Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor

Article abstract of DOI:10.1016/j.bmc.2013.11.030

The β₂-adrenergic receptor (β₂-AR) agonist [³H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (K_i values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [³H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the K_i values obtained using [³H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [³H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [³H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [³H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

Full text of DOI:10.1016/j.bmc.2013.11.030

Bioorganic & Medicinal Chemistry 22 (2014) 234–246
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Comparative molecular field analysis of fenoterol derivatives
interacting with an agonist-stabilized form of the b2-adrenergic
receptor
Anita Plazinska a, , Karolina Pajak a, , Ewelina Rutkowska a, , Lucita Jimenez b, Joseph Kozocas b,
Gary Koolpe b, Mary Tanga b, Lawrence Toll c, Irving W. Wainer d, Krzysztof Jozwiak a,
⇑
a Medical University of Lublin, Lublin, Poland
b SRI International, Menlo Park, CA, United States
c Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, United States
d Laboratory of Clinical Investigation, National Institute on Aging Intramural Research Program, Baltimore, MD, United States
a r t i c l e i n f o
a b s t r a c t
The b2-adrenergic receptor (b2-AR) agonist [3H]-(R,R0)-methoxyfenoterol was employed as the marker
ligand in displacement studies measuring the binding affinities (Ki values) of the stereoisomers of a series
of 40-methoxyfenoterol analogs in which the length of the alkyl substituent at a0 position was varied from
0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the
inverse agonist [3H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP
accumulation, measured as EC50 values, were determined in HEK293 cells expressing the b2-AR. The data
indicate that the highest binding affinities and functional activities were produced by methyl and ethyl
substituents at the a0 position. The results also indicate that the Ki values obtained using [3H]-(R,R0)-
methoxyfenoterol as the marker ligand modeled the EC50 values obtained from cAMP stimulation better
than the data obtained using [3H]-CGP-12177 as the marker ligand. The data from this study was
combined with data from previous studies and processed using the Comparative Molecular Field Analysis
approach to produce a CoMFA model reflecting the binding to the b2-AR conformation probed by
[3H]-(R,R0)-40-methoxyfenoterol. The CoMFA model of the agonist-stabilized b2-AR suggests that the
binding of the fenoterol analogs to an agonist-stabilized conformation of the b2-AR is governed to a
greater extend by steric effects than binding to the [3H]-CGP-12177-stabilized conformation(s) in which
electrostatic interactions play a more predominate role.
Article history:
Received 3 September 2013
Accepted 16 November 2013
Available online 23 November 2013
Keywords:
b2-Adrenoceptor selective agonist
b2-Adrenoceptor conformations
Agonist-stabilized conformations
Antagonist-stabilized conformations
[3H]-(R,R0)-40-methoxyfenoterol
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
(EC50cAMP) and cadiomyocyte contractility (EC50cardio) as the func-
tional markers.1,3 In the first stage of the project 26 compounds
(R,R0)-Fenoterol ((R,R0)-1, Table 1) is a potent and selective
agonist of the b2-adrenergic receptor (b2-AR), which has entered
clinical trials for the treatment of congestive heart failure. The
potential therapeutic utility of (R,R0)-1 is based upon its 43-fold
selectivity for the b2-AR relative to the b1-AR, determined as bind-
ing affinities (Ki values) that were calculated using the b2-AR
antagonist [3H]-CGP-12177 as the marker ligand.1 (R,R0)-1 also
selectively couples to Gs proteins2 and produces a 300% increase
in contractile response in isolated rat ventricular myocytes.1,2
Using (R,R0)-1 as the scaffold, a project was initiated to study the
effect of stereochemistry and the structure of the aminoalkyl moi-
ety of the molecule on b2-AR binding affinity and selectivity as well
as activity using induced stimulation of cAMP accumulation
were synthesized and the Kib2-AR data used to develop a Compara-
tive Molecular Field Analysis (CoMFA) model1, which was then
used for virtual in silico prediction of binding affinities for a set
of 21 new molecular structures.3 Six of the designed molecules
were synthesized and their experimentally determined Kib2-AR val-
ues were in good agreement with the theoretical prediction and
the data obtained using the additional compounds were used to
refine the CoMFA model. While the expected Kib2-AR values were
observed for the designed compounds, two of the derivatives,
(R,R0)-52 and (R,R0)-54 had unexpectedly high b2-AR subtype
selectivity with Kib1-AR/Kib2-AR of 334 and 573, respectively.3 The
functional activities of a subset of the compounds were also deter-
mined and relationships between Kib2-AR and EC50cAMP and Kib2-AR
and EC50cardio were observed for (R,R0)-1, (R,R0)-2 and (R,R0)-54 sug-
gesting that the CoMFA model could be used to design compounds
for use in the treatment of congestive heart failure. However, when
⇑
Corresponding author. Tel.: +48 81 7584862.
E-mail address: krzysztof.jozwiak@umlub.pl (K. Jozwiak).
Equal contributors.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.030

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
235
Table 1
Chemical structures of discussed FEN derivatives
Compound
a0 carbon atom number
Structure
Old compounds
OH
OH
HO
NH
NH
(R,R0)-1
(R,S0)-1
1
CH3
OH
OH
HO
CH3
OH
OH
OH
OH
HO
HO
NH
NH
(R,R0)-52
(R,S0)-52
2
OH
OH
OH
OH
OH
HO
NH
(R,R0)-54
(R,S0)-54
1
CH3
O
OH
CH3
OH
OH
HO
HO
NH
NH
CH3
O
OH
OH
CH3
(R,R0)-2
(R,S0)-2
(S,R’)-2
(S,S’)-2
1
CH3
O
CH3
OH
OH
OH
HO
HO
HO
NH
NH
NH
CH3
CH3
CH3
O
OH
OH
OH
CH3
O
CH3
O
CH3
(continued on next page)

236
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
Table 1 (continued)
Compound
a0 carbon atom number
Structure
New compounds
OH
OH
OH
OH
OH
OH
OH
OH
HO
NH
(R,R0)-64
2
3
3
0
H3C
O
OH
CH3
HO
HO
HO
HO
HO
HO
HO
NH
(R,S0)-64
(R,S0)-65
(R,R0)-65
(R,R0)-66
(R,S0)-66
(R)-67
H3C
O
OH
OH
OH
OH
OH
OH
OH
CH3
NH
H3C
CH3
O
CH3
NH
H3C
CH3
O
CH3
NH
O
CH3
NH
CH3
O
CH3
NH
CH3
O
CH3
NH
(S)-67
O
CH3
(R,R0)-52 was studied, the compound had a >100-fold lower activity
in the cardiomyocyte contractility model while the relationship be-
tween Kib2-AR and EC50cAMP was consistent with the other tested
compounds. The results suggested that the substitution of an ethyl
group on the aminoalkyl moiety of (R,R0)-1 had a profound effect
on the interaction of (R,R0)-52 with a conformation(s) of b2-AR
and that this effect was not reflected by the Kib2-AR values obtained
using [3H]-CGP-12177 as the marker ligand or the CoMFA model
constructed using these Kib2-AR values.
values were determined using [3H]-CGP-12177 and [3H]-(R,R0)-2 as
the marker ligands. [3H]-(R,R0)-2 was used for this study as it has
been previously demonstrated that this radioligand binds with
high affinity to an agonist conformation of the b2-AR4, and that
Kib2-AR values for the fenoterol analogs, including (R,R0)-52, deter-
mined using [3H]-(R,R0)-2 modeled their EC50cAMP values better
than the data obtained using [3H]-CGP-12177.4 The EC50cAMP values
of the newly synthesized compounds were also determined. The
data were used to refine the CoMFA model developed using the
Kib2-AR values obtained with [3H]-CGP-12177 as the marker ligand
and to develop a new CoMFA model with the Kib2-AR values ob-
tained when [3H]-(R,R0)-2 was the marker ligand. The new CoMFA
model reflects binding to an agonist-stabilized conformation of the
b2-AR. The results demonstrate that there are subtle but significant
differences between the two models and suggest that the use of
Based on the data obtained with (R,R0)-52, we have explored the
effect of steric bulk at the a0-carbon of the aminoalkyl moiety on
binding affinity and functional activity using 40-methoxyfenoterol
(2) as the scaffold. The alkyl length of the substituent on the
a0-carbon of the aminoalkyl moiety was varied from 0 (compound
67) to 3 carbon atoms (compounds 65, 66), Table 1, and the Kib2-AR

