. Anal. (C20H25NO2.HCl.H2O) C, H, N. (±)-cis-N-Benzyl-1-(6-cyclohexyl-6-phenyl-1,4-dioxan-2-yl)methanamine

(14a).

This

compound was prepared starting from 30a (0.77 g, 1.99 mmol) following the procedure described for 12a: an oil was obtained (0.29 g; 40% yield). 1H NMR (CDCl3): δ 0.60-2.51 (m, 12H, cyclohexyl, NH, exchangeable with D2O), 2.70 (m, 2H, CH2N), 3.37 (dd, J = 11.1, 10.9 Hz, 1H, HA 3-CH2), 3.42 (d, J = 11.5 Hz, 1H, HA 5-CH2), 3.85 (m, 3H, HB 3-CH2, CH2Ar), 4.25 (m, 1H, 2CH), 4.49 (d, J = 11.5 Hz, 1H, HB 5-CH2), 7.21-7.43 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from EtOH: mp 141-142 °C. Anal. (C24H31NO2.HCl.1.5H2O) C, H, N. (±)-trans-N-Benzyl-1-(6-cyclohexyl-6-phenyl-1,4-dioxan-2-yl)methanamine

(14b).

This

compound was prepared starting from 30b (2.32 g, 6.01 mmol) following the procedure described for 12a: an oil was obtained (1.6 g; 73% yield). 1H NMR (CDCl3): δ 0.60-1.84 (m, 12H, cyclohexyl, NH, exchangeable with D2O), 2.60 (m, 2H, CH2N), 3.30 (m, 2H, CH2Ar), 3.61 (m, 2H, 3-CH2), 3.79 (m, 2H, 2-CH, HA 5-CH2), 4.52 (d, J = 12.0 Hz, 1H, HB 5-CH2), 7.21-7.42 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2PrOH: mp 131-132 °C. Anal. (C24H31NO2.HCl.0.5H2O) C, H, N. ACS Paragon Plus Environment


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(±)-N-Benzyl-1-(6,6-diphenyl-1,4-dioxan-2-yl)methanamine (15). This compound was prepared starting from 3127 (1.9 g, 5.0 mmol) following the procedure described for 12a: an oil was obtained (1.13 g; 63% yield). 1H NMR (CDCl3): δ 1.84 (br s, 1H, NH, exchangeable with D2O), 2.69 (dd, J = 12.4, 3.9 Hz, 1H, HA CH2N), 2.83 (dd, J = 12.4, 7.3 Hz, 1H, HB CH2N), 3.59 (m, 2H, 3-CH2), 3.75-3.93 (m, 4H, 2-CH, HA 5-CH2, CH2Ar), 4.62 (d, J = 12.3 Hz, 1H, HB 5-CH2), 7.237.60 (m, 15H, ArH). The free base was transformed into the oxalate salt that was crystallized from MeOH: mp 228-229 °C. Anal. (C24H25NO2.H2C2O4.0.5H2O) C, H, N. (±)-N-Benzyl-2-(6,6-diphenyl-1,4-dioxan-2-yl)ethanamine (16). A solution of 8 (1.0 g, 3.53 mmol) and benzaldehyde (0.80 g, 7.54 mmol) in benzene (100 mL) was refluxed for 8 h. The reaction mixture was filtered and, after evaporation of the solvent, the residue was dissolved in ethanol (100 mL) and NaBH4 (0.35 g, 9.25 mmol) was added. The reaction was stirred at room temperature for 3 h, acidified with 2N HCl, extracted with CHCl3 and dried over Na2SO4. After evaporation of the solvent the residue was purified by column chromatography eluting with CHCl3/MeOH (99:1) to obtain an oil (1.07 g; 81% yield). 1H NMR (CDCl3): δ 1.58 (m, 2H, CH2), 1.77 (br s, 1H, NH, exchangeable with D2O), 2.81 (m, 2H, CH2N), 3.41 (dd, J = 11.2, 10.9 Hz, 1H, HA 3-CH2), 3.55 (d, J = 12.3 Hz, 1H, HA 5-CH2), 3.69 (m, 2H, 2-CH, HB 3-CH2), 3.78 (m, 2H, CH2Ar), 4.60 (d, J = 12.3 Hz, 1H, HB 5-CH2), 7.15-7.42 (m, 15H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 168-170 °C. Anal. (C25H27NO2.HCl.0.5H2O) C, H, N. (±)-cis-N-Benzyl-1-(5-phenyl-1,4-dioxan-2-yl)methanamine

(17a).

