Comparative Molecular Field Analysis of Fenoterol Derivatives: A Platform Towards Highly Selective and Effective \xce\xb22 Adrenergic Receptor Agonists
Purpose to use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the \xce\xb22 adrenergic receptor. Results Out of 21 computationally designed structures 6 compounds were synthesized and characterized for \xce\xb22-AR binding affinities, subtype selectivities and functional activities. Conclusion the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with Ki\xce\xb22-AR = 0.28 \xc2\xb5M, Ki\xce\xb21-AR/Ki\xce\xb22-AR = 573, EC50cAMP = 3.9 nM, EC50cardio = 16 nM. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.
1. Introduction<!>2. Results<!>3. Discussion<!>4.1. \xce\xb22-AR binding assays<!>4.2. \xce\xb22-AR mediated stimulation of cAMP accumulation<!>4.3. Cardiomyocyte contractility<!>4.4. Comparative Molecular Field Analysis (CoMFA)<!>4.5. Chemistry<!>Synthesis of (R,R)- and (R,S)-fenoterol analogues<!>General Procedure for the Preparation of Aryl Ketones<!>1-(4-(Benzyloxy)phenyl)butan-2-one<!>1-(Naphthalen-2-yl)propan-2-one<!>1-(4-Methoxynaphthalene-1-yl)propan-2-one<!>General Procedure for the Preparation of N-Benzylaminoalkanes<!>rac-N-Benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine<!>rac-N-Benzyl-1-(naphthylen-2-yl)propan-2-amine<!>rac-N-Benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine<!>General Procedure for enantiomeric resolution of N-benzylaminoalkanes<!>(R)-(\xe2\x88\x92)-N-Benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine<!>(S)-(+)-N-Benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine<!>(R)-(\xe2\x88\x92)-N-Benzyl-1-(naphthylen-2-yl)propan-2-amine<!>(S)-(+)-N-Benzyl-1-(naphthylen-2-yl)propan-2-amine<!>(R)-(\xe2\x88\x92)-N-Benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine<!>(S)-(+)-N-Benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine<!>Preparation of Fenoterol Analogs<!>(R,R-(\xe2\x88\x92)-5-(1-Hydroxy-2-(1-hydroxyphenyl)butan-2-ylamino)ethyl) benzene-1,3-diol [(R,R)-52]<!>(R,S)-(+)-5-(1-Hydroxy-2-(1-hydroxyphenyl)butan-2-ylamino)ethyl) benzene-1,3-diol [(R,S)-52]<!>(R,R)-5-(1-Hydroxy-2-(1-(naphthalen-2-yl)propan-2-ylamino)ethyl) benzne-1,3-diol [(R,R)-53]<!>(R,S)-5-(1-Hydroxy-2-(1-(naphthalen-2-yl)propan-2-ylamino)ethyl) benzne-1,3-diol [(R,S)-53]<!>(R,R)-(\xe2\x88\x92)-5-(1-Hydoxy-2-(1-(4-methoxynaphthalen-1-yl)propan-2-ylamino)ethyl)benzene-1,3-diol [(R,R)-54]<!>(R,S)-(+)-5-(1-Hydoxy-2-(1-(4-methoxynaphthalen-1-yl)propan-2-ylamino)ethyl)benzene-1,3-diol [(R,S)-54]<!>
<p>In the initial studies in this series, (R,R)-fenoterol, Fig. 1, a potent and selective β2-adrenoceptor (β2-AR) agonist was identified as a potential drug for the treatment of congestive heart failure.1,2 (R,R)-Fenoterol is currently entering initial clinical trials for use in this disease. Based on the potential therapeutic use of (R,R)-fenoterol, the stereoisomers of fenoterol and a series of fenoterol analogues were synthesized and tested for β2-AR activity and selectivity.3 Three of the 26 compounds investigated in the latter study had submicromolar affinity for the β2-AR, a >40-fold selectivity for the β2-AR relative to the β1-AR and were full agonists of the β2-AR. One of these compounds, (R,R)-methoxyfenoterol was also identified as a potential candidate for drug development and preliminary pharmacokinetic and toxicity studies have been initiated.</p><p>The binding affinities, Ki values, of the 26 fenoterol analogues were used to develop a comparative molecular field analysis model, CoMFA(n = 26), to describe and predict the binding of the stereoisomers of fenoterol and fenoterol analogues to the β2-AR.3 The CoMFA(n = 26) model explained the role of stereoconfiguration and structural modifications of studied molecules on observed β2-AR affinities. The model was also consistent with earlier findings from other laboratories, 4,5 which indicated that the β2-AR selectivity of the fenoterol analogues was due to interactions of the aminoalkyl portion of the molecules with different sites within the transmembrane (TM) components of the β2-AR. These interactions included hydrogen bond formation between the p-oxygen moiety on the phenyl ring in fenoterol and methoxyfenoterol and tyrosine 308 (Y308) in TM7 and/or histidine 296 (H296) in TM6 and π–π and/or π-hydrogen bond interactions between the aromatic system of the ligand and aromatic residues located in this area of the binding site. In addition, the data from the previous study3 and an earlier study6 indicated that the binding process of the fenoterol analogues with the β2-AR included the interaction of the chiral center of the aminoalkyl portion with a sterically restricted site on the receptor.</p><p>In this study, the CoMFA(n = 26) model was used to design 21 additional fenoterol analogues as potential selective and potent β2-AR agonists. Based on the CoMFA(n = 26) model and the indicated binding interactions, three sites on the aminoalkyl portion of the fenoterol molecule were chosen for alteration, Fig 1. The modifications were utilized to explore the effect on β2-AR binding affinity, selectivity and agonist activity of the steric bulk at the second chiral center, of a 1-napthyl versus a 2-napthyl substituent, and of the effect of a substituent at the 4-position of a 1-naphthyl substituent. A subset of 6 compounds, composed of the (R,R)- and (R,S)- isomers of 3 of the designed compounds, was synthesized using the previously described approach.3 The β2-AR and β1 -AR binding affinities and the EC50 values associated with induced cAMP accumulation (EC50cAMP) were determined. The (R,R)-isomers of the subset were tested for their activity in a previously described cardiomyocyte contractility model3 and their EC50cardio values determined.</p><!><p>In the previous study, the CoMFA(n = 26) model was derived using the experimentally determined β2-AR binding affinities of a training set of 26 fenoterol derivatives and stereoisomers.3 The validity parameters of the model were high (R2 = 0.920, Q2 = 0.847, F = 60.380) and the standard error of prediction was low (SEP=0.309). In the current study, the CoMFA(n = 26) model was used to estimate the pKi values for the (R,R)-isomers of a set of 12 new molecular structures, and the (R,S)-, (S,R)- and (S,S)-isomers of three of these compounds for a set of 21 unique compounds, 52–63, Table 1.