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
237
multiple models can be beneficial in the design of new chemical
entities with specific pharmacological properties.
presented in Table 2. All of the tested compounds were full
b2-AR agonists and the most active analogs were (R,R0)-2, (R,S0)-2
and (R,R0)-64 with calculated EC50 values of 0.3 nM, (2 nM and
1.63 nM, respectively. The results are consistent with the observa-
tion that the use of [3H]-(R,R0)-2 as the marker ligand in displace-
ment studies probes binding to an agonist conformation of the
b2-AR and models EC50cAMP values better than the data obtained
using [3H]-CGP-12177.4
2. Results
2.1. Binding affinity of studied derivatives
Eight derivatives of compound 2 were synthesized by modify-
ing the alkyl substituent at the a0 position from 0 to 3 carbon atom
count (Table 1). The binding affinities of the synthesized com-
pounds to b2-AR were determined using assays displacing either
the antagonist radioligand [3H]-CGP-12177 or the selective ago-
nist, [3H]-(R,R0)-2.1,4,5 When [3H]-CGP-12177 was the marker
ligand, (R,R0)-2 and (R,R0)-64 had equal potency, 474 nM and
420 nM, while significantly lower affinities were observed for the
other derivatives with an (R,R0) or equivalent configuration,
2,675 nM, 7,604 nM and not quantifiable for (R)-67 (R,R0)-66 and
(R,S0)-65, respectively. It should be noted that the inclusion of
(R,S0)-65 in this cohort is based on the change in the R and S
2.2. Docking of ligands into b2-AR
Molecular models of studied derivatives were docked into the
crystal model of the b2-AR binding site obtained in studies of the
receptor cocrystallized with the agonist BI-167107 and the Gs
protein. This model, PDB id: 3SN6, is commonly considered repre-
sentative of an active state of the receptor6. Docking simulations
show that the orientation of docked FEN (fenoterol) molecules
corresponds well with the orientation of the BI-167107 molecule,
Supplemental Figure S1. Comparison of the docking of (R,R0)-2 with
BI-167107 indicates that both molecules assume very similar
orientations within the binding site and thus have very similar li-
gand–receptor interactions. The most important interactions are
those linking the protonated amino group and b-hydroxyl moiety
of both ligands via a very strong network of hydrogen bonds with
D113 and N312. In addition, the 3,5-hydroxyphenyl moiety of
(R,R0)-2 interacts via hydrogen bonds with S203 and S207 residues
located on TM5 while the 40-methoxy moiety on the other end of
the molecule forms a hydrogen bond with K305 residue on helix
7 (Fig. 1a).
Docking simulations attempted to identify optimal positions of
the derivatives with increased alkyl chain length. Figure 1b and c
show the lowest energy conformations obtained in docking of
(R,R0)-64 and (R,S0)-64, respectively. It can be seen that in compar-
ison to the docking position assumed by (R,R0)-2, increased bulki-
ness of the alkyl moiety significantly affects the location within
the binding site and possible ligand – receptor interactions. In this
agonist conformation, both (R,R0)-64 and (R,S0)-64 cannot assume
positions allowing all interactions analogous to (R,R0)-2. In particu-
lar, the 3,5-dihydroxyphenyl ring of (R,R0)-64 is not positioned to
designation at the
a’ position based on the Cahn–Ingold–Prelog
stereochemistry convention and not a change in actual configura-
tion, (see Table 1). The measured affinities of (R,R0)-2 and (R,R0)-
64 determined using [3H]-(R,R0)-2 as the marker ligand were
4.09 nM and 35.7 nM, respectively, indicating that while the affin-
ities of the two compounds were similar, the magnitude of the
binding affinity of (R,R0)-2 was more affected by the change in mar-
ker ligand than (R,R0)-64. However, this may only be a reflection of
the fact that the calculated affinity of (R,R0)-2 is a Kd value. The Ki
values for (R,S0)-65, (R,R0)-66, and (R)-67 could be determined using
[3H]-(R,R0)-2 as the marker ligand and were 3420 nM, 250 nM and
101 nM, respectively.
The data indicate that the optimal length of the alkyl moiety at-
tached to the a0 position of the molecular scaffold is one or two
carbon atoms, that is a methyl or ethyl group.
The observed trend in the effect of stereochemistry on receptor
affinities was confirmed in studies of the potency and maximal
activity for stimulation of cAMP accumulation HEK-293 cells stably
transfected with human b2-AR gene. The EC50 values and % of stim-
ulation in relation to isoproterenol, the reference full agonist, are
Table 2
Binding affinities of studied compounds
Compound
b2-AR binding affinity determined in displacement of
Induced cAMP accumulation
[3H]-CGP-12177
[3H]-(R,R0)-2
Ki (nM)
0.46 0.06
HS
Ki (nM)
3.69 1.36
HS
EC50 (nM)
% Stimulation
Propranolol
Isoproterenol
(R,R0)-1
1.24 0.1
0.85 0.06
0.92 0.1
0.81 0.1
1.01 0.01
2.07 0.4
1.07 0.09
1.06 0.02
0.98 0.05
1.01 0.14
1.28 0.09
2.30 0.32
1.00 0.11
ND
ND
ND
0.90 0.03
ND
1.80 0.70
ND
1.88 0.34
0.78 0.07
0.76 0.1
0.97 0.1
0.93 0.1
0.87 0.2
0.86 0.1
0.90 0.2
0.80 0.9
1.00 0.05
0.86 0.13
1.68 0.54
0.82 0.04
0.86 0.05
1.41 0.47
1.45 0.56
1.38 0.32
0.89 0.14
1.59 0.50
0.89 0.03
192 24
345 33.8a
3695 245a
1273 81a
5758 833a
277 11a
2.44 0.28
4.00 0.75b
183 30.0b
39.1 5.38b
294 45.1b
13.3 2.72b
12.7 1.83b
4.09 0.55b
26.1 2.4b
91.3 32b
0.20 0.09
0.30 0.09a
4.70 0.50a
2.80 0.90a
16.60 4.90a
3.90 1.80a
4.00 1.29a
0.30 0.23b
2.00 0.45b
7.20 1.01b
33.20 11.48b
1.63 0.51
1,010 240
26,200 6,500
6,100 1,500
462 161
6,100 1,300
10.19 1.79
210 96
100.00
123
9
(R,S0)-1
131 30
(R,R0)-52
(R,S0)-52
(R,R0)-54
(R,S0)-54
(R,R0)-2
(R,S0)-2
(S,R’)-2
(S,S0)-2
(R,R0)-64
(R,S0)-64
(R,R0)-65
(R,S0)-65
(R,R0)-66
(R,S0)-66
(R)-67
125
98
6
4
106 11
118 30
317 6a
474 40a
135.70 11.03
120.10 18.97
129.00 12.67
156.40 35.93
156 33
145.7 8.8
103.5 7.8
207 56
147.2 2.7
197 32
114.2 8.4
131 43
1930 140a
5270 510a
15,900 2700a
420 120
2870 234b
35.7 4.3
4,300 1,000
29,200 4,400
3,420 320
250 41
4,740 700
101 33
1,150 410
20,100 4950
>100,000
>100,000
7600 230
>100,000
2670 240
>100,000
(S)-67
a
3
Data obtained from Ref.
Data obtained from Ref.
.
b
4
.

238
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
(A)
(B)
(C)
Figure 2. CoMFA model constructed for affinity data determined using [3H]-(R,R0)-2
displacement assay for 32 FEN derivatives assayed in the current and in previous
reports.1,3 The fields are color coded in the following manner: green and yellow
represent regions of steric (bulk) interactions (favorable and unfavorable, respec-
tively), electrostatic interactions with a positive charge (or H-bond donor) are blue,
and electrostatic interactions with a negative charge (or H-bond acceptor) are red.
Molecular model of (R,R0)-64 is rendered in stick mode for orientation purpose.
0
reaches 5 ÅA). Instead, the 40-methoxy group forms a hydrogen bond
with W313 residue of TM7 (the interaction is not shown on
Fig. 1c).
Analogous observations as above can be made in docking the
other derivatives, 65, 66 and 67. Either increasing the chain to pro-
pyl for (R,S0)-65 and (R,R0)-66, or reducing it to hydrogen as in case
of (R)-67 prevents the molecules from occupying positions that
exercise all interactions as observed in docking of (R,R0)-2, data
not shown. This negative effect is increasingly profound in docking
simulation of these derivatives with other stereochemistries. This is
in agreement with experimental data where (R,R0)-64, (R,S0)-65,
(R,R0)-66 or (R)-67 are more potent within the pair of stereoisomers.
Since the binding affinity of (R,R0)-64 is very similar to those of
(R,R0)-2 or (R,R0)-1 the results suggest that the receptor most likely
assumes slightly different conformation to adopt the molecule
with a0ethyl moiety.
Figure 1. The lowest energy conformations of (A) (R,R0)-2, (B) (R,R0)-64 and (C)
(R,S0)-64 obtained in docking simulations to molecular model of b2-AR binding site.
For clarity of both figures, TM1, TM2, and extracellular loop 2 were hidden, and the
remaining transmembrane segments are color coded as follows: TM3, red; TM4,
green; TM5, magenta; TM6, yellow; and TM7, blue. Only the residues forming
2.3. 3D-QSAR
The affinity data determined for new derivatives were subjected
to 3D-QSAR modeling. Previous CoMFA models generated based on
affinity data determined using the [3H]-CGP-12177 displacement
assay underlined the importance of the stereochemical configura-
tion of a derivative and the type of modification at the aminoalkyl
part of the molecule. If currently studied compounds explore addi-
tional dimensions of chemical modification this fact should be re-
flected in new CoMFA models. Additionally, the binding data for a
significant number of FEN derivatives were determined using the
radiolabeled agonist [3H]-(R,R0)-2. This presents the unique oppor-
tunity to compare our previous data with a second 3D-QSAR model
basedon pKi valuesdetermined to an agonist receptor conformation.
The number of data points in the current model (pKi values
determined using [3H]-(R,R0)-2 derived data) is n = 32 (18 in cur-
rent report, Table 1 and 14 previously presented3). The newly gen-
erated model maintains high statistical significance as reflected by
R2 = 0.699, F = 191.7, SEP = 0.458, Q2 = 0.86.
hydrogen bonds (shown as green arrows) with
explicitly. All aliphatic hydrogen atoms are hidden.
a ligand molecule are shown
form strong hydrogen bonds with S203 and S207 on TM5, while
the 40-methoxyphenyl ring of the molecule is bent preventing
therefore the formation of a strong hydrogen bond with K305 (in
all three cases distances between donor and acceptor atoms exceed
0
3.5 ÅA). The amino group of (R,R0)-64 does form coulombic interac-
tions with D113 (marked with a yellow arrow on Fig. 1b) but does
not form a hydrogen bond with another possible partner, N312.
This reduced hydrogen bond formation can explain the 10 fold
decrease in binding affinity of (R,R0)-64 when tested using
[3H]-(R,R0)-2 to measure binding in an agonist conformation. In
docking of (R,S0)-64, the amino group of the ligand cannot interact
with D113 but forms a hydrogen bond with N312; the 3,5-dihy-
droxyphenyl ring is closer to TM5 allowing stronger hydrogen
The field distribution of the CoMFA [3H]-(R,R0)-2 model is pre-
sented in Figure 2. The model confirms previous observations that
the stereochemistry of a molecule plays a very important role in
the observed affinity. Both chiral centers of studied molecules are
bonds with serine residues (distances between donor and acceptor
0
atoms are between 2.6 and 2.9 ÅA); 40-methoxyphenyl ring is bent
deeper into the cavity, entirely out of range for K305 (distance