This

compound

was

prepared starting from 32a (2.75 g, 7.89 mmol) following the procedure described for 12a: an oil was obtained (0.45 g; 20% yield). 1H NMR (CDCl3): δ 1.62 (br s, 1H, NH, exchangeable with D2O), 2.70 (dd, J = 12.3, 4.5 Hz, 1H, HA CH2N), 3.20 (dd, J = 12.3, 9.2 Hz, 1H, HB CH2N), 3.783.94 (m, 7H, 2-CH, 3-CH2, 6-CH2, CH2Ar), 4.62 (dd, J = 7.7, 3.3 Hz, 1H, 5-CH), 7.20-7.43 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 262-263 °C. Anal. (C18H21NO2.H2C2O4) C, H, N. ACS Paragon Plus Environment

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(±)-trans-N-Benzyl-1-(5-phenyl-1,4-dioxan-2-yl)methanamine (17b). This compound was prepared starting from 32b (2.0 g, 5.74 mmol) following the procedure described for 12a: an oil was obtained (0.94 g; 58% yield). 1H NMR (CDCl3): δ 1.74 (br s, 1H, NH, exchangeable with D2O), 2.76 (m, 2H, CH2N), 3.61 (m, 2H, HA 3-CH2, HA 6-CH2), 3.84 (s, 2H, CH2Ar), 3.89-4.01 (m, 3H, 2-CH HB 3-CH2, HB 6-CH2), 4.57 (dd, J = 10.4, 2.5 Hz, 1H, 5-CH), 7.20-7.40 (m, 10H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2PrOH: mp 253-254 °C. Anal. (C18H21NO2.HCl.0.5H2O) C, H, N. (±)-N-Benzyl-1-(5,5-diphenyl-1,4-dioxan-2-yl)methanamine (18). This compound was prepared starting from 3327 (1.14 g, 3.0 mmol) following the procedure described for 12a: an oil was obtained (1.03 g; 96% yield). 1H NMR (CDCl3): δ 1.80 (br s, 1H, NH, exchangeable with D2O), 2.59 (m, 2H, CH2N), 3.40 (m, 2H, CH2Ar), 3.65-3.95 (m, 4H, 2-CH, 3-CH2, HA 6-CH2), 4.61 (d, J = 12.4 Hz, 1H, HB 6-CH2), 7.20-7.49 (m, 15H, ArH). The free base was transformed into the hydrochloride

salt

that

was

crystallized

from

MeOH:

mp

222-224

°C.

Anal.

(C24H25NO2.HCl.0.5H2O) C, H, N. (±)-N-Benzyl-2-(5,5-diphenyl-1,4-dioxan-2-yl)ethanamine (19). This compound was prepared starting from 11 (1.2 g, 4.23 mmol) following the procedure described for 16: an oil was obtained (0.60 g; 38% yield). 1H NMR (CDCl3): δ 1.54 (m, 3H, CH2, NH, exchangeable with D2O), 2.69 (m, 2H, CH2N), 3.35 (dd, J = 11.7, 10.5 Hz, 1H, HA 3-CH2), 3.62 (dd, J = 11.7, 2.8 Hz, 1H, 3CH2), 3.70 (d, J = 12.4 Hz, 1H, HA 6-CH2), 3.75 (s, 2H, CH2Ar), 3.83 (m, 1H, 2-CH), 4.60 (d, J = 12.4 Hz, 1H, HB 6-CH2), 7.19-7.50 (m, 15H, ArH). The free base was transformed into the hydrochloride salt that was crystallized from 2-PrOH: mp 234-235 °C. Anal. (C25H27NO2.HCl) C, H, N. (±)-cis-N-Benzyl-1-(2-phenyl-1,3-dioxan-4-yl)methanamine (20). This compound was prepared starting from 3639 (1.9 g, 5.45 mmol) following the procedure described for 12a: an oil was obtained (0.94 g; 61% yield). 1H NMR (CDCl3): δ 1.47 (m, 1H, HA 5-CH2), 1.80 (br s, 1H, NH, exchangeable with D2O), 1.89 (m, 1H, HB 5-CH2), 2.79 (m, 2H, CH2N), 3.82 (m, 2H, CH2Ar), ACS Paragon Plus Environment