</p><p>The (R,R)- and (R,S)-isomers of three of the designed fenoterol analogues, 52, 53, 54, were synthesized using a previously described synthetic scheme in which the epoxide formed from (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin was coupled with the (R)- or (S)-enantiomer of the appropriate N-benzylamino-alkanes.3 The chirality of the first asymmetric center was set as (R) based on the data from the previous study that fenoterol analogues with this configuration had higher affinities and activities at the β2-AR relative to the corresponding analogues with a (S)-configuration at this carbon.3</p><p>The binding affinities of the synthesized compounds were determined using membranes derived from HEK cells stably transfected with human β2-AR and β1-AR as previously described3 and the corresponding Ki values were calculated, Table 2. The maximum difference, Δ value, between computationally predicted and experimentally determined affinities, expressed as pKi values (− logKi), was − 0.6 and in some cases did not exceed the standard error of experimental determination, Table 1. The external validation of the CoMFA(n = 26) model with the newly synthesized test set was calculated and the results, R2PRED = 0.583, SPRESS = 0.337, where PRESS is predictive residual sum of squares, Σ(Δ2)7, indicated that the CoMFA(n = 26) model had a high predictive power for the computational design of congeneric β2-AR agonists based upon fenoterol.</p><p>The β2-AR and β1-AR binding affinities, Ki(β2-AR) and Ki(β1-AR), and relative binding affinities, Ki(β1-AR)/Ki(β2-AR), of the (R,R)- and (R,S)- isomers of 52, 53 and 54, were determined, Table 2. In addition, the β2-AR agonist activities of the (R,R)-isomers of the previously reported compounds 1, 2, 5 and 52, 53, 54 were examined using a previously described cardiomyocyte contractility model, Table 3.3 In these studies, the agonist-stimulated β2-AR contractility response was distinguished from that of β1-AR activity using ICI 118,551, a β2-AR specific antagonist.</p><p>The data demonstrate that the substitution of the methyl group by an ethyl moiety at the chiral center on the aminoalkyl portion of the fenoterol backbone, 1 and 52, respectively, reduced the calculated binding affinities to the β2-AR. The Ki(β2-AR) value for (R,R)-52 was ~4-fold greater than that of (R,R)-1 and the value for (R,S)-52 was 1.5-fold greater than that of (R,S)-1, Table 2. The binding affinities to the β1-AR were also affected, as the Ki(β1-AR) of (R,R)-52 was 28-fold higher then that of (R,R)-1 and the Ki(β1-AR) for (R,S)-52 was 6-fold greater than that of (R,S)-1. The greater reductions in the affinity to the β1-AR were reflected in the relative binding affinities as the Ki(β1-AR)/Ki(β2-AR) for the (R,R)-isomers increased from 43 (1) to 334 (52) and from 5 (1) to 20 (52) for the (R,S)-isomers. Thus, a modest increase in the steric bulk at the chiral center on the aminoalkyl portion of the molecule increased the relative energies of the agonist-β2-AR and agonist-β1-AR complexes, irrespective of the configuration at that site. These results are consistent with the existence of a sterically restricted site on the β2-AR and suggest that the site is also present in the β1-AR and that the steric interaction has a stronger effect on the binding to the β1-AR.</p><p>The substitution of an ethyl moiety for the methyl group produced a slight but significant increase in the EC50cAMPvalues of (R,R)- and (R,S)-52 relative to (R,R)- and (R,S)-1, Table 3. However, the increase in steric bulk produced a profound decrease in the activity of (R,R)-52 in the cardiomyocyte contractility model as the EC50cardio of (R,R)-52 was 8,551 nM, a 128-fold increase relative to (R,R)-1, EC50cardio = 83 nM, Table 3. Thus it appears that steric interactions involving the chiral center on the aminoalkyl portion of the fenoterol molecule had a greater effect in β2-AR associated cardiomyocyte contractility system than in the displacement binding or cAMP stimulation studies. It is interesting to note that the activity of (R,R)-52 in the cardiomocyte contractility model was antagonized by the β2-AR selective antagonist ICI 118,551, but (R,R)-52 did not antagonize the activity of zinterol in this model (data not shown).</p><p>When the effect of the change in the orientation of a naphthyl moiety on the amionoalkyl portion of the molecule from the 1- to the 2-position on the ring, 5 and 53, was investigated, the data indicate that this change had only a slight effect on the binding affinities to the β2-AR and β1-AR, Table 2. In these studies, the differences between the Ki(β2-AR) for the (R,R)- and (R,S) isomers of 53 and the corresponding isomers of 5 were less than 2-fold as was the difference in the Ki(β1-AR) for (R,S)-53 and (R,S)-5. The greatest effect was seen in the Ki(β1-AR) of (R,R)-53 which was ~4-fold higher relative to that of (R,R)-5, and reflected in an increase in the relative binding affinities, Ki(β1-AR)/Ki(β2-AR) from 14 (5) to 40 (53). The results suggest that the change from a 1-naphthyl to a 2-naphthyl substituent on the fenoterol molecule had a relatively small effect on the π–π and/or π-hydrogen bond interactions between the naphthyl substituent and aromatic residues located in this area of the binding site of the β2-AR. The data also suggest that these interactions occur between the agonists and the β1-AR.</p><p>While the change from a 1-naphthyl to a 2-naphthyl substituent has less than a 2-fold affect on the Ki(β2-AR) of the (R,R)-isomers, a greater effect was seen in the EC50cAMPvalues as the EC50cAMPof (R,R)-53 was 30-fold lower than that of (R,R)-5, Table 3. However, the inverse was observed with cardiomyocyte contractility as the EC50cardio of (R,R)-53 was 3-fold higher than that of (R,R)-5, Table 3, but it is not clear if this difference has any pharmacological significance. There was less than a 3-fold difference in the EC50cAMP values (R,S)-53 and (R,S)-5, Table 3.