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
239
asymmetrically surrounded by molecular fields: b carbon atom by
a combination of electrostatic fields and a0 carbon atom by a com-
bination of steric fields. In the first case, the red field representing
interactions with negative charge (or hydrogen bond acceptor) is
located in front of the molecule, while a blue, electropositive field
is behind the molecule in Fig. 2. The model, thus, illustrates that
the b-hydroxyl moiety of FEN and analogs can donate a proton to
make a hydrogen bond while in the (R) stereoconfiguration, and
such an interaction is disfavored in the (S) configuration of the chi-
ral center. With regards to the other chiral center, both sides of the
a0 carbon atom, are filled by steric fields: the large yellow unfavor-
able field behind the molecule on Fig. 2, underlines the fact that
pointing the alkyl moiety in that direction (i.e., the (S0) stereocon-
figuration of the center for methyl, ethyl and n-propyl moieties;
the (R0) configuration for i-propyl) negatively affects the binding
affinity. On the other hand, green sterically favorable field in front
of the chiral center suggests that the (R0) configuration of these
analogs derivatives has a positive effect on observed pKi values.
Interestingly, the green field is associated with another smaller yel-
low field at a greater distance from the chiral center what clearly
supports the observation that increased size of alkyl moiety (i.e.,
propyl chains) significantly reduces the affinity these derivatives
even if it is in preferred stereoconfiguration.
Another region of interest in this CoMFA model is the aminoal-
kyl part of these molecules where structural modifications are con-
centrated. This region of the model is represented by several steric
fields, among them one significant favorable (green) field within
the plane of the phenyl ring of the molecule (Fig. 2). This field likely
corresponds with positive effects associated with occurrence of a
naphthyl ring present in several analogs (previously described in
detail3). There is also one electropositive field in this region of
the model (blue, on the bottom right of Fig. 2.), which represents
the positive effect of forming a hydrogen bond with an acceptor
atom (either oxygen or nitrogen) in 40-position of a derivative.
The model has high statistical significance and may be consid-
ered as a 3D representation of derivatives0 affinity to an active form
of the receptor. Supporting material includes a Table S2 collecting
pKi values predicted by the model for all derivatives including
those not yet tested in the [3H]-(R,R0)-2 displacement assay.
Ki(R,R0), which differed from the ꢀ5-fold differences calculated
for the (R,R0)- and (R,S0)-isomers of 2 and 52. The Ki values of
(R,R0)-66 and (R)-67 were also lower than their respective stereo-
isomers, indeed no measurable binding affinities were observed
for (R,S0)-66 and (S)-67. The data suggest that both the 40-methoxy
moiety and the stereochemical configuration of the alkyl moiety at
the a0 carbon affect the binding interactions with the conforma-
tions probed by [3H]-CGP-12177.
When the methyl moiety on (R,R0)-2 was replaced by a hydro-
gen atom, the Ki value of (R)-67 was increased by 50-fold relative
to (R,R0)-2 and decreased by ꢀ3-fold relative to the Ki value of
(R)-7, the N-desmethyl analog of (R,R0)-1.1 These results suggest
that the presence of some steric bulk at the a0 carbon has a positive
effect on the binding affinity of the area(s) probed using [3H]-CGP-
12177 as the marker ligand. However, the data obtained with the
other analogs indicate that this effect is limited as the substitution
of an n-propyl moiety, (R,R0)-66, significantly reduced the Ki value
to >7000 nM and no measurable binding affinities were observed
with the derivatives containing an i-propyl substituent, (R,R0)-65
and (R,S0)-65 or for (R,S0)-66 and (S)-67.
Previous studies of the binding affinities of FEN analogs to the
b2-AR utilizing [3H]-(R,R0)-2 as the marker ligand have demon-
strated that the calculated Ki values are significantly lower than
the values calculated using [3H]-CGP-12177 as the marker ligand.4
The same results were obtained in this study including the ability
to determine Ki values for (R,R0)-65, (R,S0)-65, (R,S0)-66 and (S)-67,
Table 2. The same effect of the stereochemical configuration at
the a0 carbon was also observed as lower Ki values were observed
for (R,R0)-2, (R,R0)-64, (R,S0)-65 and (R,R0)-66 relative to their respec-
tive stereoisomers. In this regards, it is important to note that the
configuration at the a0 carbon of (R,S0)-65 is the same as the config-
urations in (R,R0)-64 and (R,R0)-66 as the only change is in the
Cahn–Ingold–Prelog nomenclature produced by the a0-i-propyl
moiety which changes the priority at this chiral center from (R)
to (S). The Ki value for (R)-67 was also 10-fold lower than (S)-67,
consistent with the importance of the configuration at the b-OH
carbon.
The Ki values for compounds 64, 65, 66 and 67 determined
using [3H]-(R,R0)-2 were combined with the Ki values of 22
previously studied FEN analogs4 to create a new CoMFA model cor-
relating molecular fields with the affinity data determined using
[3H]-(R,R0)-2. The resulting model, Fig. 2, confirms the importance
of a proper stereochemistry at both chiral centers of the FEN scaf-
fold: (1) the stereoconfiguration of the b-OH carbon corresponding
to the (R)-configuration is preferred due to the possibility of donat-
ing a hydrogen bonding partner from the b-OH group; (2) the ste-
reoconfiguration of the a0 carbon atom corresponding to the (R0)
configuration is preferred due to a steric reason as the model
clearly indicates that the opposite configuration of this chiral cen-
ter is sterically unfavored in terms of binding affinity. In addition,
the binding data and the modeling results suggest that for FEN
analogs with a 40-methoxy moiety the optimal binding affinity is
obtained with either a methyl (2) or ethyl (64) moiety at a0 posi-
tion and an (R,R0)-stereoconfiguration, while the molecules with
no alkyl chain (67) or propyl chains (65 or 66) show drastically re-
duced affinities.
The extensive 3D-QSAR modeling performed on a larger cohort
of FEN derivatives offers the opportunity to compare the new CoM-
FA model generated using the agonist [3H]-(R,R0)-2 and the previ-
ously reported model generated from affinity data determined
using the antagonist [3H]-CGP-12177.1,3 The two models are very
similar in regions responsible for stereoselectivities with similar
fields distribution can be identified in vicinities of the two chiral
centers. There are, however, significant differences in the third re-
gion of structural variation, the aminoalkyl tail of the derivatives.
While CoMFA{[3H]-CGP-12177} models show mainly electrostatic
3. Discussion
Previous studies of b2-AR agonists built upon a catecholamine
scaffold established that compounds with an R configuration at
the b-OH carbon are more active than the corresponding isomers
with an S configuration at this position.1,3 Data obtained in the ini-
tial studies of the stereoisomers of 2 were consistent with this
observation as the compounds with an (R-) configuration at the
b-OH carbon, (R,R0)-2 and (R,S0)-2, had higher binding affinities
than (S,R0)-2 and (S,S0)-2 and were more pharmacologically active
in both stimulation of cAMP accumulation and in cardiomyocyte
contractility.1–3 The results also indicated that the stereochemical
orientation the a0-alkyl moieties had an effect on b2-AR interac-
tions as the relative binding affinity and pharmacological activity
of (R,R0)-2 was greater than (R,S0)-2 and only (R,R0)-2 selectively sig-
naled through Gs proteins.1–3 Based on these results, only the
(R,R0)- and (R,S0)-isomers of the test compounds were synthesized
and studied. The compounds were produced by changing the alkyl
chain at the a0 position from 0 to 3 carbon atoms using compound
2 as the scaffold. In these studies, the binding affinities of the test
compounds to the b2-AR were determined using [3H]-CGP-12177
and [3H]-(R,R0)-2 as the marker ligands.
When [3H]-CGP-12177 was used as the marker ligand, the Ki va-
lue of (R,R0)-64 was equivalent to (R,R0)-2 and 3-fold lower than
(R,R0)-52. In addition, there was a 50-fold difference between the
binding affinities of (R,S0)-64 and (R,R0)-64, calculated as Ki(R,S0)/

240
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
fields in this region, the CoMFA{[3H]-(R,R0)-2} model indicates rel-
ative prevalence of steric fields in the vicinity of the aminoalkyl
part of derivatives. The results from the 3D-QSAR studies suggest
that steric non-polar effects dominate in interactions of studied
ligands with the COMFA{[3H]-(R,R0)-2} model, while prevalence
of electrostatic interactions can be assigned to the COMFA{[3H]-
CGP-12177} model.
was conducted on membranes derived from HEK cells containing
human b2-AR (provided by Dr. Brian Kobilka, Stanford Medical
Center, Palo Alto, CA). Cells were grown in Dulbecco’s Modified Ea-
gle Medium (DMEM) containing 10% fetal bovine serum (FBS) and
0.05% penicillin–streptomycin with 400 lg/mL G418. The binding
assays contained 0.3 nM [3H]-CGP-12177 and 60
l
g of cell mem-
branes in a volume of 1.0 mL. Nonspecific binding was determined
using 10 M propranolol. Bound radioactivity was counted on a
In a recent publication, Kim et al.7 discussed three NMR-probed
states of the b2-AR, which were designated as S1, S2 and S3. S1 and
S2 were associated with the inverse agonist carazolol and the par-
tial agonist salmeterol, respectively, and were described as two
distinct inactive-state conformers, while S3 was identified as an
agonist-activated intermediate. The results from our previous
studies of the binding of FEN analogs to the b2-AR1,3 and the data
from this study are consistent with the hypothesis that Ki values
determined using [3H]-CGP-12177 and [3H]-(R,R0)-2 represent
binding affinities to different states of the b2-AR with [3H]-(R,R0)-
2 reflecting binding to an agonist-stabilized conformation (S3)
and [3H]-CGP-12177 probing antagonist-stabilized populations
(S1,S2) as well as S3.4 As part of these studies we characterized
the thermodynamics of the binding process of (R,R0)-1, (R,S0)-1,
(S,R0)-1 and (S,S0)-1 to the b2-AR. Van0t Hoff analysis of the data ob-
tained using [3H]-CGP-12177 as the marker determined that bind-
ing of (R,R0)-1 and (R,S0)-1 were exclusively entropy driven while
the binding of (S,R’)-1 and (S,S’)-1 were predominantly enthalpy
driven.3 When [3H]-(R,R0)-2 was used as the radioligand, the bind-
ing for all four stereoisomers of 1 were entropy-controlled.4 The
data from the thermodynamic studies of the stereoisomer of 1
are consistent with the results presented by Kim et al.7 which indi-
cated that the activation to S3 is enthalpically unfavorable and
entropically favored. Therefore, results from the 3D-QSAR studies
suggest that the COMFA{[3H]-(R,R0)-2} model reflects an agonist-
stabilized state of the b2-AR (S3) and the COMFA{[3H]-CGP-
12177} model is associated with conformation states reflecting
antagonist-stabilized forms of the b2-AR. The difference between
the models is also reflected by the pharmacological activities of
the compounds tested in this study. All of the analogs were full
b2-AR receptor agonists with respect to the stimulation of cAMP
accumulation, relative to isoproterenol, Table 2. However, when
[3H]-CGP-12177 was used as the marker ligand no significant bind-
ing was observed for (R,R0)-65, (R,S0)-65, (R,S0)-66 and (S)-67,
Table 2.
l
Wallac plate liquid scintillation counter (PerkinElmer Life and Ana-
lyticalSciences, Waltham, MA) and expressed in counts per minute.
Competition curves with standard and unknown compounds in-
cluded 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.9
4.2. b2-AR binding assays using [3H]-(R,R0)-2 as a marker and
determination of the stimulation of cAMP accumulation
Binding affinities and cAMP accumulation measurement exper-
iments were conducted using exactly the same protocol as de-
scribed in Toll et al.4
4.3. CoMFA analysis
The CoMFA [3H]-(R,R0)-2 model was generated using methodol-
ogy implemented in Sybyl-X 2.0 (TRIPOS Inc., St. Louis, MO). The
molecular models of structures were prepared in HyperChem v.
6.03 (HyperCube Inc., Gainesville, FL) using Model Build procedure
to ensure the same conformation of the common scaffold.10 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 (–Cb–CH2–NH–C a0–CH2–) as a
common substructure. A PREDICT procedure implemented in CoM-
FA package was used to compute the estimated pKi values. Two
types of molecular fields (steric and electrostatic) were sampled
on the grid (2 Å spacing) lattice surrounding each structure.
4.4. Docking simulations
In the present study we used the automated docking simula-
tions of the flexible ligands (stereoisomers of fenoterol and its
derivatives) to the crystal model of b2-AR. The crystallographic
structure of b2-AR with bounded agonist, BI-167107 was obtained
from Protein Data Bank (PDB id: 3SN6).6 In order to stabilize the
active state of the receptor, b2-AR was crystallized in the complex
with Gs protein. In the present study the stereoisomers of fenoterol
and its derivatives were used as a molecular probe to identify dif-
ferences in stereo-recognition of structurally similar agonists.
Models of ligands were built using the HyperChem 6.03 (Hyper-
Cube Inc., Gainesville, FL) software and the in-built Model Build
procedure. Molecules were optimizing by using the AM1 semi-
empirical potential11 implemented in the HyperChem v. 6.03. The
Molegro Virtual Docker (MVD v. 2010.4.0.0) software was em-
ployed for docking ligands (n = 74) to the rigid model of b2-AR.
The MolDock SE as a search algorithm was used, and the number
of runs was set to 100. The parameters of docking procedure are
as follows: population size of 50, maximum iteration of 1500, en-
ergy threshold of 100.00, and maximum number of steps of 300.
The maximum number of poses to generate was increased to 10
from a default value of 5. Docking procedure was performed for
the ligands with protonated amine group. The appropriate charge
was added in MVD. The estimation of ligand–receptor interactions
was described by the MVD-related scoring functions: MolDock
Score, Rerank Score, Hbond Score, Similarity Score, Docking Score.
The scoring functions include the influence of several factors such
One of the objectives of the current study was the determina-
tion of the pharmacological profile of (R,R0)-64. This objective
was based upon the previous observations that (R,R0)-52 was
essentially inactive in the stimulation of rat cardiomyocyte
contractility3 while potently inhibiting proliferation of 1321N1
astrocytoma cells (IC50 1.4 nM).5 In addition, in the rat, the oral bio-
availability of (R,R0)-2 is 3-fold higher than that of (R,R0)-1, indicat-
ing that the substitution of
a 4’-methoxy moiety reduced
presystemic glucuronidation.8 Initial studies using the previously
described procedures1,3,5 demonstrated that (R,R0)-64 has no activ-
ity in the cardiomyocyte contractility model and effectively inhib-
its the proliferation of 1321N1 astrocytoma cells with an IC50 of
1.5 nM (unpublished data). The data suggest that (R,R0)-64 and
the COMFA{[3H]-(R,R0)-2} model can be used for the design and
optimization of orally available anti-cancer agents which have no
cardiovascular effects.
4. Experimental section
4.1. b2-AR binding assays using [3H]-CGP-12177 as a marker
Compounds synthesized in this study were tested at least three
times in triplicate to determine their binding affinities at the b2-AR
following a previously described approach.1 In brief, b2-AR binding