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3.96-4.11 (m, 2H, 6-CH2), 4.28 (m, 1H, 4-CH), 5.54 (s, 1H, 2-CH), 7.21-7.55 (m, 10H, ArH). The free base was transformed into the oxalate salt that was crystallized from EtOH: mp 195-196 °C. Anal. (C18H21NO2.H2C2O4) C, H, N. (±)-N-Benzyl-1-(2,2-diphenyl-1,3-dioxan-4-yl)methanamine (21). This compound was prepared starting from 3740 (0.50 g, 1.18 mmol) following the procedure described for 12a: an oil was obtained (0.19 g; 45% yield). 1H NMR (CDCl3): δ 1.39 (m, 1H, HA 5-CH2), 1.70 (br s, 1H, NH, exchangeable with D2O), 1.91 (m, 1H, HB 5-CH2), 2.82 (m 2H, CH2N), 3.88 (m, 2H, CH2Ar), 4.02-4.21 (m, 3H, 4-CH, 6-CH2), 7.19-7.58 (m, 15H, ArH). The free base was transformed into the oxalate salt that was crystallized from 2-PrOH: mp 228-229 °C. Anal. (C24H25NO2.H2C2O4) C, H, N. 1-(Allyloxy)-2-phenylbutan-2-ol (24). 2237 (8.9 g, 60.05 mmol) was added dropwise to a stirred solution of freshly cut sodium (0.45 g, 19.56 mmol) in allyl alcohol (45 mL) at room temperature. After 1 h at room temperature the reaction was refluxed for 20 h. Most of the unreacted allyl alcohol was then separated by distillation at atmospheric pressure. After cooling to room temperature, 6N H2SO4 (1 mL) was added to the residual solution to neutralize the sodium alkoxide, and solvent removal was continued to afford an oil which was purified by column chromatography eluting with cyclohexane/EtOAc (9:1) to obtain an oil (9.9 g; 80% yield). 1H NMR (CDCl3): δ 0.79 (t, J = 7.4 Hz, 3H, CH3), 1.85 (m, 2H, CH2), 2.80 (br s, 1H, OH, exchangeable with D2O), 3.60 (m, 2H, CH2O), 3.99 (d, J = 5.6 Hz, 2H, OCH2), 5.19 (m, 2H, C=CH2), 5.83 (m, 1H, CH=C), 7.21-7.49 (m, 5H, ArH). 2-(Allyloxy)-1-cyclohexyl-1-phenylethanol (25). This compound was prepared starting from 2338 (6.5 g, 32.13 mmol) following the procedure described for 24: an oil was obtained (6.44 g; 77% yield). 1H NMR (CDCl3): δ 0.83-1.95 (m, 11H, cyclohexyl), 2.83 (br s, 1H, OH, exchangeable with D2O), 3.79 (m, 2H, CH2O), 3.98 (m, 2H, OCH2), 5.19 (m, 2H, C=CH2), 5.82 (m, 1H, CH=C), 7.20-7.43 (m, 5H, ArH).


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(±)-cis-(6-Phenyl-1,4-dioxan-2-yl)methyl 4-methylbenzenesulfonate (28a). Tosyl chloride (7.6 g, 39.86 mmol) was added to a solution of 26a27 (2.93 g, 15.09 mmol) in anhydrous pyridine (40 mL). The reaction was stirred at 0 °C for 3 h, left in the fridge for 20 h and then poured into ice and concentrated HCl. The aqueous solution was extracted with CHCl3, which was washed with 2N HCl, saturated NaHCO3 solution, water and dried over Na2SO4. After evaporation of the solvent the residue was purified by column chromatography eluting with cyclohexane/EtOAc (9:1) to obtain an oil (1.4 g; 27% yield). 1H NMR (CDCl3): δ 2.41 (s, 3H, CH3), 3.37 (m, 2H, CH2O), 3.85 (m, 2H, 3-CH2), 3.99-4.14 (m, 3H, 2-CH, 5-CH2), 4.64 (dd, J = 10.4, 2.7 Hz, 1H, 6-CH), 7.26-7.42 (m, 7H, ArH), 7.80 (d, J = 8.3 Hz, 2H, ArH). (±)-trans-(6-Phenyl-1,4-dioxan-2-yl)methyl 4-methylbenzenesulfonate (28b). This compound was prepared starting from 26b27 (2.9 g, 14.93 mmol) following the procedure described for 28a. an oil was obtained (1.70 g; 33% yield). 1H NMR (CDCl3): δ 2.44 (s, 3H, CH3), 3.48 (m, 2H, CH2O), 3.77 (m, 2H, 3-CH2), 4.04 (m, 1H, 2-CH), 4.29 (dd, J = 10.3, 6.3 Hz, 1H, HA 5-CH2), 4.41 (dd, J = 10.3, 6.9 Hz, 1H, HB 5-CH2), 4.64 (dd, J = 8.8, 3.1 Hz, 1H, 6-CH), 7.26-7.39 (m, 7H, ArH), 7.78 (d, J = 8.2 Hz, 2H, ArH). (±)-cis-2-Ethyl-6-(iodomethyl)-2-phenyl-1,4-dioxane