</p><p>The data from the initial studies of the aminoalkyl analogues of fenoterol, indicated that optimum binding affinities and β2-AR selectivities were observed with an electronegative substituent in the 4-position of the benzyl moiety or with a 1-naphthyl moiety.3 In this study, a series of 4-substituted-1-naphthyl analogues of fenoterol, 54 – 63, were docked in the CoMFA(n = 26) model and their pKi values predicted, Table 1. The results indicated that the presence of an electronegative substituent increased the binding affinity of the compound and that the pKi values of the (R,R)-isomers of 54 – 59 would be greater than the corresponding values of 1, 2 and 5. It is of interest to note that 4-amino-1-naphthylfenoterol (60) had the lowest predicted pKi which is consistent with the previous study in which the Ki(β2-AR) of (R,R)-4-aminobenzylfenoterol (4) was greater than the corresponding values for 1, 2 and 5.3</p><p>In this study, (R,R)-54 and (R,S)-54 were synthesized and tested. The data obtained with (R,R)-54 indicate that the β2-AR binding affinity of this compound was equivalent to (R,R)-5 and slightly stronger than (R,R)-2, Table 2. The same trend was observed with the (R,S)-isomers as the Ki(β2-AR) values of (R,S)-54 and (R,S)-5 were equivalent and both were about 6-fold stronger than (R,S)-2. The results suggest that when both a p-methoxy moiety and a 1-naphthyl moieties are present in the molecule, interactions with the naphthyl group appear to play a more significant role in the stabilization of the agonst-β2-AR complex.</p><p>The Ki(β1-AR) value of (R,R)-54 was significantly weaker than (R,R)-2 (19-fold) and (R,R)-5 (141-fold), while there was no significant difference between (R,S)-54 and the (R,S)-isomers of 2 and 5, Table 2. The weaker β1-AR binding affinity for (R,R)-54 was reflected in the relative binding affinity ratios, Ki(β1-AR)/Ki(β2-AR) which increased to 573, as compared to 47 and 14, for (R,R)-2 and (R,R)-5, respectively. The results indicate that both the p-methoxy and 1-naphthyl moieties in (R,R)-54 had a destabilizing effect in the binding interactions with the β1-AR relative to the (R,R)-isomers of 2 and 5. This effect was not observed in the relative binding of the (R,S)-isomers of 54, 2 and 5 to the β1-AR suggesting that the S-configuration in the aminoalkyl portion of the molecule altered the role of the 4-oxy and/or 1-naphthyl substituents in the binding of (R,S)-54 to the β1-AR.</p><p>While the Ki(β2-AR) values of the (R,R)-isomers of 2, 5 and 54 were essentially equivalent, the EC50cAMP values of 5 and 54 were significantly higher, 40-fold and 10-fold, respectively, than the EC50cAMPvalue of 2, Table 3. If the EC50cAMP value of (R,R)-1 is also considered, then the data suggest that fenoterol analogues containing a p-oxy-phenyl moiety have a greater effect on cAMP stimulation than fenoterol analogues containing a 1-naphthyl moiety, 5, and that the addition of a 4-methoxy substituent in the naphthyl ring has a positive effect on the EC50cAMP value, 54. It is of interest to note that there is no significant difference in the EC50cAMP values of (R,R)-2 and (R,R)-53, which contains a 2-naphthyl ring. This suggests that the difference in the EC50cAMP values between 2 and 5 is a function of the relative position of the naphthyl group within the β2-AR binding area rather than the lack of a p-oxy-substituent.</p><p>When the effect on cardiomyocyte contractility was investigated, the relative EC50cardio values were 54 < 5 < 2, although the difference between 54 and 2 was only ~10-fold. These results indicate that the presence of the 1-naphthyl ring and the 4-methoxy substituent had a synergistic effect in this model system.</p><p>No significant differences were observed in the cAMP stimulation produced by the (R,S)-isomers of 2, 5, and 54, Table 3. For these compounds, the relative differences between the (R,R)- and (R,S)-isomers of each compound mirrored the relative differences in Ki(β2-AR) values, i.e. (R,R)-2 < (R,S)-2; (R,R)-5 > (R,S)-5; (R,R)-54 ≈ (R,S)-54, Table 2,Table 3. These results suggest that the displacement binding studies and the induced stimulation of cAMP accumulation may reflect the same binding process.</p><p>The pKi data from this study was used to refine the CoMFA(n = 26) model. A total of 32 structures (26 original and 6 newly synthesized) were used as the training set for a revised 3D-QSAR model, CoMFA(n = 32). Partial Least Square methodology extracted four statistically significant components of the model, and all of the validation parameters were high and indicated that the model had a stronger predictive power than the CoMFA(n = 26) model, Table 4.</p><p>The CoMFA(n = 32) model is presented in Fig. 2. For visual clarity only (R,R)-54 is included in the figure. The interaction fields are color coded in the following manner: green represents a region of favorable steric (bulk) interactions, unfavorable steric interactions are denoted by yellow, electrostatic interactions with a positive charge (or H-bond donors) are blue, and electrostatic interactions with a negative charge (or H-bond acceptors) are red.</p><p>As previously demonstrated with the CoMFA(n=26) model, the CoMFA(n = 32) model explains the source of stereoselectivity observed with this group of compounds. At the first center of chirality, an R-configuration directs the hydroxyl moiety towards the negative charge field (red), a favorable interaction, while an S-configuration at this center directs the hydroxyl moiety towards the positive charge field, a destabilizing interaction. Steric fields are associated with both chiral centers and an S-configuration at both of the sites directs substituents toward an unfavorable steric interaction site, denoted by the yellow fields.