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
241
as van der Waals forces, electrostatic interactions and solvent
terms.12 The values of these functions are used to discriminate
among orientations and molecules and to interpret of ligand poses
in the binding cavity. The predicted positions of ligands in the cav-
ity of b2-AR are characterized by a simultaneous lowering of the
scoring function values; this corresponds to the high values of
the ligand binding energy. The analysis of docking results was fo-
cused on the scoring function values obtained for complexes fulfill-
ing all the three following conditions: (1) the lowest value of
MolDock Score; (2) the 3,5-dihydroxyphenyl moiety of the ligand
interacts with serine residues on the fifth trasmembrane domain
(TM5); (3) the protonated nitrogen atom of ligand creates the salt
bridge with D113 (TM3). According to the docking procedure, all
studied ligands (fenoterol derivatives) and the BI-167107 molecule
(for the validation of docking procedure), were docked to the bind-
ing cavity limited by the sphere, the centre of which corresponds to
the position of agonist molecule in the BI-167107-b2-AR complex
(PDB id: 3SN6). This sphere contains the amino acid residues which
play an important role in creating the ligand-b2-AR attractive inter-
action e.g.: D113, N293, S203, S204, S207, Y308, K305 and the sec-
ond extracellular loop (ECL2). The significance of these residues
was confirmed in functional and biophysical studies.
modifications by coupling the chiral amines to (R)- or (S)-2-(3,5-
bis(benzyloxy)phenyl)oxirane (R)-73 or (S)-73.
4.5.1. General preparation of 2-alkyloxiranes, (R)-2-
Isopropyloxirane represented [(R)-78]
4.5.1.1. (S)-2-Chloro-3-methylbutanoic acid [(S)-76].
To
12.5 g (107 mmol) of D-(ꢁ)-valine dissolved in 134 mL of 6 N HCl
with heat then cooled to 0 °C was added 11.8 g (171 mmol,
1.6 equiv) of sodium nitrite in small portions over 2–3 h (hours).
The solution was stirred at 0 °C for 6 h then warmed to rt (room
temperature) and extracted into DCM (dichloromethane), the ex-
tracts washed with water, dried (MgSO4), filtered, and evaporated
to give a colorless oil, 12.6 g (86%). 1H NMR: (300 MHz, CDCl3) d
1.07 (d, J = 6.6 Hz, 3H), 1.09 (d, J = 6.6 Hz, 3H), 2.36 (m, J = 6.0 Hz,
1H), 4.20 (d, J = 6.0 Hz, 1H) ppm.
4.5.1.2.
(S)-2-Chloro-3-methylbutan-1-ol
[(S)-77].
To
12.55 g (92 mmol) of (S)-76 in 80 mL of hexanes was added at rt,
20.2 mL (102 mmol, 1.1 equiv) of BH3SMe2 (borane dimethylsul-
fide) (5 M in Et2O). The reaction mixture refluxed for 1 h, cooled,
and poured into 100 mL of ice-cold methanol and stirred overnight.
The solvent was removed to give 11.26 g (100%) of a colorless oil,
which was used without further purification. 1H NMR: (300 MHz,
CDCl3) d 1.02 (d, J = 6.0 Hz, 3H), 1.04 (d, J = 6.0 Hz, 3H), 1.76 (br s,
1H), 2.05 (m, 1H), 3.49 (s, 1H), 3.77 (m, 2H), 3.91 (m, 1H) ppm.
4.5. Chemistry of new derivatives
All reactions were carried out using commercial grade reagents
and anhydrous 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 either a Varian Mercury VMX 300-MHz or a Varian
XL-400 MHz spectrophotometer. In reporting the NMR multiplici-
ties, 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 Ther-
mo LCQ Fleet MS system equipped with an electrospray ionization
(ESI) probe. High-resolution mass spectra (HRMS) were obtained
by the Old Dominion University Mass Spectrometry Service
(Norfolk, VA), also with an ESI probe. Analytical HPLC data were
obtained using a Waters 2690 Separations Module with PDA detec-
tion. Merck silica gel (230–400 mesh) was used for open column
chromatography.
Chiral amines (72a–c) were accessed using a modified prepara-
tion for a similar substrate13, the major modifications being the
stoichiometry of the initial aryl–alkylation reaction was changed
to 1:1 with respect to the chiral oxirane and using excess
borane-etherate. In early attempts, specifically for 1-(4-methoxy-
phenyl)butane-2-ol (68a), 4-iodoanisole was used in place of 4-
bromoanisole, but yield variability on scaling-up resulted in our
using 4-bromoanisole. We also found that preparing the mesylate
in dichoromethane in the presence of triethylamine to be prefera-
ble to methylsulfonation in pyridine. Note: All traces of dichloro-
methane should be carefully removed prior to treatment with
sodium azide to prevent the formation of potentially explosive
diazidomethane.14
4.5.1.3. (R)-2-Isopropyloxirane[(R)-78].
To11.2 g(91.3 mmol)
of (S)-77 chilled to ꢁ70 °C was added 10.25 g (183 mmol) of finely
ground potassium hydroxide. The slurry was allowed to warm to rt,
then distilled (bp 71–73 °C) giving 4.79 g (61%) of (R)-78. 1H NMR:
(300 MHz, CDCl3) d 0.96 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H),
1.47 (m, 1H), 2.51 (m, 1H), 2.71 (m, 2H) ppm.
4.5.1.4. (R)-2-Chloro-3-methylbutanoic acid [(R)-76].
Pre-
pared from 46.8 g (0.40 mol) of L-(+)-valine to give 44.0 g (80%).
1H NMR: (300 MHz, CDCl3) d 1.07 (d, J = 6.6 Hz, 3H), 1.09 (d,
J = 6.6 Hz, 3H), 2.36 (m, J = 6.0 Hz, 1H), 4.20 (d, J = 6.0 Hz, 1H) ppm.
4.5.1.5. (R)-2-Chloro-3-methylbutan-1-ol [(R)-77].
Prepared
from 12.3 g (90.0 mmol) of (R)-76 to give 10.7 g (97%). Bp 85–
88 °C/18 mm; 1H NMR: (300 MHz, CDCl3) d 1.02 (d, J = 6.0 Hz,
3H), 1.04 (d, J = 6.0 Hz, 3H), 1.76 (br s, 1H), 2.05 (m, 1H), 3.49 (s,
1H), 3.77 (m, 2H), 3.91 (m, 1H) ppm.
4.5.1.6. (S)-2-Isopropyloxirane [(S)-78].
Prepared from
10.7 g (87.6 mmol) of (R)-77 to give 5.78 g (77%). 1H NMR:
(300 MHz, CDCl3) d 0.96 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H),
1.47 (m, 1H), 2.51 (m, 1H), 2.71 (m, 2H) ppm.
4.5.1.7. (S)-2-Chloropentanoic acid [(S)-79].
Prepared from
10.0 g (85.4 mmol) of L-(+)-norvaline to give 9.58 g (82%). 1H
NMR: (300 MHz, CDCl3) d 0.97 (t, J = 7.2 Hz, 3H), 1.53 (m, 2H),
2.00 (m, 2H), 4.33 (dd, J = 6.0, 7.6 Hz, 1H) ppm.
4.5.1.8. (S)-2-Chloropentan-1-ol [(S)-80].
Prepared from 9.58 g
(70.1 mmol) of (S)-79 to give 7.14 g (83%). Bp 79–80 °C/20 mm; 1H
NMR: (300 MHz, CDCl3) d 0.94 (t, J = 7.2 Hz, 3H), 1.52 (m, 2H), 1.72
(m, 2H), 1.81 (br s, 1H), 3.66 (m, 1H), 3.79 (m, 1H), 4.04 (m, 1H) ppm.
In the case of the ethyl analog, the chiral oxirane is commer-
cially available. For the i-propyl and n-propyl analogs, the oxiranes
were prepared from valine and norvaline isomers, respectively, via
a Sandmeyer type chlorination with retention of absolute configu-
ration15,16 followed by reduction of the acid to the chlorohydrin,
then epoxidation with base resulting in an inversion of absolute
configuration. Benzylamine 72d was prepared using established
benzylation chemistry on the corresponding primary amine
precursor.
4.5.1.9. (R)-2-Propyloxirane [(R)-81].
Prepared from 7.14 g
(58.3 mmol) of (S)-80 to give 3.88 g (77%). Bp 78–79 °C; 1H NMR:
(300 MHz, CDCl3) d 0.91 (t, J = 7.2 Hz, 3H), 1.40 (m, 2H), 1.52 (m,
2H), 2.46 (m, 1H), 2.74 (m, 1H), 2.89 (m, 1H) ppm.
With the new amines in hand, fenoterol analogs 64–67 were
4.5.1.10. (R)-2-Chloropentanoic acid [(R)-79].
Prepared
prepared using our previously reported synthesis1,3 with minimal
from 10.0 g (85.4 mmol) D-(ꢁ)-norvaline to give 8.05 g (69%). 1H