(29a)

and

(±)-trans-2-Ethyl-6-

(iodomethyl)-2-phenyl-1,4-dioxane (29b). A solution of mercury(II) acetate (1.64 g, 5.15 mmol) in H2O (7.56 mL) and acetic acid (0.025 mL) was added to a stirred solution of 24 (1.0 g, 4.85 mmol). The reaction mixture was heated to reflux for 45 min, then allowed to stand overnight at room temperature. After the reaction mixture was filtered, a solution of KI (1.06 g, 6.39 mmol) in H2O (10 mL) was added to the filtrate and ((6-ethyl-6-phenyl-1,4-dioxan-2-yl)methyl)mercury(II) iodide separated as an oil, which was dissolved in CHCl3 (10 mL). A solution of I2 (1.01 g, 3.98 mmol) in CHCl3 was added and the reaction mixture was heated to boiling and then allowed to stand at room temperature for 18 h. The organic phase was washed with 10% Na2SO3, 10% KI, and dried over Na2SO4. After evaporation of the solvent, the residue was purified by column chromatography eluting with petroleum ether/EtOAc (99:1). The cis diastereomer eluted first as an ACS Paragon Plus Environment


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oil (0.56 g; 35% yield). 1H NMR (CDCl3): δ 0.71 (t, J = 7.4 Hz, 3H, CH3), 1.75 (m, 1H, HA CH2), 2.62 (m, 1H, HB CH2), 3.21 (m, 3H, CH2I, HA 3-CH2), 3.35 (d, J = 11.4 Hz, 1H, HA 5-CH2), 3.80 (d, J = 11.4 Hz, 1H, HB 5-CH2), 3.98 (m, 1H, 2-CH), 4.03 (dd, J = 11.0, 2.9 Hz, 1H, HB 3-CH2), 7.22-7.41 (m, 5H, ArH). The second fraction was the trans diastereomer as an oil (0.76 g; 47% yield). 1H NMR (CDCl3): δ 0.69 (t, J = 7.6 Hz, 3H, CH3), 1.68 (m, 2H, CH2), 3.01 (d, J = 6.2 Hz, 2H, CH2I), 3.22 (dd, J = 11.1, 10.7 Hz, 1H, HA 3-CH2), 3.57 (d, J = 12.1 Hz, 1H, HA 5-CH2), 3.60 (m, 1H, 2-CH), 3.81 (dd, J = 11.1, 2.8 Hz, 1H, HB 3-CH2), 4.45 (d, J = 12.1 Hz, 1H, HB 5-CH2), 7.23-7.50 (m, 5H, ArH). (±)-cis-2-Cyclohexyl-6-(iodomethyl)-2-phenyl-1,4-dioxane (30a) and (±)-trans-2-cyclohexyl-6(iodomethyl)-2-phenyl-1,4-dioxane (30b). These compounds were prepared starting from 25 (5.0 g; 19.20 mmol) following the procedure described for 29a and 29b. The cis diastereomer eluted first as an oil (0.74 g; 10% yield). 1H NMR (CDCl3): δ 0.63-2.43 (m, 11H, cyclohexyl), 3.10 (d, J = 6.2 Hz, 2H, CH2I), 3.18 (dd, J = 11.1, 10.5 Hz, 1H, HA 3-CH2), 3.40 (d, J = 11.5 Hz, 1H, HA 5CH2), 4.00 (dd, J = 11.1, 2.9 Hz, 1H, HB 3-CH2), 4.09 (m, 1H, 2-CH), 4.41 (d, J = 11.5 Hz, 1H, HB 5-CH2), 7.24-7.34 (m, 5H, ArH). The second fraction was the trans diastereoisomer as an oil (4.15 g; 56% yield). 1H NMR (CDCl3): δ 0.63-1.84 (m, 11, cyclohexyl), 3.04 (d, J = 5.9 Hz, 2H, CH2I), 3.23 (dd, J = 11.0, 10.6 Hz, 1H, HA 3-CH2), 3.60 (m, 2H, 2-CH, HA 5-CH2), 3.78 (dd, J = 11.0, 2.8 Hz, 1H, HB 3-CH2), 4.50 (d, J = 12.1 Hz, 1H, HB 5-CH2), 7.22-7.44 (m, 5H, ArH). (±)-cis-(5-Phenyl-1,4-dioxan-2-yl)methyl 4-methylbenzenesulfonate (32a). This compound was prepared starting from 27a27 (3.9 g, 20.08 mmol) following the procedure described for 28a: an oil was obtained (2.8 g; 40% yield). 1H NMR (CDCl3): δ 2.43 (s, 3H, CH3), 3.56 (dd, J = 12.2, 9.1 Hz, 1H, HA 3-CH2), 3.65 (dd, J = 12.2, 3.3 Hz, 1H, HB 3-CH2), 3.79-3.98 (m, 3H, CH2O, 2-CH), 4.27 (dd, J = 10.3, 5.7 Hz, 1H, HA 6-CH2), 4.43 (dd, J = 10.3, 7.5 Hz, 1H, HB 6-CH2), 4.58 (dd, J = 9.0, 3.3 Hz, 1H, 5-CH), 7.20-7.42 (m, 7H, ArH), 7.83 (d, J = 8.2 Hz, 2H, ArH). (±)-trans-(5-Phenyl-1,4-dioxan-2-yl)methyl 4-methylbenzenesulfonate (32b). This compound was prepared starting from 27b27 (3.5 g, 18.02 mmol) following the procedure described for 28a: ACS Paragon Plus Environment