</p><p>The interactions of the aminoaryl portion of the fenoterol analogues with the β2-AR and the relative stabilities of the agonist-receptor complexes were also refined in the CoMFA(n = 32) model. These interactions are illustrated by the proposed stabilizing interactions associated with the 4-methoxy-1-naphthyl substituent of (R,R)-54, Fig. 2. In this model, the substituent is placed in the vicinity of electrostatic interactions with a positive charge or H-bond donors (blue regions), suggesting hydrogen bond interaction with the oxygen atom. Additional stabilizing interactions are suggested by the presence of two large electronegative fields (red regions) surrounding both faces of the naphthyl ring of the (R,R)-54 indicating the possibility of π–π interactions with aromatic residues within the binding site. Steric favoring regions (green regions) are also present at both edges of the naphthyl ring, which most probably reflects the preference of 1-naphthyl versus 2-naphthyl in the β2-AR binding. These regions may also affect the conformational mobility of a molecule during the optimization of electrostatic and π–π interactions. The three interaction sites are independent and appear to act cooperatively in the binding of (R,R)-54.</p><!><p>The results of this study demonstrate that the CoMFA(n = 26) model can be used to design selective β2-AR agonists based upon fenoterol or compounds that contain interactive sites equivalent to those found on the aminoalkyl portion of the fenoterol molecule. This design strategy is reflected by the synthesis and properties of the β2-AR agonist (R,R)-54, which is highly selective (Kiβ1/Kiβ2 = 573) and effective (EC50 = 16 nM, cardiomyocyte contractility).</p><p>The revised molecular model, CoMFA(n = 32) is consistent with the earlier model3 and with the previously reported models describing the molecular basis of β2-AR selectivity,4–6 although a recent paper has questioned the role that Y308 plays in this process.8 The data from this study supports the hypothesis that hydrogen bond formation between the p-oxygen moiety on the aromatic substituents of the (R,R)-isomers of 1, 2, 52, and 54 and Y308 in TM7 and/or H296 in TM6 contribute to the >40-fold β2-AR selectivity. The role of Y308 in β2-AR selective binding was also supported by the data from a study of the binding of salmeterol to chimeric β1/β2ARs and alanine-substituted β2-AR mutants.5 In these studies, the Ki(β2-AR) changed from 1.5 nM in the wild-type receptor to 184 nM in the Y308A mutant, the largest effect observed in the series of alanine-substituted β2-AR mutants used in the study.</p><p>The data also indicate that the >300-fold β1-AR/β2-AR selectivity of (R,R)-52 and (R,R)-54 was primarily due to a large decrease in β1-AR binding affinities, Table 2. It is reasonable to assume that the weaker affinities reflect the loss of hydrogen bond interactions due to the replacement of Y308 and H296 in the β2-AR by F359 and K347, respectively, in the β1-AR.6 This hypothesis is supported by binding studies with the wild-type β1-AR and β2-AR and salmeterol and a salmeterol derivative that lacked an ether linkage found in the parent compound.5 The Ki(β2-AR) of salmeterol and the derivative were 1.5 nM and 220 nM, respectively, while the Ki(β1-AR) values were both ~2,300 nM. The differences in the Ki(β2-AR) values were reflected in the Ki(β1-AR)/ Ki(β2-AR) ratios which were >1,000 for salmeterol and ~10 for the derivative. The authors of this study concluded that the large difference in the relative binding ratios was a result of the inability of the derivative to interact with Y308 in the β2-AR due to the absence of the oxygen atom of the ether linkage.</p><p>The >500-fold β2-AR selectivity observed with (R,R)-54 and the >40-fold β2-AR selectivity observed with compounds that do not contain p-oxygen moieties, i.e. (R,S)-5, (R,R)-53, (R,S)-53, Table 2, indicate that π–π and/or π-hydrogen bond interactions also play a role in the relative stabilities of the two β-ARs. The contribution of π–π and/or π-hydrogen bond interactions in the binding process is indicated by the two electronegative fields in the CoMFA(n = 32) model, which are located on both faces of the docked (R,R)-54, Fig. 2. The electronegative fields in the CoMFA(n = 32) model reflect a network of π–π interactions which include Y308 in the β2-AR and other aromatic residues such as H296, Y316, F289, F193, F290 and W109 which are in close proximity to Y308.9 The higher Ki(β1-AR) values for the same compounds suggest that π–π interaction networks based upon F325 and other aromatic residues also exist in the β1-AR, but that the interactions are weaker than the corresponding ones in the β2-AR. In addition, the presence of extended electronegative fields in both β-ARs also explains the lack of a significant difference in the affinities of the 1-naphthyl and 2-naphthyl analogues, except for the interaction of (R,R)-54 with the β1-AR. In this case, the 4-methoxy substituent may play a more significant role in the increased Ki(β1-AR) value than the 1-naphthyl ring.</p><p>In the initial study involving the stereoisomers of fenoterol and fenoterol analogues3 and with the compounds synthesized in this study, the β2-AR binding affinities of the (R,R)-isomers were greater than the corresponding (R,S)-isomers. The results support the hypothesis that the stereochemistry of the chiral carbon on the aminoalkyl moiety positions this portion of the molecule relative to the interactive moieties in TM3, TM6 and TM7 and that this effect reflects interactions with a sterically-restricted site on the β2-AR.3 These data indicate that this effect was more pronounced with compounds containing a phenyl ring with a p-hydroxy or p-methoxy substituent, i.e. 1, 2, 52, Table 2. This may reflect the fact that the hydrogen bonding interactions between the p-substituents on 1, 2 and 52 and the β2-AR occur at more defined sites, i.e. Y308 and H296, than the sites binding to non-oxygen containing fenoterol analogues, i.e. a diffuse π–π interaction area. Therefore, subtle changes in the binding interactions produced by alterations in the relative positions of the (R,R)- and (R,S)-isomers should have a greater effect in 1, 2, and 52 than 5 and 53. The minimal differences between the Ki(β2-AR) values of (R,R)-54 and (R,S)-54 suggests that the π–π interactions play a relatively larger role in the stabilization of the 54-β2-AR complex than the hydrogen bonding interactions.