242
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
NMR: (300 MHz, CDCl3) d 0.97 (t, J = 7.2 Hz, 3H), 1.53 (m, 2H), 2.00
(m, 2H), 4.33 (dd, J = 6.0, 7.6 Hz, 1H) ppm.
(50.0 mmol) of (R)-78 and 9.2 mL (75 mmol) of BF3ꢂEt2O. Column
chromatography on silica with hexanes/DCM gave 6.08 g (63%).
1H NMR: (300 MHz, CDCl3) d 0.99 (t, J = 6.9 Hz, 6H), 1.44 (br s,
1H), 1.74 (m, 1H), 2.53 (m, 1H), 2.80 (m, 1H), 3.55 (m, 1H), 3.80
(s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
4.5.1.11. (R)-2-Chloropentan-1-ol [(R)-80].
Prepared from
8.05 g (58.9 mmol) of (R)-79 to give 7.02 g (95%). 1H NMR:
(300 MHz, CDCl3) d 0.94 (t, J = 7.2 Hz, 3H), 1.52 (m, 2H), 1.72 (m,
2H), 1.81 (br s, 1H), 3.66 (m, 1H), 3.79 (m, 1H), 4.04 (m, 1H) ppm.
4.5.3.3. (S)-1-(4-Methoxyphenyl)pentan-2-ol [(S)-68c].
Pre-
pared from 8.10 g (43.3 mmol) 4-bromoanisole, 17.3 mL
(43.3 mmol) of n-BuLi (2.5 M in hexanes), 3.11 g (36.1 mmol) of
(S)-81 and 6.7 mL (54 mmol) of BF3ꢂEt2O. Column chromatography
on silica with hexanes/DCM gave 6.20 g (88%). 1H NMR: (300 MHz,
CDCl3) d 0.94 (t, J = 6.6 Hz, 3H), 1.47 (m, 5H), 2.57 (m, 1H), 2.79 (m,
1H), 3.75 (m, 1H), 3.79 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
4.5.1.12. (S)-2-Propyloxirane [(S)-81].
Prepared from 7.02 g
(57.3 mmol) of (R)-80 to give 3.11 g (63%). bp 78–79 °C; 1H NMR:
(300 MHz, CDCl3) d 0.91 (t, J = 7.2 Hz, 3H), 1.40 (m, 2H), 1.52 (m,
2H), 2.46 (m, 1H), 2.74 (m, 1H), 2.89 (m, 1H) ppm.
4.5.2. Preparation of chiral alcohols
4.5.2.1. (S)-1-(4-Methoxyphenyl)butan-2-ol[(S)-68a].
Under
an argon atm, a solution of 5.85 g (25 mmol) 4-iodoanisole in
120 mL of dry THF was cooled to ꢁ70 °C internal (CO2-iPrOH bath).
A solution of 10.0 mL (25 mmol) of n-butyllithium (n-BuLi) (2.5 M
in hexanes) was added slowly over 20 min. The mixture was stirred
for 10 min, then 1.1 mL (12.5 mmol, 0.5 equiv) of (S)-(ꢁ)-1,2-epox-
ybutane was added followed by the addition of 2.3 mL (19 mmol,
0.75 equiv) of BF3ꢂEt2O. The solution was stirred for 20 min then
removed from the cooling bath and quenched by the slow addition
of saturated NH4Cl solution (ꢀ40 mL). Partitioned between Et2O
and 100 mL of added water, washed with brine, dried (Na2SO4),
filtered, and evaporated. Column chromatography on silica with 1:3
hexanes:DCM gave 1.5 g (72%). 1H NMR: (300 MHz, CDCl3) d 0.99 (t,
J = 7.2 Hz, 3H), 1.50 (m, 2H), 1.59 (br s, 1H), 2.59 (m, 1H), 2.76 (m,
1H), 3.68 (m, 1H), 3.79 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
4.5.3.4. (R)-1-(4-Methoxyphenyl)pentan-2-ol [(R)-68c].
Pre-
pared from 10.1 g (53.8 mmol) 4-bromoanisole, 21.5 mL
(53.8 mmol) of n-BuLi (2.5 M in hexanes), 3.86 g (44.8 mmol) of
(R)-81 and 8.3 mL (67 mmol) of BF3ꢂEt2O. Column chromatography
on silica with hexanes/DCM gave 7.68 g (74%). 1H NMR: (300 MHz,
CDCl3) d 0.94 (t, J = 6.6 Hz, 3H), 1.47 (m, 5H), 2.57 (m, 1H), 2.79 (m,
1H), 3.75 (m, 1H), 3.79 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
4.5.4. General procedure for the preparation of mesylates (69a–
c)
To the chiral alcohol (68a–c) in dry DCM (concn 0.15 M) at
ꢁ70 °C was added over 1 min triethylamine (1.2 equiv) followed
by the slow addition of methanesulfonyl chloride (1.2 equiv). The
mixture was allowed to warm to rt over 1–2 h and stirring was
continued for an additional 1 h. The reaction mixture was poured
into 100 mL of ice water, the organics separated, washed with
brine, dried (MgSO4), filtered, and evaporated. The residues were
used without further purification.
4.5.2.2. (R)-1-(4-Methoxyphenyl)butan-2-ol [(R)-68a].
Pre-
pared as for (S)-68a from 3.25 g (13.9 mmol) 4-iodoanisole,
6.0 mL (13.9 mmol) of n-BuLi (2.3 M in hexanes), 1.2 mL
(13.9 mmol, 1 equiv) of (R)-(+)-1,2-epoxybutane and 2.6 mL
(20.9 mmol, 1.5 equiv) of BF3ꢂEt2O. Column chromatography on sil-
ica with 1:3 hexanes:DCM gave 1.6 g (64%). 1H NMR: (300 MHz,
CDCl3) d 0.99 (t, J = 7.2 Hz, 3H), 1.50 (m, 2H), 1.59 (br s, 1H), 2.59
(m, 1H), 2.76 (m, 1H), 3.68 (m, 1H), 3.79 (s, 3H), 6.86, (d,
J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H) ppm.
4.5.5. (S)-1-(4-Methoxyphenyl)butan-2-yl methanesulfonate
[(S)-69a]
Prepared from 1.92 g (10.7 mmol) of (S)-68a, 0.9 mL (12 mmol)
of triethylamine, and 1.68 mL (12 mmol) of methansulfonyl chlo-
ride gave 2.66 g (97%). 1H NMR: (300 MHz, CDCl3) d 1.03 (t,
J = 7.5 Hz, 3H), 1.78 (m, 2H), 2.53 (s, 3H), 2.91 (d, J = 6.6 Hz, 2H),
3.79 (s, 3H), 4.73 (p, J = 6.6 Hz, 1H), 6.85 (d, J = 8.4 Hz, 2H), 7.15
(d, J = 8.4 Hz, 2H) ppm.
4.5.3. General procedure for the preparation of alcohols (68b-c)
Under an argon atm, 4-bromoanisole was dissolved in dry THF
(concn 0.6 M) and cooled to an internal temperature of ꢁ70 °C. A
solution of 2.5 M n-BuLi in hexanes (1 equiv) was added slowly
taking care to maintain the low internal temperature. The chiral
oxirane (0.83–0.9 equiv) was added over 5 min followed by the
slow addition of BF3ꢂEt2O (1.25 equiv). Stirring was continued for
30 min, at which point the reaction was removed from the cooling
bath and quenched by the slow addition of NH4Cl. The reaction
mixture was partitioned with ether, washed with brine, dried
(MgSO4), filtered, and evaporated. The residues were purified on
silica gel with a gradient of 60–100% DCM in hexanes.
4.5.6. (R)-1-(4-Methoxyphenyl)butan-2-yl methanesulfonate
[(R)-69a]
Prepared from 1.89 g (10.5 mmol) of (R)-68a, 1.8 mL (13 mmol)
of triethylamine, and 0.98 mL (13 mmol) of methanesulfonyl chlo-
ride gave 2.70 g (100%). 1H NMR: (300 MHz, CDCl3) d 1.03 (t,
J = 7.5 Hz, 3H), 1.78 (m, 2H), 2.53 (s, 3H), 2.91 (d, J = 6.6 Hz, 2H),
3.79 (s, 3H), 4.73 (p, J = 6.6 Hz, 1H), 6.85 (d, J = 8.4 Hz, 2H), 7.15
(d, J = 8.4 Hz, 2H) ppm.
4.5.7. (S)-1-(4-Methoxyphenyl)-3-methylbutan-2-yl
methanesulfonate [(S)-69b]
4.5.3.1.
68b].
(S)-1-(4-Methoxyphenyl)-3-methylbutan-2-ol
Prepared from 5.42 g (29.0 mmol) of 4-bromoanisole,
[(S)-
Prepared from 3.93 g (20.2 mmol) of (S)-68b, 3.4 mL (24 mmol)
of triethylamine, and 1.9 mL (24 mmol) of methanesulfonyl chlo-
ride gave 5.37 g (98%). 1H NMR: (300 MHz, CDCl3) d 1.03 (m, 6H),
2.05 (m, 1H), 2.40 (s, 3H), 2.88 (m, 2H), 3.78 (s, 3H), 4.68 (m,
1H), 6.85 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H) ppm.
11.6 mL (29.0 mmol) of n-BuLi (2.5 M in hexanes), 2.24 g
(26.0 mmol) of (S)-78 and 4.8 mL (39 mmol) of BF3ꢂEt2O. Column
chromatography on silica with hexanes/DCM gave 3.93 g (77%).
1H NMR: (300 MHz, CDCl3) d 0.99 (t, J = 6.9 Hz, 6H), 1.44 (br s,
1H), 1.74 (m, 1H), 2.53 (m, 1H), 2.80 (m, 1H), 3.55 (m, 1H), 3.80
(s, 3H), 6.86, (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
4.5.7.1. (R)-1-(4-Methoxyphenyl)-3-methylbutan-2-yl methane-
sulfonate [(R)-69b].
Prepared from 6.07 g (31.2 mmol) of (R)-
4.5.3.2.
68b].
(R)-1-(4-Methoxyphenyl)-3-methylbutan-2-ol
Prepared from 10.3 g (55.0 mmol) of 4-bromoanisole,
[(R)-
68b, 5.2 mL (37 mmol) of triethylamine, and 2.9 mL (37 mmol) of
methanesulfonyl chloride gave 8.50 g (100%). 1H NMR: (300 MHz,
CDCl3) d 1.03 (m, 6H), 2.05 (m, 1H), 2.40 (s, 3H), 2.88 (m, 2H),
22.0 mL (55.0 mmol) of n-BuLi (2.5 M in hexanes), 4.31 g