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an oil was obtained (1.82 g; 29% yield). 1H NMR (CDCl3): δ 2.50 (s, 3H, CH3), 3.52 (m, 2H, 3CH2), 3.85 (m, 2H, CH2O), 3.90-4.14 (m, 3H, 6-CH2, 2-CH), 4.52 (dd, J = 9.2, 3.0 Hz, 1H, 5-CH), 7.20-7.43 (m, 7H, ArH), 7.83 (d, J = 8.3 Hz, 2H, ArH). (±)-2-(6,6-Diphenyl-1,4-dioxan-2-yl)acetonitrile (34). KCN (3.0 g, 46.07 mmol) and NaI (3.55 g, 23.68 mmol ) were added to a solution of 3127 (4.5 g, 11.84 mmol) in DMSO (10 mL). The reaction was heated to 100 °C for 2 h. The mixture was diluted with water, extracted with Et2O and dried over Na2SO4. After evaporation of the solvent, the residue was purified by column chromatography eluting with cyclohexane/EtOAc (95:5) to obtain an oil (1.40 g; 42% yield). 1H NMR (CDCl3): δ 2.56 (m, 2H, CH2CN), 3.60 (m, 2H, HA 3-CH2, HA 5-CH2), 3.83 (dd, J = 11.2, 2.8 Hz, 1H, HB 3-CH2), 3.92 (m, 1H, 2-CH), 4.63 (d, J = 12.5 Hz, 1H, HB 5-CH2), 7.21-7.57 (m, 10H, ArH). (±)-2-(5,5-Diphenyl-1,4-dioxan-2-yl)acetonitrile (35). This compound was prepared starting from 3327 (1.5 g, 3.95 mmol) following the procedure described for 34: an oil was obtained (0.5 g; 45% yield). 1H NMR (CDCl3): δ 2.42 (m, 2H, CH2CN), 3.40 (dd, J = 11.1, 10.8 Hz, 1H, HA 3CH2), 3.80 (m, 2H, HB 3-CH2, HA 6-CH2), 4.06 (m, 1H, 2-CH), 4.62 (d, J = 12.6 Hz, 1H, HB 6CH2), 7.20-7.51 (m, 10H, ArH).

ASSOCIATED CONTENT Supporting Information Available Elemental analysis for compounds 4-21 (Table 1S); Electronic properties of chair and twist forms for some representative 1,3- and 1,4-dioxanes as computed by DFT calculations (Table 2S); Molecular formula strings and associated biochemical and biological data of 4-21 (Table 3S); NOE spectra for compounds 5a, 5b, 6a, and 6b (Figures 1S-4S); MEP surface as derived by density functional theory (DFT) calculations for the simple 4-aminomethyl-1,3-dioxane and 2aminomethyl-1,4-dioxane in chair and twist (Figure 5S); Pharmacology; Computational details.



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AUTHOR INFORMATION Corresponding Author *Phone: +390737402237. Fax: +390737637345. E-mail: [email protected]. ┬

These authors contributed equally to this work

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from the University of Camerino (Fondo di Ateneo per la Ricerca 2011-2012 and Fondo di Ateneo per la Ricerca 2014-2015) and Westfälische WilhelmsUniversität Münster. We express our gratitude to Prof. Mario Giannella and Prof. Maria Pigini for their suggestions and comments for this manuscript. ABBREVIATIONS USED iGluRs, ionotropic glutamate receptors; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; CNS, central nervous system; SAR, structure-activity relationship; NOE, Nuclear Overhauser Effect; [3H]DTG, [3H]-di-o-tolylguanidine; LDH, lactate dehydrogenase; SRB, sulforhodamine B; GI50, Grow Inhibition 50; TGI, Total Growth Inhibition; LC50, Lethal Concentration 50; DFT, Density Functional Theory; MEP, molecular electrostatic potential; rmsd, root-mean-square deviation.