</p><p>In this study, the Ki(β1-AR) values of the (R,R)-isomers of 1, 2, 5, and 53 were lower than the corresponding (R,S)-isomers although the calculated differences were < 5-fold, Table 2. The opposite effect was observed with 52 and 54 where the Ki(β1-AR) of the (R,R)-isomers were 4-fold and 10-fold larger, respectively, Table 2. The Ki(β1-AR)/Ki(β2-AR) ratios of 52 and 54 were also significantly greater than those observed with the other fenoterol analogues, by 334 and 573 fold, respectively. The results obtained with 52 may reflect the effect of the increase in steric bulk at the chiral center and the resulting destabilization of the 52-β1-AR complexes, irrespective of the chirality, as both isomers have Ki(β1-AR) values > 100 µM. This suggests that the postulated sterically restricted site on the β2-AR also exist in the β1-AR.</p><p>When the results obtained with 54 are compared to 2 and 5, it is evident that for (R,S)-54, the presence of both a 4-methoxy moiety and a 1-naphthyl moiety had little effect on the Ki(β1-AR) relative to the presence of only a 4-methoxy moiety or a 1-naphthyl moiety, (R,S)-2 and (R,S)-5, respectively, Table 2. The more significant effect is seen with (R,R)-54 as the calculated Ki(β1-AR) increased from 21.9 µM (2) and 3.35 µM (5) to 160.5 µM. The results indicate that the presence of the 4-methoxy and 1-napthyl moieties act in a synergistic manner to reduce the stability of the (R,R)-54-β1-AR complex. The mechanism associated with this phenomenon is currently unknown.</p><p>The 12 compounds used in this study stimulated cAMP accumulation with EC50(cAMP) values that ranged from 0.30 nM to 16.60 nM, Table 3, and all of these compounds can be classified as full β2-AR agonists. There was no direct relationship between the Ki(β2-AR) and EC50(cAMP) values probably due to the fact that the binding studies used a radiolabeled antagonist. Planned studies with a radiolabeled fenoterol analog may produce a better correlation between binding affinity and stimulation of cAMP accumulation. The fact that fenoterol analogs stimulate cAMP affinity at considerably lower concentrations than they stimulate cardiomyocyte contractility suggests either a far greater degree of amplification in the cAMP activity pathway, or potentially a different signal transduction pathway altogether.</p><p>In the cardiomyocyte contractility model system, 8 of the 9 compounds studied had stimulatory effects with EC50cardio values ranging from 16 ((R,R)-54) to 575 nM ((R,S)-1), Table 3. The clear outlier was (R,R)-52 with an EC50cardio value of 8,551 nM. When the Ki(β2-AR) and EC50cardio values for the 8 compounds were compared, there was a statistically significant relationship between the values, r2 = 0.8804, p = 0.006. The data indicate that in a series of fenoterol analogues a Ki(β2-AR) of ≤0.500 µM should correspond to a EC50cardio of <0.200 µM and that the CoMFA models described in these studies are capable of designing compounds which are active agonists in the β2-AR cardiomyocyte contractility model. The EC50cardio value obtained with (R,R)-52 was similar to previous data obtained with (S,R)-1 and (S,R)-2, EC50(cardio) = 2,340 nM and 3,160 nM, respectively.10 It is of interest to note that all three compounds can be classified as full β2-AR agonists with respect to cAMP stimulation, EC50(cAMP) = 2.80 ((R,R)-52), 8.50 nM ((S,R)-1), 7.20 nM ((S,R)-2). The data indicate that the chirality at both of the chiral centers on the fenoterol backbone and the steric bulk at the second chiral center play limited roles in cAMP stimulation, but a major role in cardiomyocyte contractility. The role of the chiral centers in cardiomyocyte contractility has been previously demonstrated in which (R,R)-1 and (R,R)-2 were shown to preferentially activate GS signaling while the corresponding (S,R)-isomers activated both GS and Gi proteins.10 Our initial data indicate that the cardiomyocyte contractility of (R,R)-52 is consistent with the selectivity of the (S,R)-isomers as this activity is sensitive to pertussis toxin, which indicates that this compound activates both GS and Gi proteins (data not shown).</p><p>The data from this study are consistent with the hypothesis that there are two binding areas within the β2-AR molecule. One area is the site that interacts with the "catechol" portion of an agonist and is created by residues in TM3, TM5 and TM6, (the "first" binding area).7,11–15 The "second" binding area is created by residues in TM3, TM6 and TM7 and interacts with the aminoalkyl side chain of the fenoterol analogues.3–6 The results also suggest that the binding process between the fenoterol analogues and the β2-AR involves a number of multi-step interactions. In addition, these interactions and their functional consequences vary based upon the composition and stereochemistry of the fenoterol analogue.</p><p>The binding of an agonist to the β2-AR has been previously described as a multi-step sequential process that produces a series of conformational changes in the receptor. 14,15 This description reflects the extensive study of the binding of agonists to the "first" binding area and it is important to note that the majority of the compounds used in these studies did not have large amino alkyl side chains containing oxygen or aromatic moieties, c.f. ref. 13. Thus it is reasonable to assume that with compounds such as the fenoterol analogues, the sequential binding mechanism will also include interactions with the "second" binding area and that these interactions will also result in distinct conformational changes in the β2-AR molecule. However, the data from the current study are unable to differentiate between these biding interactions. This is in part due to the fact that the binding studies were conducted using [3H]CGP-12177, a highly selective β2-AR antagonist, which, unfortunately, does not contain a fenoterol-like alkylamino moiety.16 This issue is being addressed in binding studies using a [3H]-labeled fenoterol analogue as the marker ligand and in molecular modeling and thermodynamic studies, which have also been initiated.</p><!