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
243
3.78 (s, 3H), 4.68 (m, 1H), 6.85 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz,
2H) ppm.
J = 6.6 Hz, 1H), 3.80 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
4.5.7.2. (S)-1-(4-Methoxyphenyl)pentan-2-yl methanesulfonate
4.5.14. (S)-1-(2-Azidopentyl)-4-methoxybenzene [(S)-70c]
Prepared from 10.79 (39.6 mmol) of (R)-69c and 5.14 g
(79.0 mmol) of NaN3. Chromatography on silica gel with hex-
anes/DCM gave 7.07 g (81%). 1H NMR: (300 MHz, CDCl3) d 0.93 (t,
J = 6.6 Hz, 3H), 1.50 (m, 4H), 2.76, (d, J = 6.9 Hz, 2H), 3.47 (p,
J = 6.6 Hz, 1H), 3.80 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
[(S)-69c].
Prepared from 6.20 g (31.9 mmol) of (S)-68c, 5.3 mL
(38 mmol) of triethylamine, and 3.0 mL (38 mmol) of methanesul-
fonyl chloride gave 7.66 g (88%). 1H NMR: (300 MHz, CDCl3) d 0.94
(t, J = 7.2 Hz, 3H), 1.47 (m, 2H), 1.67 (m, 2H), 2.53 (s, 3H), 2.91 (d,
J = 7.5 Hz, 2H), 3.79 (s, 3H), 4.79 (p, J = 6.0 Hz, 1H), 6.85 (d,
J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
4.5.8. (R)-1-(4-Methoxyphenyl)pentan-2-yl methanesulfonate
[(R)-69c]
4.5.15. General procedure for the preparation of primary
amines (71a–c)
Prepared from 7.68 g (39.5 mmol) of (R)-68c, 6.6 mL (47 mmol)
of triethylamine, and 3.7 mL (47 mmol) of methanesulfonyl chlo-
ride gave 10.76 g (100%). 1H NMR: (300 MHz, CDCl3) d 0.94 (t,
J = 7.2 Hz, 3H), 1.47 (m, 2H), 1.67 (m, 2H), 2.53 (s, 3H), 2.91 (d,
J = 7.5 Hz, 2H), 3.79 (s, 3H), 4.79 (p, J = 6.0 Hz, 1H), 6.85 (d,
J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
The azide was dissolved in absolute EtOH (concn = 0.22 M) and
added was palladium on carbon. The mixture was hydrogenated
with 1 atm of hydrogen gas for 12–48 h. The reaction was filtered
through Celite and concentrated.
4.5.16. (R)-1-(4-Methoxyphenyl)butan-2-amine [(R)-71a]
Prepared from 1.56 g (7.60 mmol) of (R)-70a and 40 mg of 5%
Pd/C (Sigma) giving 1.36 g (100%). 1H NMR: (300 MHz, CDCl3) d
0.97 (t, J = 7.2 Hz, 3H), 1.36 (m, 1H), 1.51 (m, 1H), 1.65 (br s, 1H),
2.38 (m, 1H), 2.74 (m, 1H), 2.86 (m, 1H), 3.79 (s, 3H), 6.84 (d,
J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H) ppm.
4.5.9. General procedure for the preparation of azides (70a–c)
The mesylate was combined with an excess of sodium azide
(NaN3) in dry DMF (concn = 0.1 M) and stirred at 35 °C for 36 h.
Caution: All traces of dichloromethane should be carefully re-
moved from the mesylate prior to treatment with sodium azide
to prevent the formation of potentially explosive diazidome-
thane.14 The reaction was partitioned between Et2O and water,
washed with brine, dried (MgSO4), filtered, and evaporated. The
residues were purified on silica gel with a gradient of 60–100%
DCM in hexanes.
4.5.17. (S)-1-(4-Methoxyphenyl)butan-2-amine [(S)-71a]
Prepared from 1.80 g (8.77 mmol) of (S)-70a and 360 mg of 10%
Pd/C (Strem Co, 50% paste in water) giving 1.33 g (85%). 1H NMR:
(300 MHz, CDCl3) d 0.97 (t, J = 7.2 Hz, 3H), 1.36 (m, 1H), 1.51 (m,
1H), 1.65 (br s, 2H), 2.38 (m, 1H), 2.74 (m, 1H), 2.86 (m, 1H),
3.79 (s, 3H), 6.84 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H) ppm.
4.5.10. (R)-1-(2-Azidobutyl)-4-methoxybenzene [(R)-70a]
Prepared from 2.66 g (10.3 mmol) of (S)-69a, 2.60 g (40 mmol)
of NaN3. Chromatography on silica gel with hexanes/DCM gave
1.56 g (71%). 1H NMR: (300 MHz, CDCl3) d 1.01 (t, J = 7.2 Hz, 3H),
1.55 (m, 2H), 2.75 (d, J = 7.5 Hz, 2H), 3.39 (p, J = 6.0 Hz, 1H), 3.80
(s, 3H), 6.85 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H) ppm.
4.5.18. (R)-1-(4-Methoxyphenyl)-3-methylbutan-2-amine [(R)-
71b]
Prepared from 3.62 g (16.5 mmol) of (R)-70b and 50 mg of 5%
Pd/C (Sigma) giving 3.13 g (98%). 1H NMR: (300 MHz, CDCl3) d
0.97 (t, J = 6.6 Hz, 6H), 1.21 (br s, 2H), 1.65 (m, 1H), 2.34 (m, 1H),
2.76 (m, 2H), 3.79 (s, 3H), 6.84 (d, J = 8.4 Hz, 2H), 7.11 (d,
J = 8.4 Hz, 2H) ppm.
4.5.11. (S)-1-(2-Azidobutyl)-4-methoxybenzene [(S)-70a]
Prepared from 2.70 g (10.5 mmol) of (R)-69a and 3.07 g
(47.2 mmol) of NaN3. Chromatography on silica gel with hex-
anes/DCM gave 1.84 g (84%). 1H NMR: (300 MHz, CDCl3) d 1.01 (t,
J = 7.2 Hz, 3H), 1.55 (m, 2H), 2.75 (d, J = 7.5 Hz, 2H), 3.39 (p,
J = 6.0 Hz, 1H), 3.80 (s, 3H), 6.85 (d, J = 8.4 Hz, 2H), 7.13 (d,
J = 8.4 Hz, 2H) ppm.
4.5.19. (S)-1-(4-Methoxyphenyl)-3-methylbutan-2-amine [(S)-
71b]
Prepared from 5.94 g (27.1 mmol) of (S)-70b and 50 mg of 5%
Pd/C (Sigma) giving 5.23 g (100%). 1H NMR: (300 MHz, CDCl3) d
0.97 (t, J = 6.6 Hz, 6H), 1.21 (br s, 2H), 1.65 (m, 1H), 2.34 (m, 1H),
2.76 (m, 2H), 3.79 (s, 3H), 6.84 (d, J = 8.4 Hz, 2H), 7.11 (d,
J = 8.4 Hz, 2H) ppm.
4.5.11.1.
[(R)-70b].
(R)-1-(2-Azido-3-methylbutyl)-4-methoxybenzene
Prepared from 5.37 g (19.7 mmol) of (S)-69b and
1.95 g (30 mmol) of NaN3. Chromatography on silica gel with hex-
anes/DCM gave 3.63 g (84%). 1H NMR: (300 MHz, CDCl3) d 1.01 (t,
J = 6.6 Hz, 6H), 1.83 (m, 1H), 2.68 (m, 1H), 2.81 (m, 1H), 3.34 (m,
1H), 3.80 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
4.5.20. (R)-1-(4-Methoxyphenyl)pentan-2-amine [(R)-71c]
Prepared from 5.46 g (24.9 mmol) of (R)-70c and 50 mg of 5%
Pd/C (Sigma) giving 4.81 g (100%). 1H NMR: (300 MHz, CDCl3) d
0.93 (t, J = 6.6 Hz, 3H), 1.35 (m, 2H), 1.45 (m, 2H), 1.72 (br s, 2H),
2.42 (m, 1H), 2.75 (m, 1H), 2.95 (m, 1H), 3.79 (s, 3H), 6.84 (d,
J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H) ppm.
4.5.12. (S)-1-(2-Azido-3-methylbutyl)-4-methoxybenzene [(S)-
70b]
Prepared from 8.50 g (31.2 mmol) of (R)-69b and 4.06 g
(62.5 mmol) of NaN3. Chromatography on silica gel with hexanes/
DCM gave 5.94 g (87%). 1H NMR: (300 MHz, CDCl3) d 1.01 (t,
J = 6.6 Hz, 6H), 1.83 (m, 1H), 2.68 (m, 1H), 2.81 (m, 1H), 3.34 (m,
1H), 3.80 (s, 3H), 6.86 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H) ppm.
4.5.21. (S)-1-(4-Methoxyphenyl)pentan-2-amine [(S)-71c]
Prepared from 7.07 g (32.2 mmol) of (S)-70c and 50 mg of 5%
Pd/C (Sigma) giving 6.23 g (100%). 1H NMR: (300 MHz, CDCl3) d
0.93 (t, J = 6.6 Hz, 3H), 1.35 (m, 2H), 1.45 (m, 2H), 1.72 (br s, 2H),
2.42 (m, 1H), 2.75 (m, 1H), 2.95 (m, 1H), 3.79 (s, 3H), 6.84 (d,
J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H) ppm.
4.5.13. (R)-1-(2-Azidopentyl)-4-methoxybenzene [(R)-70c]
Prepared from 7.66 (28.1 mmol) of (S)-69c and 4.15 g
(63.8 mmol) of NaN3. Chromatography on silica gel with hex-
anes/DCM gave 5.46 g (81%). 1H NMR: (300 MHz, CDCl3) d 0.93 (t,
J = 6.6 Hz, 3H), 1.50 (m, 4H), 2.76, (d, J = 6.9 Hz, 2H), 3.47 (p,
4.5.22. General procedure for the preparation of N-
benzylamines (72a–c)
A flask was charged with the primary amine (71a–c) in absolute
EtOH (concn 0.15 M) and 1 equiv of benzaldehyde. The solution