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the development of novel M3 muscarinic receptor antagonists. J. Med. Chem. 2012, 55, 1783– 1787. (30) Mammoli, V.; Bonifazi, A.; Del Bello, F.; Diamanti, E.; Giannella, M.; Hudson, A. L.; Mattioli, L.; Perfumi, M.; Piergentili, A.; Quaglia, W.; Titomanlio, F.; Pigini, M. Favourable involvement of α2A-adrenoreceptor antagonism in the I2-imidazoline binding sites-mediated morphine analgesia enhancement. Bioorg. Med. Chem. 2012, 20, 2259–2265. (31) Bonifazi, A.; Piergentili, A.; Del Bello, F.; Farande, Y.; Giannella, M.; Pigini, M.; Amantini, C.; Nabissi, M.; Farfariello, V.; Santoni, G.; Poggesi, E.; Leonardi, A.; Menegon, S.; Quaglia, W. Structure–activity

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in

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R

O1

5 2 3 4

O

H H N

R O1

6

2 3

5 4

O

Dexoxadrol: R = C 6H5 (K i NMDA = 19 ± 2.5) Etoxadrol: R = C 2H 5 (K i NMDA = 22 ± 3.9)

NHR 1

1: R = C 2H 5; R1 = H (trans) (K i NMDA = 24 ± 2.0; K i σ1 = 955 ± 145; K i σ2 = >10000) 2: R = H; R1 = CH2 C6H 5 (trans) (K i NMDA>10000; K i σ1 = 19 ± 1.0; K i σ2 = 92 ± 40) 3: R = C 2H 5; R1 = CH 2C6H5 (trans) (NMDA>10000; K i σ1 = 27 ± 2.0; K i σ2 = 903 ± 190)

Chart 1. Chemical Structures of Dexoxadrol, Etoxadrol, and Compounds 1-3.

O

R

5 4 3 6 1 2

O

O

NH2

n

R O

4a: R = H, n = 1 (cis) 4b: R = H, n = 1 (trans) 5a: R = C2H5, n = 1 (cis) 5b: R = C2H5, n = 1 (trans) 6a: R = C6H11, n = 1 (cis) 6b: R = C6H11, n = 1 (trans) 7: R = C6H5, n = 1 8: R = C6H5, n = 2

NH2

n

9a: R = H, n = 1 (cis) 9b: R = H, n = 1 (trans) 10: R = C6H5, n = 1 11: R = C6H5, n = 2

O R O

O

NH n

R O

NH n

17a: R = H, n = 1 (cis) 17b: R = H, n = 1 (trans) 18: R = C6H5, n = 1 19: R = C6H5, n = 2

12a: R = H, n = 1 (cis) 12b: R = H, n = 1 (trans) 13a: R = C2H5, n = 1 (cis) 13b: R = C2H5, n = 1 (trans) 14a: R = C6H11, n = 1 (cis) 14b: R = C6H11, n = 1 (trans) 15: R = C6H5, n = 1 16: R = C6H5, n = 2 RO O

NH

20: R = H (cis) 21: R = C6H5

Chart 2. Chemical Structures of Compounds 4-21.



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Scheme 1a R O

O

5a (cis) 5b (trans)

NH2

R

OH O 26a: R = 6-C6H5 (cis) to 28a, 28b, 26b: R = 6-C6H5 (trans) c) 32a, 32b 27a: R = 5-C6H5 (cis) to 29a, 29b, b) 27b: R = 5-C6H5 (trans) 30a, 30b O O R f), g) h) CN R O X from 31, 33 from 29a, 29b O 34: R = 6,6-(C6H5)2 28a: R = 6-C6H5; X = OTs (cis) 35: R = 5,5-(C6H5)2 28b: R = 6-C6H5; X = OTs (trans) 29a: R = 6-C2H5,6-C6H5; X = I (cis) i) 29b: R = 6-C2H5,6-C6H5; X = I (trans) 30a: R = 6-C6H11,6-C6H5; X = I (cis) O 30b: R = 6-C6H11,6-C6H5; X = I (trans) R 31: R = 6,6-(C6H5)2; X = I O NH2 32a: R = 5-C6H5; X = OTs (cis) O R 24: R = C2H5 25: R = C6H11

22: R = C2H5 23: R = C6H11

O

O

OH

a)

32b: R = 5-C6H5; X = OTs (trans) 33: R = 5,5-(C6H5)2; X = I

8: R = 6,6-(C6H5)2 11: R = 5,5-(C6H5)2

d)

j)

O H N

R

O 12a: R = 6-C6H5 (cis) 12b: R = 6-C6H5 (trans) 13a: R = 6-C2H5,6-C6H5 (cis) 13b: R = 6-C2H5,6-C6H5 (trans) 14a: R = 6-C6H11,6-C6H5 (cis) 14b: R = 6-C6H11,6-C6H5 (trans) 15: R = 6,6-(C6H5)2 17a: R = 5-C6H5 (cis) 17b: R = 5-C6H5 (trans) 18: R = 5,5-(C6H5)2