><p>Compounds synthesized in this study were tested up to three times each to determine their binding affinities at the β1- and β2-ARs following a previously described approach.3 In brief, β2-AR binding was conducted on membranes derived from HEK cells containing human β2 -AR (provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto, CA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.05% penicillin-streptomycin with 400 µg/mL G418. β1-AR binding was conducted using rat cortical membranes. The binding assays contained 0.3 nM or 1.4 nM [3H]CGP-12177 in a volume of 1.0 ml for binding to β2- and β1-ARs respectively. Nonspecific binding was determined using 10 µM propranolol. To block the β2AR sites present in the cortical membrane preparation, 30 nM ICI 118–551 was added to the assay buffer. Competition curves with standard and unknown compounds included at least six concentrations, in triplicate. IC50 values and Hill coefficients were calculated using Prism software. Ki values were calculated using the Chang-Prusoff transformation.17</p><!><p>The HEK cells expressing β2-ARs were grown on 96-well plates in DMEM containing 10% FBS, 0.05% penicillin-streptomycin, and 400 µg/ml G418. When the cells reached confluence, the medium was removed and each well was rinsed with 0.1 ml of Krebs-HEPES buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 1.2 mM MgSO4, 25 mM HEPES, and 10 mM glucose, pH 7.3). The test compound was diluted in Krebs-HEPES buffer containing 0.1% ascorbic acid, 10 µM pargyline, and 50 µM 3-isobutyl-1-methylxantine (IBMX), and 0.1 ml of the solution was added to each well. In the agonist assay, the plates were preincubated for 10 min at room temperature (RT) with buffer alone; then test compound diluted in buffer was added to the wells. In the antagonist assay, the plates were preincubated (for 20 min at 37°C) with test compound diluted in buffer; then buffer plus 30 nM formoterol was added. The plates were incubated for an additional 10 min with the test compound. After incubation, the medium was removed and 0.1 ml of 0.5 M formic acid added. After a minimum of 1 h, the supernatant was removed and lyophilized. cAMP was quantified using the protein kinase binding assay of Gilman.18 The amount of protein per well was determined and used to calculate the amount of cAMP/mg/well.</p><!><p>These experiments were carried out as previously described.3 In brief, single ventricular myocytes were isolated from 2–4 month old rat hearts by a standard enzymatic technique. The isolated cells were resuspended in HEPES buffer solution [20 mM, pH 7.4] containing, NaCl (137 mM), KCl (5.4 mM), MgCl2 (1.2 mM), NaH2PO4 (1.0 mM), CaCl2 (1.0 mM), and glucose (20 mM). All experiments were performed within 8 h of cell isolation.</p><p>The cells were placed on the stage of an inverted microscope (Zeiss model IM-35, Zeiss, Thornwood, NY), perfused with the HEPES- buffered solution at a flow rate of 1.8 ml/min, and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored by an optical edge-tracking method using a photodiode array (Model 1024 SAQ, Reticon, Boston, MA) with a 3 ms time resolution. Cell contraction was measured by the percent shortening of cell length following electrical stimulation.</p><!><p>SYBYL 8.0. (TRIPOS Inc., St. Louis, MO) was used for 3D QSAR modeling. The previously described CoMFA model3 was used for pKi prediction for the set of newly designed derivatives. Briefly, molecular models of structures were prepared in HyperChem v. 6.03 (HyperCube Inc., Gainesville, FL) using ModelBuild procedure to ensure the same conformation of the common scaffold. The models were extracted to SYBYL and the Gasteiger – Huckel atomic charges were calculated. The models were aligned with molecules of original training set using the two asymmetric carbon atoms in the core of the fenoterol molecule (-C*-CH2-NH-C*-CH2-) as a common substructure. A PREDICT procedure implemented in CoMFA package was used to compute the estimated pKi values.</p><p>The revised CoMFA(n = 32) model was developed using a training set that included the new structures and pKi values examined in this study, i.e. the (R,R)-and (R,S) isomers of 52, 53, 54. Two types of molecular fields (steric and electrostatic) were sampled on the grid (2 Å spacing) lattice surrounding each structure. Distance-dependent dielectric constant was used in electrostatic calculations and energetic cutoffs of 30 kcal/mol for both the steric and the electrostatic energies were set.</p><p>The application of the partial Least Square correlation procedure to the resultant database extracted four significant components and validation parameters for this solution (CoMFA(n = 32)), which are presented in Table 4. For the sake of comparison the validity parameters obtained for the original solution (CoMFA(n = 26))3 are also included in Table 4.</p><!><p>All reactions were carried out using commercial grade reagents and solvents. Ultraviolet spectra were recorded on a Cary 50 Concentration spectrophotometer. Optical rotations were done at 25 °C on a Rudolph Research Autopol IV. NMR Spectra were recorded on a Varian Mercury VMX 300-MHz spectrophotometer. In reporting the NMR multiplicities, we used the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; m, multiplet; apt., apparent; and br, broad. Low resolution mass spectra (MS) were obtained on a Finnigan LCQDuo LC MS/MS system equipped with an electrospray ionization (ESI) probe. High resolution mass spectra (HRMS) were obtained by the University of Minnesota Mass Spectrometry Service, also with an ESI probe. . Analytical HPLC data were obtained using a Waters 2690 Separations Module with PDA detection. Method (a): Varian Sunfire 100 × 4.6 mm C18 column. Method (b): ThermoHypersil BDS 100 × 4.6 mm C18 column. Method (c): Chiralpak IA 250 × 10 mm. Merck silica gel (230–400 mesh) was used for open column chromatography.</p><!><p>The fenoterol analogues used in this study were prepared following a previously reported synthetic scheme in which (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin or (S)-(+)-3',5'-dibenzyloxyphenylbromohydrin was condensed with the appropriate free N-benzylaminoalkane.3 The present work only utilized (R)-(−)-3',5'-dibenzyloxy-phenylbromohydrin, which was synthesized as previously reported.3 The syntheses of the new N-benzylaminoalkanes and fenoterol analogues are reported below.</p><!