244
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
was stirred at reflux under argon atm for 2 h monitoring by TLC.
The reaction was cooled to 0 °C under argon and an excess of so-
dium triacetoxyborohydride (Na(OAc)3BH) was added in portions.
After stirring under argon atm for 16 h the reaction mixture was
concentrated, partitioned between DCM and 10% K2CO3 solution
(5–6 equiv), dried (K2CO3), filtered, and evaporated. The residue
was either purified by column chromatography with 10–30% EtOAc
in DCM or by recystallization from optically active mandelic acids.
Chiral-HPLC Method A: Chirobiotic V column, 250 ꢃ 4.6 mm;
1.0 mL/min; Gradient program 20–40% MeOH over 40 min/
20 mM KH2PO4; Det: 225 nm. Chiral-HPLC Method B: Chiralpak
AD-H column, 250 ꢃ 4.6 mm; 0.25 mL/min; isocratic 7:93:0.5
iPrOH/hexane/DEA; Det: 279 nm.
(m, 4H), 1.52 (br s, 1H), 2.70 (m, 3H), 3.72 (d, J = 13.2 Hz, 1H),
3.78 (d, J = 13.2 Hz, 1H), 3.80 (s, 3H), 6.83 (d, J = 8.4 Hz, 2H), 7.08
(d, J = 8.4 Hz, 2H), 7.38-7.15 (m, 5H) ppm; MS m/z (rel): 284
(100, M+H); Chiral-HPLC Method B: Rt = 17.2 min (97.1% R).
4.5.28. (S)-N-Benzyl-1-(4-methoxyphenyl)pentan-2-amine [(S)-
72c]
Prepared from 6.23 (32.2 mmol) of (S)-71c, 3.43 g (32.3 mmol)
of benzaldehyde and 12.7 g (60.0 mmol) of Na(OAc)3BH. Chroma-
tography on silica gel with DCM/EtOAc gave 6.84 g (79%). 1H
NMR: (300 MHz, CDCl3) d 0.94 (t, J = 7.5 Hz, 3H), 1.42 (m, 4H),
1.52 (br s, 1H), 2.70 (m, 3H), 3.72 (d, J = 13.2 Hz, 1H), 3.78 (d,
J = 13.2 Hz, 1H), 3.80 (s, 3H), 6.83 (d, J = 8.4 Hz, 2H), 7.08 (d,
J = 8.4 Hz, 2H), 7.38–7.15 (m, 5H) ppm; MS m/z (rel): 284 (100,
M+H); Chiral-HPLC Method B: Rt = 16.4 min (95.8% S).
4.5.23. (R)-N-Benzyl-1-(4-methoxyphenyl)butan-2-amine [(R)-
72a]
Prepared from 1.36 g (7.60) mmol of (R)-71a, 806 mg
(7.60 mmol) of benzaldehyde and 3.22 g (15.2 mmol) of Na(OAc)3-
BH. Chromatography on silica gel with DCM/EtOAc gave 1.69 g
(82%). 1H NMR: (300 MHz, CDCl3) d 0.94 (t, J = 7.5 Hz, 3H), 1.48
(m, 3H), 2.67 (m, 3H), 3.69 (d, J = 13.2 Hz, 1H), 3.79 (d,
J = 13.2 Hz, 1H), 3.80 (s, 3H), 6.83 (d, J = 8.4 Hz, 2H), 7.08 (d,
J = 8.4 Hz, 2H), 7.40-7.20 (m, 5H) ppm; MS m/z (rel): 270 (100,
M+H); Chiral-HPLC Method A: Rt = 19.3 min (98.0% R).
4.5.29. N-Benzylidene-2-(4-methoxyphenyl)ethanamine (82)
A 50 mL round bottom flask fitted with a Dean–Stark trap con-
taining sodium sulfate in its side arm, was charged with 1.73 g
(11.5 mmol) of 4-methoxyphenethylamine, 40 mL of toluene and
1.19 g (11.3 mmol) of benzaldehyde. The mixture was refluxed un-
der argon atm for 48 h, cooled, and the toluene removed to give
2.63 g (97%) of an orange solid that was used in the next step with-
out further purification. 1H NMR (300 MHz, CDCl3): d 2.96 (t, 2H,
J = 7.5 Hz), 3.81 (t, 2H, J = 7.5 Hz), 3.83 (s, 3H), 6.83 (d, 2H,
J = 6.6 Hz), 7.16 (d, 2H, J = 6.6 Hz), 7.26–7.42 (m, 3H), 7.69–7.71
(m, 2H) ppm; 13C NMR (75 MHz, CDCl3): d 36.6, 55.2, 63.4, 113.7,
128.0, 129.9, 130.5, 132.0, 136.2, 157.9, 161.4 ppm.
4.5.24. (S)-N-Benzyl-1-(4-methoxyphenyl)butan-2-amine [(S)-
72a]
Prepared from 1.04 g (5.80) mmol of (S)-71a, 613 mg
(5.80 mmol) of benzaldehyde and 3.07 g (14.5 mmol) of Na(OAc)3-
BH. Chromatography on silica gel with DCM/EtOAc gave 400 mg
(26%). 1H NMR: (300 MHz, CDCl3) d 0.94 (t, J = 7.5 Hz, 3H), 1.48
(m, 3H), 2.67 (m, 3H), 3.69 (d, J = 13.2 Hz, 1H), 3.79 (d,
J = 13.2 Hz, 1H), 3.80 (s, 3H), 6.83 (d, J = 8.4 Hz, 2H), 7.08 (d,
J = 8.4 Hz, 2H), 7.40-7.20 (m, 5H) ppm; MS m/z (rel): 270 (100,
M+H); Chiral-HPLC Method A: Rt = 17.5 min (99% S).
4.5.30. N-Benzyl-2-(4-methoxyphenyl)ethanamine (72d)
A 1.56 g (6.53 mmol) sample of 82 was dissolved in 25 mL of
ethanol and degassed with argon. Added in portions was 3.45 g
(16.3 mmol, 3 eq) mg Na(OAc)3BH, and the mixture was allowed
to stir overnight. The solvent was removed, the residue partitioned
between 30 mL of chloroform 180 mL of 5% K2CO3 solution, fol-
lowed by 50 mL of 10% K2CO3 and 50 mL of water. The organic
layer was dried (Na2SO4), evaporated and purified on silica gel
(CHCl3–MeOH) giving 1.41 g (90%) of a pale yellow oil. 1H NMR
(400 MHz, CDCl3): d 2.75 (t, 2H, J = 7.2 Hz), 2.85 (t, 2H, J = 7.2 Hz),
3.76 (s, 3H), 3.78 (s, 2H) 6.81 (d, 2H, J = 8.4 Hz), 7.10 (d, 2H,
J = 8.8 Hz), 7.21-7.32 (m, 5H) ppm; 13C NMR (75 MHz, CDCl3): d
35.45, 50.77, 53.90, 52.25, 113.83, 126.87, 128.07, 128.37, 129.62,
132.08, 140.39, 158.03 ppm; MS m/z (ESI+): 242 (M+H).
4.5.25. (R)-N-Benzyl-1-(4-methoxyphenyl)-3-methylbutan-2-
amine [(R)-72b]
Prepared from 3.13 g (16.2) mmol of (R)-71b, 1.68 g
(16.2 mmol) of benzaldehyde and 7.00 g (33.0 mmol) of Na(OAc)3-
BH. Chromatography on silica gel with DCM/EtOAc gave 3.24 g
(72%). 1H NMR: (300 MHz, CDCl3) d 1.00 (m, 6H), 1.20 (br s, 1H),
1.90 (m, 1H), 2.54 (m, 1H), 2.63 (m, 1H), 2.74 (m, 1H), 3.66 (d,
J = 13.2 Hz, 1H), 3.76 (d, J = 13.2 Hz, 1H), 3.82 (s, 3H), 6.86 (d,
J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.42–7.18 (m, 5H) ppm;
MS m/z (rel): 284 (100, M+H); Chiral-HPLC Method A: Rt = 26.0 min
(98.4% R).
4.5.31. General procedure for the preparation of N-alkyl
modified fenoterol analogs (64–67)
(R)-2-(3,5-Bis(benzyloxy)phenyl)oxirane (R)-73 was combined
with (0.95–1 equiv) of the appropriate chiral N-benzylamine
72a–d in toluene and then evaporated under high vacuum. The
residue was heated at the temperature and time indicated in the
specific procedure. The amber residue was applied to a column
of silica gel (1:75) eluting with EtOAc/hexanes to give the pure
74a–d. Unless otherwise noted, the entire residue was deprotected
by hydrogenating at 50 psi over 10% wt Pd/C (Strem Co 50% paste
in water, 1:5 wt:wt). Filtered through CeliteÒ, washed the cake
well with EtOH and evaporated to a dry residue. The specific rota-
tion of the free amine was taken in MeOH and then the base was
immediately converted to the hemi-fumarate salt by heating with
0.5 equiv of the diacid in MeOH (concn = 0.1 M) to give the feno-
terol salt (64–67).
4.5.26. (S)-N-Benzyl-1-(4-methoxyphenyl)-3-methylbutan-2-
amine [(S)-72b]
Prepared from 5.23 g (27.1 mmol) of (S)-71b, 2.88 g
(27.1 mmol) of benzaldehyde and 10.6 g (50.0 mmol) of Na(OAc)3-
BH. Chromatography on silica gel with DCM/EtOAc gave 6.47 g
(84%). 1H NMR: (300 MHz, CDCl3) d 1.00 (m, 6H), 1.20 (br s, 1H),
1.90 (m, 1H), 2.54 (m, 1H), 2.63 (m, 1H), 2.74 (m, 1H), 3.66 (d,
J = 13.2 Hz, 1H), 3.76 (d, J = 13.2 Hz, 1H), 3.82 (s, 3H), 6.86 (d,
J = 8.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 7.42–7.18 (m, 5H) ppm;
MS m/z (rel): 284 (100, M+H); Chiral-HPLC Method A: Rt = 28.3 min
(99% S).
4.5.27. (R)-N-Benzyl-1-(4-methoxyphenyl)pentan-2-amine [(R)-
72c]
Prepared from 4.83 g (25.0 mmol) of (R)-71c, 2.66 g
(25.0 mmol) of benzaldehyde and 10.4 g (49.0 mmol) of Na(OAc)3-
BH. Chromatography on silica gel with DCM/EtOAc gave 4.74 g
(67%). 1H NMR: (300 MHz, CDCl3) d 0.94 (t, J = 7.5 Hz, 3H), 1.42
4.5.31.1.
phenyl)butan-2-yl)amino)ethyl)benzene-1,3-diol
64]. A mixture of 768 mg (2.31 mmol) of (R)-73 and 591 mg
(2.19 mmol) of (R)-72a was heated at 120 °C for 30 h and further
treated using the general procedure above to give 470 mg (55%)
(R,R0)-(-)-5-(1-Hydroxy-2-((1-(4-methoxy-
[(R,R0)-