O R O

N H

16: R = 6,6-(C6H5)2 19: R = 5,5-(C6H5)2

e) O R

NH2 O 4a: R = 6-C6H5 (cis) 4b: R = 6-C6H5 (trans) 6a: R = 6-C6H11,6-C6H5 (cis) 6b: R = 6-C6H11,6-C6H5 (trans) 7: R = 6,6-(C6H5)2 9a: R = 5-C6H5 (cis) 9b: R = 5-C6H5 (cis) 10: R = 5,5-(C6H5)2 a

Reagents: (a) Na, allyl alcohol; (b) (CH3COO)2Hg; H2O, KI, I2; (c) TsCl, pyridine; (d) Benzylamine, CH3OCH2CH2OH, ∆; (e) 10% Pd/C, 4.4% HCOOH/CH3OH; (f) Potassium phtalimide salt, K2CO3, DMF; (g) NH2NH2, THF; (h) KCN, NaI, DMSO, ∆; (i) LiAlH4, THF, ∆; (j) Benzaldehyde, benzene, NaBH4, CH3CH2OH. ACS Paragon Plus Environment

Page 41 of 51

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Scheme 2a

a

Reagents: (a) Benzylamine, CH3OCH2CH2OH, ∆.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 51

Table 1. NMDA Receptor (PCP Binding Site) and σ1 and σ2 Receptor Affinities, expressed as Ki, of 4-11 and the Reference Compounds (S)-(+)-Ketamine, PCP, Dexoxadrol, Etoxadrola and Inhibition of Cell Death, expressed as IC50, by NMDA Receptor (PCP Binding Site) Antagonists 6a, 6b, 7, (S)-(+)-Ketamine, PCP, and Dexoxadrol O R O

O

NH 2

n

R O

4-8

NH2

n

9-11

Ki ± SEM [nM] Compd

R

n

IC50± SEM [nM]

σ1

σ2

NMDA

guinea pig

rat

pig

NMDA

4a (cis)

H

1

>10000

>10000

>10000

4b (trans)

H

1

>10000

>10000

>10000

5a (cis)

C 2 H5

1

>10000

>10000

>10000

5b (trans)

C 2 H5

1

>10000

>10000

>10000

6a (cis)

C6H11

1

>10000

>10000

893 ± 40

71.2 ± 16.3

6b (trans)

C6H11

1

>10000

2500 ± 450

413 ± 20

17.7 ± 5.2

7

C 6 H5

1

>10000

5800 ± 680

712 ± 99

24.2 ± 6.4

8

C 6 H5

2

>10000

>10000

>10000

9a (cis)

H

1

>10000

>10000

>10000

9b (trans)

H

1

>10000

>10000

>10000

10

C 6 H5

1

1400 ± 90

>10000

>10000

11

C 6 H5

2

>10000

>10000

>10000

(S)-(+)-ketamine

419 ± 42

7.94

PCP

28 ± 4.6

11.8 ± 4.9

dexoxadrol

19 ± 2.5

9.7 ± 3.8

etoxadrol

22 ± 3.9


Page 43 of 51

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a

Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.56 The

affinity estimates were derived from displacement of [3H]-(+)-pentazocine, [3H]DTG in the presence of 500 nM (+)pentazocine, and [3H]-(+)-MK-801 binding for, σ1, σ2, and PCP binding site of the NMDA receptor, respectively. Each experiment was performed in triplicate. Ki and IC50 values were from three to five experiments.



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Page 44 of 51

Table 2. NMDA Receptor (PCP Binding Site) and σ1 and σ2 Receptor Affinities, expressed as Ki, of 12-21 and the Reference Compounds, (+)-Pentazocine, and DTGa

Ki ± SEM [nM] Compd

n

σ1

σ2

NMDA

guinea pig

rat

pig

12a (cis)

H

1

155 ± 12

>10000

>10000

12b (trans)

H

1

542 ± 62

4300 ± 160

>10000

13a (cis)

C2H5

1

543 ± 48

>10000

>10000

13b (trans)

C2H5

1

607 ± 62

>10000

>10000

14a (cis)

C6H11

1

1500 ± 55

4900 ± 230

>10000

14b (trans)

C6H11

1

2700 ± 270

>10000

>10000

15

C6H5

1

4900 ± 190

1600 ± 96

>10000

16

C6H5

2

502 ± 43

>10000

>10000

17a (cis)

H

1

192 ± 27

698 ± 37

>10000

17b (trans)