><p>The appropriate phenylacetic acid and alkyl anhydride were combined in pyridine and heated to reflux under argon for 6 h. Solvents were removed and the residue was partitioned between dichloromethane and 1N NaOH. The organic fraction was washed with water, dried (Na2SO4), filtered, and evaporated. Silica gel chromatography (1:30) eluting with 1/1 dichloromethane/hexanes affords the pure aryl ketone.</p><!><p>Prepared from 7.5 g (31 mmol) of 4-benzyloxyphenylacetic acid (Aldrich) and 17.2 mL of propionic anhydride in 17 mL of pyridine. Yield 1.92 g (24%). 1H NMR (CDCl3) δ̣ 1.00 (t, 3H, J = 7.5 Hz), 2.44 (q, 2H, J = 7.5, Hz), 3.59 (s, 2H), 5.02 (s, 2H), 6.90 (d, 2H, J = 8.7 Hz), 7.09 (d, 2H, J = 8.7 Hz), 7.25–7.40 (m, 5H) ppm.</p><!><p>Prepared from 25.0 g (134 mmol) of 2-naphthylacetic acid (Aldrich) and 100 mL of acetic anhydride in 100 mL of pyridine. Yield 3.68 g (15%). 1H NMR (CDCl3) δ 2.19 (s, 3H), 3.86 (s, 2H), 7.35 (dd, 1H, J = 2.1, 8.7 Hz), 7.45–7.50 (m, 2H), 7.68 (s, 1H), 7.79–7.85 (m, 3H), ppm.</p><!><p>Prepared from 5.04 g (23.3 mmol) of 4-methoxy-1-naphthylacetic acid (ACB Blocks Ltd.) and 30 mL of acetic anhydride in 30 mL of pyridine. Yield 1.54 g (31%). 1H NMR (CDCl3) δ 2.09 (s, 1H), 4.01 (s, 3H), 4.02 (s, 2H), 6.78 (d, 1H, J = 7.8 Hz), 7.29 (d, 1H, J = 7.8), 7.47–7.56 (m, 2H), 7.78–7.82 (m, 1H), 8.30–8.33 (m, 1H) ppm.</p><!><p>To the appropriate aryl ketone in dichloromethane (c = 0.5 M), cooled to 0 °C was added glacial acetic acid (1 eq), followed by benzylamine (1 eq) and Na(AcO)3BH (1.4 eq). The reaction mixture was warmed to RT and stirred under argon for 20 h. Cooled reaction mixture (ice bath) and added dropwise 10% NaOH (5 eq), then extracted into dichloromethane, washed with brine, dried (Na2SO4), filtered, and evaporated. The resulting product was used without further purification.</p><!><p>Prepared from 1.88 g (7.4 mmol) of 1-(4-(benzyloxy)phenyl)butan-2-one, 0.81 mL (7.4 mmol) of benzylamine, 2.40 g (11.3 mmol) of Na(OAc)3BH and 0.5 mL of acetic acid. Yield 2.55 g (100%). 1H NMR (CDCl3) δ 0.93 (t, 3H, J = 7.2 Hz), 1.45–1.55 (m, 2H), 2.60–2.75 (m, 2H, 1H), 3.76 (dd, 2H, J = 6.6, 9.9 Hz), 5.03 (s, 2H), 6.88 (d, 2H, J = 8.7 Hz), 7.06 (d, 2H, J = 8.7 Hz), 7.18–7.45 (m, 10 H) ppm. MS (ESI+) m/z (rel): 346 (100, M+H).</p><!><p>Prepared from 3.68 g (20 mmol) of 1-(naphthalen-2-yl)propan-2-one, 2.18 mL (20 mmol) of benzylamine, 6.35 g (30 mmol) of Na(OAc)3BH and 1.3 mL of acetic acid. Yield 5.04 g (92%). 1H NMR (CDCl3) δ 1.07 (d, 3H, J = 6.0 Hz), 2.70–2.91 (m, 2H), 2.97 (sextet, 1H, J = 6.6 Hz), 3.39 (s, 1H, NH), 3.75 (dd, 2H, J = 13.2, 34.2 Hz), 7.10–7.24 (m, 5H, 1H), 7.34–7.39 (m, 2H), 5.30 (s, 1H), 7.67–7.77 (m, 3H) ppm. MS (ESI+) m/z (rel): 276 (100, M+H).</p><!><p>Prepared from 1.48 g (6.9 mmol) of 1-(4-methoxynaphthalene-1-yl)propan-2-one, 0.75 mL (6.9 mmol) of benzylamine, 2.20 g (10.4 mmol) of Na(OAc)3BH and 0.45 mL of HOAc. Yield 1.96 g (93%). 1H NMR (CDCl3) δ̣ 1.15 (d, 3H, J = 6.0 Hz), 2.98–3.15 (m, 2H), 3.20–3.24 (m, 1H), 3.82 (dd, 3H, J = 13.8, 47.7 Hz), 3.99 (s, 3H), 6.73 (d, 1H, J = 7.8 Hz), 7.17–7.26 (m, 5H, 1H), 7.45–7.48 (m, 2H), 7.88–7.91 (m, 1H), 8.28–8.32 (m, 1H) ppm. MS (ESI+) m/z (rel): 306 (100, M+H).</p><!><p>The appropriate racemic N-benzylaminoalkane was combined with 1 eq of either optically active mandelic acid or naproxen in 1:2 iPrOH/MeOH (c= 0.5 M), refluxed until full dissolution, then cooled to RT. The crystals were filtered, collected, and recrystallized twice from MeOH (c = 0.3 M) to afford the optically active salts. The salts were converted to the free amine by partitioning between 10% K2CO3 and CHCl3, drying organic extracts (Na2SO4), filtering and evaporating.</p><!><p>Prepared from 2.56 g (7.4 mmol) of rac-N-benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine and 1.12 g (7.4 mmol) of (R)-(−)-mandelic acid (Aldrich). Yield 433 mg (33%), [α]D = −18.0 (0.7% MeOH).</p><!><p>Prepared from 2.11 g (6.1 mmol) of rac-N-benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine and 928 mg (6.1 mmol) of (S)-(+)-mandelic acid (Aldrich). Yield 407 mg (39%), [α]D = +21.4 (0.8% MeOH).</p><!><p>Prepared from 1.05 g (3.8 mmol) of rac-N-benzyl-1-(naphthylen-2-yl)propan-2-amine and 875 mg (3.8 mmol) of (S)-(+)-naproxen (Aldrich). Yield 310 mg (61%), [α]D = −11.2 (0.7% MeOH).</p><!><p>Prepared from 2.10 g (7.6 mmol) of rac-N-benzyl-1-(naphthylen-2-yl)propan-2-amine and 1.76 g (7.6 mmol) of (R)-(−)-naproxen (Aldrich). Yield 630 mg (60%), [α]D = +12.0 (0.7% MeOH).</p><!><p>Prepared from 1.04 g (3.4 mmol) of rac-N-benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine and 520 mg (3.4 mmol) of (S)-(+)-mandelic acid. Yield 389 mg (77%), [α]D = −12.1 (1.0% MeOH).</p><!><p>Prepared from 1.95 g (6.4 mmol) of rac-N-benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine and 971 mg (6.4 mmol) of (R)-(−)-mandelic acid. Yield 988 mg (67%), [α]D = +11.7 (1.0% MeOH).</p><!><p>In this study, the fenoterol analogues were prepared using a previously described approach.3 The key step involved the coupling of the epoxide formed from (R)-(−)-3',5'-dibenzyloxy-phenylbromohydrin with the (R)- or (S)-enantiomer of the appropriate N-benzylaminoalkanes. The epoxide was formed by combining (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin (1 eq) with K2CO3 (1.4 eq) in 1:1 THF/MeOH (c = 0.3 M) and stirring for 2 h under argon at RT. The solvent was removed and the residue partitioned between toluene and H2O, the toluene fraction dried (Na2SO4), filtered, and evaporated. The residue was dissolved with 0.95 eq of the appropriate free N-benzylaminoalkane in toluene and evaporated again under high vacuum to remove trace H2O. The resulting colorless residue was heated to 120 °C under argon for 24–48 h, monitoring by 1H NMR and mass spectrometry to afford the coupled product. The residue was dissolved in EtOH (c = 0.07 M) with heat and transferred to a Parr flask, where it was hydrogenated at 50 psi of hydrogen over 10% (wt) Pd/C (10 mg cat/65 mg bromohydrin) for 24 h to afford the debenzylated product, as confirmed by 1H NMR and mass spectrometry. The mixture was filtered through Celite®, the filter cake rinsed with iPrOH, and then concentrated. The free base was purified on silica gel (1:20) eluting with 15% methanol/chloroform. The resulting material was converted to the fumarate salt by heating with 0.5 eq of fumaric acid in 1:1 iPrOH/EtOH (c = 0.2 M) to afford the 0.5 fumarate salt of the fenoterol analog.</p><!><p>Prepared from 282 mg (0.82 mmol) of (R)-(−)-N-benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine and 260 mg (0.78 mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 175 mg (60%). 1H NMR (300 MHz, CD3OD): δ 0.950 (t, 3H, J=7.5 Hz), 1.67 (m, 2H), 2.83–3.18 (m, 4H), 3.33–3.40 (m, 1H), 3.37 (s, 4H), 4.82 (m, 1H), 6.24 (d, 1H, J = 2.1 Hz), 6.37 (d, 2H, J = 1.8 Hz), 6.73 (s, 2H, fum), 6.76 (d, 2H, J = 8.4 Hz), 7.05 (d, 2H, J = 8.7 Hz) ppm. 13C NMR (75 MHz, CD3OD): δ̣ 9.43, 23.28, 36.56, 52.29, 62.16, 70.02, 103.4, 105.3, 116.7, 127.8, 131.3, 136.5, 144.6, 157.6, 159.9, 172.3 ppm. UV (MeOH) λmax 206 nm (ε 22,500), 223 (12,300), 278 (2,460); [α]D = −15.6 (free amine, 0.5% MeOH); HPLC: (a) H2O/ACN/TFA 70/30/0.01, 1.0 mL/min, 278 nm, tR=3.57 min, 97.1% pure. (c) ACN/MeOH/TFA 90/10/0.05, 2.0 mL/min, 278 nm, tR=5.26 min, 97.5% enantiomeric purity. HRMS (m/z) Calcd for C18H24NO4 [M+H]+ 318.1700; found 318.1699.</p><!><p>Prepared from 352 mg (1.02 mmol) of (S)-(+)-N-benzyl-1-(4-(benzyloxy)phenyl)butan-2-amine and 321 mg (0.97 mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 142 mg (38%). 1H NMR (300 MHz, CD3OD): δ 0.972 (t, 3H, J = 7.5 Hz), 1.70 (p, 2H, J = 6.9 Hz)), 2.86–3.22 (m, 4H), 3.32–3.37 (m, 1H), 3.34 (s, 4H), 4.82 (m, 1H), 6.25 (t, 1H, J=2.1 Hz), 6.36 (d, 2H, J=1.8 Hz), 6.74 (s, 2H, fum), 6.77 (d, 2H, J=8.4 Hz), 7.08 (d, 2H, J=8.7 Hz) ppm. 13C NMR (75 MHz, CD3OD): δ̣ 9.820, 24.16, 36.48, 52.30, 62.32, 69.92, 103.3, 105.3, 116.8, 127.7, 131.3, 136.1, 144.4, 157.6, 159.8, 171.3 ppm. UV (MeOH) λmax 204 nm (ε 26,900), 224 (11,500), 278 (2,320). [α]D= −7.2 (free amine, 0.5% MeOH). HPLC: (a) H2O/ACN/TFA 70/30/0.01, 1.0 mL/min, 278 nm, tR = 3.56 min, 99.5% pure. (c) ACN/MeOH/TFA 90/10/0.05, 2.0 mL/min, 278 nm, tR = 5.88 min, 99.0% enantiomeric purity. HRMS (m/z): Calcd for C18H24NO4 [M+H]+ 318.1700; found 318.1706</p><!><p>Prepared from 310 mg (1.12 mmol) of (R)-(−)-N-benzyl-1-(naphthylen-2-yl)propan-2-amine and 355 mg (1.06 mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 186 mg (44 %). 1H NMR (300 MHz, CD3OD): δ 1.06 (d, 3H, J=6.3 Hz), 2.73–2.90 (m, 4H), 3.07 (m, 1H), 3.30 (m, 3H), 4.05 (dd, 1H, J = 5.1, 8.4 Hz), 6.11 (t, 1H, J = 2.4 Hz), 6.28 (d, 2H, J = 2.1 Hz), 7.26 (dd, 1H, J = 1.5, 8.4 Hz), 7.38–7.42 (m, 2H), 7.57 (s, 1H), 7.72–7.77 (m, 3H) ppm. 13C NMR (75 MHz, CD3OD): δ̣ 15.84, 40.67, 52.70, 56.66, 70.46, 103.27, 105.26, 126.97, 127.39, 128.22, 128.64, 128.70, 129.20, 129.64, 134.04, 135.05, 135.11, 136.81, 144.95, 160.13, 173.20 ppm. UV (MeOH), λmax: 276 nm (ε 6,170), 224 (90,680), 206 (50,290; [α]D = −27.4 (0.5% MeOH). HPLC: (b) H2O/ACN/TFA 70/30/0.1, 1.0 mL/min, 275 nm, tR = 3.96 min, 95.3% purity. (c) ACN/iPrOH/DEA 95/5/0.05, 3.0 mL/min, 275 nm, tR = 8.58 min, 95.6% enantiomeric purity. . HRMS (m/z): Calcd for C21H24NO3 [M+H]+ 338.1751; found 338.1745.</p><!><p>Prepared from 626 mg (2.27 mmol) of (S)-(+)-N-benzyl-1-(naphthylen-2-yl)propan-2-amine and 719 mg (2.16mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 578 mg (68%). 1H NMR (300 MHz, CD3OD): δ 1.09 (d, 3H, J = 6.3 Hz), 2.60–2.95 (m, 4H), 3.03 (m, 1H), 3.30 (m, 3H), 4.07 (dd, 1H, J = 5.1, 8.4 Hz), 6.14 (t, 1H, J = 2.4 Hz), 6.28 (d, 2H, J = 2.1 Hz), 7.33 (dd, 1H, J = 1.8, 8.4 Hz), 7.40–7.44 (m, 2H), 7.64 (s, 1H), 7.77–7.81 (m, 3H) ppm. 13C NMR (75 MHz, CD3OD): δ̣ 16.52, 40.28, 52.82, 56.88, 70.75, 103.27, 105.29, 126.97, 127.39, 128.22, 128.64, 128.69, 129.20, 129.65, 134.03, 135.05, 135.18, 136.89, 144.95, 160.00, 173.46 ppm. UV (MeOH), λmax: 276 (ε 6,310), 224 (93,530), 204 (50,580). [α]D = −18.8 (0.5% MeOH). HPLC: (b) H2O/ACN/TFA 70/30/0.1, 1.0 mL/min, 275 nm, tR = 3.94 min, 98.6% purity. (c) ACN/iPrOH/DEA 95/5/0.05, 3.0 mL/min, 275 nm, tR = 8.04 min, 96.4% enantiomeric purity. HRMS (m/z): calcd for C21H24NO3 [M+H]+ 338.1751, found 338.1746.</p><!><p>Prepared from 390 mg (1.28 mmol) of (R)-(−)-N-benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine and 404 (1.22 mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 309 mg (60%). 1H NMR (300 MHz, CD3OD): δ 1.22 (t, 3H, J = 6.6 Hz), 3.09–3.21 (m, 3H), 3.59–3.69 (m, 2H), 3.99 (s, 3H), 4.74–4.83 (m, 1H), 6.23 (t, 1H, J = 2.4 Hz), 6.37 (dd, 2H, J = 2.4, 5.7 Hz), 6.74 (s, 1H), 6.86 (d, 1H, J = 7.8 Hz), 7.32 (d, 1H, J = 7.8 Hz), 7.48 (t, 1H, J = 6.9 Hz), 7.56 (t, 1H, J = 6.9 Hz), 8.02 (dd, 1H, J = 8.4, 12.0 Hz), 8.27 (d, 1H, J = 8.7 Hz) ppm. 13C NMR (75 MHz, CD3OD): δ 15.78, 36.66, 52.39, 55.96, 70.20, 103.4, 104.5, 105.3, 123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.5, 133.8, 135.2, 144.6, 156.6, 160.0, 168.3 ppm. UV (MeOH), λmax: 298 nm (ε 4,970), 286 (9,920), 234 (22,600), 210 (42,500). [α]D= −28.8 (free amine; 0.5% MeOH). HPLC: (a) H2O/ACN/TFA 50/50/0.01, 1.0 mL/min, 286 nm, tR = 3.087 min, 99.0% pure. (c) Gradient 0–20 % iPrOH/Hexane/0.05 % TFA, 3.0 mL/min, 275 nm, tR = 8.62 min, 95.0% enantiomeric purity. HRMS (m/z): Calcd for C22H26NO4 [M+H]+ 368.1856; found 368.1845.</p><!><p>Prepared from 422 mg (1.38 mmol) of (S)-(+)-N-benzyl-1-(4-methoxynaphthalen-1-yl)propane-2-amine and 438 (1.31 mmol) of (R)-(−)-3',5'-dibenzyloxyphenylbromohydrin. Yield 302 mg (53%). 1H nMR (300 MHz, CD3OD): δ 1.20 (t, 3H, J = 6.6 Hz), 3.07–3.21 (m, 3H), 3.52–3.75 (m, 2H), 3.97 (s, 3H), 4.69–4.83 (m, 1H), 6.24 (t, 1H, J = 2.1 Hz), 6.39 (dd, 2H, J = 2.4, 5.4 Hz), 6.74 (s, 1H), 6.84 (d, 1H, J = 7.8 Hz), 7.31 (d, 1H, J = 8.1 Hz), 7.48 (t, 1H, J = 6.9 Hz), 7.56 (t, 1H, J = 6.9 Hz), 8.01 (dd, 1H, J = 8.4, 13.5 Hz), 8.27 (d, 1H, J = 7.8 Hz) ppm. 13C NMR (75 MHz, CD3OD): δ̣ 15.77, 36.64, 52.37, 55.94, 70.46, 103.4, 104.5, 105.3, 123.8, 124.3, 124.9, 126.2, 127.4, 128.1, 129.4, 133.8, 135.5, 144.7, 156.6, 160.0, 169.0 ppm. UV (MeOH), λmax: 298 nm (ε 5,430), 286 (5,710), 233 (25,100), 210 (43,200). [α]D= −15.8 (free amine; 0.5% MeOH). HPLC: (a) H2O/ACN/TFA 50/50/0.01, 1.0 mL/min, 286 nm, tR = 3.087 min, 96.0% pure (c) Gradient 0–20 % IPrOH/Hexane/0.05% TFA, 3.0 mL/min, 275 nm, tR = 9.69 min 99.0% enantiomeric purity. HRMS (m/z): Calcd for C22H26NO4 [M+H]+ 368.1856; found 368.1844.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p>