H

1

37 ± 6.5

>10000

>10000

18

C6H5

1

7.37 ± 0.3

1000 ± 114

>10000

19

C6H5

2

297 ± 31

841 ± 50

>10000

H

1

314 ± 31

174 ± 19

>10000

C6H5

1

3000 ± 87

111 ± 21

>10000

20 (cis) 21 (+)-pentazocine DTG a

R

6.03 ± 2 64 ± 11

Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng-Prusoff equation.56

The affinity estimates were derived from displacement of [3H]-(+)-pentazocine, [3H]DTG in the presence of


Page 45 of 51

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500 nM (+)-pentazocine, and [3H]-(+)-MK-801 binding for, σ1, σ2, and PCP binding site of the NMDA receptor, respectively. Each experiment was performed in triplicate. Ki values were from three to five experiments.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 3. Structural comparison of the ligands 1-11 with the known NMDA antagonist dexoxadrol as computed by

considering

as

matching

atoms

the

three

heteroatoms plus the centroid r of the phenyl ring. The key distances between the ammonium head and the centroid of the phenyl ring are also reported (all values are expressed in Å). Compd

rmsd

dN-Ph

dexoxadrol

-

3.28

1

1.12

3.97

4a

1.21

4.47

4b

1.46

4.45

5a

0.58

3.65

5b

1.35

4.39

6a

1.01

3.87

6b

0.95

3.98

7

0.97

3.97

8

1.39

4.44

9a

1.62

4.52

9b

1.52

6.28

10

1.73

4.56

11

1.59

5.30


Page 46 of 51

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Table 4. Key pharmacophore distances to parameterize the σ1 affinity. The following distances were monitored: dN-Ph1 = distance between the nitrogen atom and the proximal hydrophobic region, dN-Ph2 = distance between the nitrogen atom and the distal hydrophobic region, dPh1-Ph2 = distance between the two hydrophobic regions, dN-HB = distance between the nitrogen atom and the H-bonding group. For each ligand, the distances were analyzed by taking into account all non-redundant conformations and, thus, they are parameterized considering the mean and range values (all values are expressed in Å) Compd

dN-Ph1 mean

dN-Ph1 range

dN-Ph2 mean

dN-Ph2 range

dPh1-Ph2 mean

dPh1-Ph2 range

dN-HB mean

dN-HB range

2

3.63

0.14

8.51

3.97

10.00

9.25

3.88

2.17

3

3.63

0.16

8.45

3.31

9.23

8.29

4.09

2.79

12a

3.62

0.18

6.18

2.08

9.76

9.2

3.17

1.11

12b

3.62

0.08

5.61

1.69

9.22

9.18

3.08

1.09

13a

3.62

0.17

5.75

4.05

7.52

8.19

3.25

1.08

13b

3.62

0.18

6.22

2.58

10.77

9.49

3.17

1.18

14a

3.62

0.17

5.61

2.99

9.61

9.01

3.05

1.15

14b

3.63

0.18

5.37

1.01

9.34

8.43

3.22

1.04

15

3.62

0.19

5.93

3.27

10.1

9.68

3.13

1.14

16

3.62

0.18

6.35

5.32

10.35

10.68

3.81

2.4

17a

3.62

0.13

5.86

3.14

9.39

9.07

3.11

1.08

17b

3.63

0.17

7.74

1.53

12.02

6.89

3.15

1.09

18

3.63

0.17

7.63

0.67

11.88

7.2

3.07

1.11

19

3.62

0.18

8.34

3.9

12.34

10.74

3.83

2.22

20

3.63

0.16

6.11

2.02

9.61

10.18

3.14

1.08

21

3.62

0.28

5.76

3.56

9.82

8.48

3.14

1.16



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ha He C6H5

Ha

5

3

4

O

O

1

6

Ha He H3C

COOH

Ha

CH3

He

NH2 . COOH

2

C6H5

O

6

3

4

1

C6H11

O

1

6

He NH2 . COOH COOH

2

Ha

C6H5

Ha

O

3

4

5b

Ha He

Ha

5

5a

5

Page 48 of 51

O 2

Ha

Ha He NH2 . COOH COOH

He C6H11

Ha

5

6

O 1

2

Ha

C6H5

6a

3

4

O

He NH2 . COOH COOH

6b

Figure 1. NOE correlations of compounds 5a, 5b and 6a, 6b.

Figure 2. Effect of 18 (30 and 50 mg/kg, s.c.) pre-treatment on morphine (4 mg/kg, s.c.) antinociception in the hot plate test. The reaction latencies were expressed as a percentage of the Maximum Possible Effect (%MPE). Values are mean+SEM of 4-14 mice. *p

Novel Potent N-Methyl-d-aspartate (NMDA) Receptor Antagonists or

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