A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
245
of hemi-fumarate salt. 1H NMR (400 MHz, CD3OD): d 0.962 (t, 3H,
J = 7.6 Hz), 1.65 (p, 2H, J = 5.6), 2.86-2.92 (m, 3H), 3.03-3.08 (m,
2H), 3.76 (s, 3H), 4.76 (dd, 1H J = 3.6, 8.8 Hz), 6.20 (t, 1H,
J = 2.0 Hz), 6.33 (d, 2H, J= 2.0 Hz), 6.70 (s, 1H, fum), 6.87 (d, 2H,
J = 8.8 Hz), 7.15 (d, 2H, J = 8.8 Hz) ppm. 13C CMR (75 MHz, CD3OD):
d 8.16, 22.48, 35.68, 51.40, 54.28, 60.69, 69.21, 101.86, 103.86,
113.92, 128.17, 129.91, 135.59, 143.53, 158.54, 158.88 ppm. UV
J = 6.8 Hz), 2.07 (hept, 1H, J = 4.0 Hz), 2.77–2.82 (m, 2H), 2.88–
2.94 (m, 2H), 3.24 (m, 1H), 3.77 (s, 3H), 4.68 (dd, 1H, J = 5.6,
7.6 Hz), 6.18 (t, 1H, J = 2.4 Hz), 6.26 (d, 2H, J = 2.0 Hz), 6.69 (s, 1H,
fumarate), 6.87 (d, 2H, J = 8.8 Hz), 7.15 (d, 2H, J = 8.8 Hz) ppm.
13C NMR (75 MHz, CD3OD): d 17.62, 18.59, 30.19, 34.45, 54.44,
55.70, 66.95, 70.89, 103.17, 105.22, 115.39, 130.28, 131.17,
136.85, 145.02, 159.91, 160.24 ppm. UV (MeOH) kmax (e): 278 nm
(MeOH): kmax
(
e
) 277 nm (3,690), 223 (20,100), 210 (26,000), 202
(3,240), 224 (17,000), 205 (33,600). MS m/z (rel): 346 (M+H base).
RP-HPLC: Ace-5 C18 column, 100 ꢃ 4.6 mm; 10–60%/10 min aceto-
nitrile/20 mM KH2PO4 (pH 4.5); 1.0 mL/min; 275 nm; Rt = 1.15 min
(6.33% fumarate), 6.49 min (92.1%), purity = 98.4%. Chiral-HPLC:
Chiral AGP column, 100 ꢃ 4.0 mm; 0.70 mL/min; 8% ACN/50 mM
ammonium acetate (pH 7), 276 nm; Rt = 7.5 min (98.8% R,S).
(6,280). MS m/z (rel): 332 (100, M+H), 663 (17). RP-HPLC: Ace-5
C18 column, 100 ꢃ 4.6 mm; 1.0 mL/min; 10–50% (5 min) acetoni-
trile/0.1%TFA/water; 275 nm; Rt = 3.44 min (94.9%), 1.47 min
(3.8% fumarate). Chiral-HPLC: Chirobiotic
V
column, 250
ꢃ 4.6 mm; 1.0 mL/min; 10–30% (40 min) MeOH/20 mM KH2PO4;
275 nm; Rt = 19.2 min (93.6%, R,R), Rt = 18.4 min (6.4%, R,S). [
a]
[a
]
D
ꢁ30.6 (1.0% free base in MeOH). HRMS m/z calcd for
D
ꢁ31.1 (7.0%, free base MeOH). HRMS m/z calcd for [C19H25NO4]H+
[C20H27NO4]H+ 346.2013, found 346.2008.
332.1856, found 332.1852.
4.5.31.5. (R,R0)-5-(1-Hydroxy-2-((1-(4-methoxyphenyl)pentan-
4.5.31.2.
butan-2-yl)amino)ethyl)benzene-1,3-diol
(R,S0)-(+)-5-(1-Hydroxy-2-((1-(4-methoxyphenyl)
[(R,S0)-64].
2-yl)amino)ethyl)benzene-1,3-diol [(R,R0)-66].
A mixture of
A
550 mg (1.65 mmol) of (R)-73 and 468 mg (1.65 mmol) of (R)-
72c was heated at 135 °C for 29 h and further treated using the
general procedure above to give 304 mg (46%) of hemi-fumarate
salt. 1H NMR (400 MHz, CD3OD): d 0.899 (t, 3H, J = 7.2 Hz), 1.26–
1.46 (m, 2H), 1.57-1.65 (m, 2H), 2.80–2.98 (m, 2H), 3.02–3.15 (m,
2H), 3.35–3.36 (m, 1H), 4.73 (dd, 1H, J = 4.0, 8.8 Hz), 6.21 (t, 1H,
J = 2.0 Hz), 6.32 (d, 2H, J = 2.4 Hz), 6.68 (s, 1H, fum), 6.89 (d, 2H,
J = 8.8 Hz), 7.15 (d, 2H, J = 8.8 Hz) ppm. 13C NMR (75 MHz, CD3OD):
d 14.12, 19.42, 33.24, 37.46, 52.50, 55.68, 60.90, 70.36, 103.29,
105.21, 115.37, 129.20, 131.32, 136.46, 144.71, 160.00, 160.41,
mixture of 380 mg (1.15 mmol) of (R)-73 and 294 mg (1.1 mmol)
of (S)-72a was heated at 120 °C for 24 h and further treated using
the general procedure above to give 376 mg (88%) of hemi-fuma-
rate salt. 1H NMR (400 MHz, CD3OD): d 0.974 (t, 3H, J = 9.6 Hz),
1.67 (m, 2H), 2.87-2.96 (m, 3H), 3.12 (dd, 1H, J = 4.4, 16.4 Hz),
3.77 (s, 3H), 4.74 (dd, 1H J = 4.0, 12.8 Hz), 6.19 (t, 1H, J = 2.8 Hz),
6.31 (d, 2H, J = 2.8 Hz), 6.70 (s, 1H, fum), 6.89 (d, 2H, J = 11.6 Hz),
7.17 (d, 2H, J = 11.6 Hz) ppm. 13C NMR (75 MHz, CD3OD): d 9.91,
24.68, 37.08, 52.85, 55.71, 62.20, 70.55, 103.24, 105.27, 115.41,
129.64, 131.34, 137.01, 144.93, 159.93, 160.34 ppm. UV (MeOH)
172.21 ppm. UV (MeOH) kmax (e): 277 nm (3,280), 224 (17,800),
kmax
(
e
): 277 nm (3,530), 223 (19,750), 204 (41,300). MS m/z
203 (40,500). MS m/z (rel): 346 (100, M+H base). RP-HPLC: Ace-5
C18 column, 100 ꢃ 4.6 mm; 10–60%/10 min ACN/20 mM KH2PO4
(pH 4.5); 1.0 mL/min; 275 nm; Rt = 6.79 min (98.8%). Chiral-HPLC:
(rel): 332 (100, M+H), 663 (17). RP-HPLC: Ace-5 C18 column, 100
ꢃ 4.6 mm; 1.0 mL/min; 10–50% (5 min) ACN/ 0.1% TFA/water;
275 nm; Rt = 3.47 min (94.3%), 1.46 min (4.04%, fumarate). Chiral-
HPLC: Chirobiotic V column, 250 ꢃ 4.6 mm; 1.0 mL/min; 10–30%
(40 min) MeOH/20 mM KH2PO4; 275 nm; Rt = 18.3 min (99%, R,S).
Chirobiotic
V
column, 250 ꢃ 4.6 mm; 1.0 mL/min; 20–40%
(40 min) MeOH/20 mM KH2PO4; 275 nm; Rt = 20.0 min (96.7%
R,R). [
a]
ꢁ32.1 (1.0% MeOH, free base). HRMS m/z calcd for
D
[
a]
D
+1.43 (7.0%, free base in MeOH). HRMS m/z calcd for [C19H25-
[C20H27NO4]H+ 346.2013, found 346.2009.
NO4]H+ 332.1856, found 332.1853.
4.5.31.6. (R,S0)-5-(1-Hydroxy-2-((1-(4-methoxyphenyl)pentan-2-
4.5.31.3. (R,R0)-5-(1-Hydroxy-2-((1-(4-methoxyphenyl)-3-meth-
yl)amino)ethyl)benzene-1,3-diol [(R,S0)-66].
A mixture of
ylbutan-2-yl)amino)ethyl)-benzene-1,3-diol [(R,R0)-65].
A
587 mg (1.77 mmol) of (R)-73 and 500 mg (1.77 mmol) of (S)-72c
was heated at 135 °C for 29 h and treated using the general proce-
dure above to give 352 mg (49%) of hemi-fumarate salt. 1H NMR
(400 MHz, CD3OD): d 0.904 (t, 3H, J = 7.2 Hz), 1.35–1.45 (m, 2H),
1.54–1.58 (m, 2H), 2.83–2.92 (m, 2+1H), 3.03-3.07 (m, 1H), 3.22
(m, 1H), 3.77 (s, 3H), 6.67 (dd, 1H, J = 3.2, 9.2 Hz), 6.19 (t, 1H,
J = 2.4), 6.29 (d, 2H, J = 2.0 Hz), 6.68 (s, 1H, fum), 6.88 (d, 2H,
J = 8.8 Hz), 7.15 (d, 2H, J = 8.8 Hz) ppm. 13C NMR (75 MHz, CD3OD):
d 14.22, 19.71, 34.36, 37.78, 52.98, 55.71, 60.77, 70.72, 103.21,
105.27, 115.40, 129.75, 131.34, 136.91, 145.02, 159.95,
mixture of 579 mg (1.74 mmol) of (R)-73 and 494 mg (1.74 mmol)
of (R)-72b was heated at 145 °C for 48 h and further treated using
the general procedure above to give 401 mg (57%) of hemi-fuma-
rate salt. 1H NMR (400 MHz, CD3OD): d 1.05 (d, 6H, J = 6.9 Hz),
2.08 (hept, 1H, J = 3.7 Hz), 2.76-2.84 (m, 2H), 2.91-3.00 (m, 2H),
3.25 (m, 1H), 3.77 (s, 3H), 4.64 (dd, 1H, J = 3.3, 10.0 Hz), 6.20 (d,
2H, J = 2.2 Hz), 6.67 (s, 1H, fumarate), 6.90 (d, 2H, J = 8.6 Hz), 7.19
(d, 2H, J = 8.6 Hz) ppm. 13C NMR (75 MHz, CD3OD): d 17.68,
18.54, 30.68, 34.42, 54.00, 55.74, 66.03, 69.90, 103.17, 105.26,
115.54, 130.38, 131.20, 136.91, 144.86, 159.85, 160.34,
160.34 ppm. UV (MeOH) kmax (e): 277 nm (3,510), 224 (18,900),
173.70 ppm. UV (MeOH) kmax
(
e
): 278 nm (3490), 223 (18,800),
203 (38,900). MS m/z (rel): 346 (100, M+H base). RP-HPLC: Ace-5
C18 column, 100 ꢃ 4.6 mm; 10–60%/10 min ACN/20 mM KH2PO4
(pH 4.5); 1.0 mL/min; 275 nm; Rt = 6.82 min (97.5%). Chiral-HPLC:
206 (22,800). MS m/z (rel): 346 (M+H base). RP-HPLC: Ace-5 C18
column, 100 ꢃ 4.6 mm; 10–60%/10 min ACN/20 mM KH2PO4 (pH
4.5); 1.0 mL/min; 275 nm; Rt = 1.15 min (5.82% fumarate),
6.44 min (92.8%), purity 98.6%. Chiral-HPLC: Chiral AGP column,
100 ꢃ 4.0 mm; 0.70 mL/min; 8% acetonitrile/50 mM ammonium
Chirobiotic
(40 min) MeOH/20 mM KH2PO4; 275 nm; Rt = 18.0 min (98.0%
R,S). [
ꢁ1.20 (1.0% MeOH, free base). HRMS m/z calcd for
[C20H27NO4]H+ 346.2013, found 346.2009.
V
column, 250 ꢃ 4.6 mm; 1.0 mL/min; 20–40%
a]
D
acetate (pH 7); 276 nm; Rt = 9.1 min (98.4% R,R). [a] +3.10 (1.0%
D
free base in MeOH); HRMS m/z calcd for [C20H27NO4]H+
346.2013, found 346.2006.
4.5.31.7.
(R)-(ꢁ)-5-(1-Hydroxy-2-((4-methoxyphenethyl)
amino)ethyl)benzene-1,3-diol [(R)-67].
A
mixture of
4.5.31.4.
methylbutan-2-yl)amino)-ethyl)benzene-1,3-diol
65]. A mixture of 644 mg (1.94 mmol) of (R)-73 and 550 mg
(R,S0)-(-)-5-(1-Hydroxy-2-((1-(4-methoxyphenyl)-3-
[(R,S0)-
642 mg (1.93 mmol) of (R)-73 and 443 mg (1.83 mmol) of 72d
was heated at 135 °C for 30 h and further treated using the general
procedure above to give 350 mg (53% overall) of the hemi-fuma-
rate salt (R)-67. 1H NMR (400 MHz, CD3OD): d 2.88–2.95 (m,
2H), 3.02 (dd, 1H, J = 9.6, 12.4 Hz), 3.09–3.18 (m, 3H), 3.76 (s,
3H), 4.76 (dd, 1H, J = 3.2, 9.2 Hz), 6.20 (t, 1H J = 2.0 Hz), 6.33 (d,
(1.94 mmol) of (S)-72b was heated at 145 °C for 48 h and further
treated using the general procedure above to give 478 mg (61%)
of hemi-fumarate salt. 1H NMR (400 MHz, CD3OD): d 1.04 (t, 6H,

246
A. Plazinska et al. / Bioorg. Med. Chem. 22 (2014) 234–246
2H, J = 2.0 Hz), 6.68 (s, 1H, fum), 6.87 (d, 2H, J = 8.4 Hz), 7.16 (d, 2H,
J = 8.4 Hz) ppm. 13C NMR (75 MHz CD3OD): d 32.43, 50.27, 55.29,
55.68, 70.26, 103.27, 105.27,115.30, 129.85, 130.79, 137.04,
Foundation for Polish Science (TEAM Programme). The article
was developed using the equipment purchased within the Project
‘The equipment of innovative laboratories doing research on new
medicines used in the therapy of civilization and neoplastic dis-
eases’ within the Operational Program Development of Eastern Po-
land 2007–2013, Priority Axis I Modern Economy, Operations I.3
Innovation Promotion.
144.81, 159.94, 160.26, 174.13 ppm. UV (MeOH) kmax (e): 277 nm
(3,040), 222 (17,180), 208 (24,800). MS m/z (rel): 304 (100, M+H
base). RP-HPLC: Eclipse XDB-C18, 150 ꢃ 4.6 mm; 10–50%/5 min
ACN/0.1%TFA-water, 1.0 mL/min, 275 nm, Rt = 5.11 min (95.66%),
2.16 min (2.80%, fumarate). Chiral-HPLC: 100 ꢃ 4.0 mm; 95/5
50 mM NH4OAc (pH 7); 0.70 mL/min; 275 nm; Rt = 15.14 min
Supplementary data
(96.8% R), 13.91 min (3.2% S); [
a]
ꢁ14.42 (0.95% free base in
D
MeOH). HRMS: m/z calcd for [C17H21NO4]H+ 304.1543, found
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.11.030.
304.1540.
4.5.31.8.
(S)-5-(1-Hydroxy-2-((4-methoxyphenethyl)amino)
mixture of 768 mg
References and notes
ethyl)benzene-1,3-diol [(S)-67].
A
1. Jozwiak, K.; Khalid, C.; Tanga, M. J.; Berzetei-Gurske, I.; Jimenez, L.; Kozocas, J.
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32601, USA.
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12. Thomsen, R.; Christensen, M. H. J. Med. Chem. 2006, 49, 3315–3321.
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(2.38 mmol) of (S)-73 and 546 mg (2.26 mmol) of 72d was heated
at 135 °C for 30 h and further treated using the general procedure
above to give 421 mg (52% overall) of the hemi-fumarate salt (S)-
67. 1H NMR (400 MHz, CD3OD): d 2.89-2.95 (m, 2H), 3.02 (dd,
1H, J = 10.0, 12.8 Hz), 3.76 (s, 3H), 3.14-3.19 (m, 3H), 4.77 (dd,
1H, J = 3.6, 9.6 Hz), 6.19 (t, 1H J = 2.4 Hz), 6.34 (d, 2H, J = 2.4 Hz),
6.83 (s, 1H, fum), 6.87 (d, 2H, J = 8.4 Hz), d 7.16 (d, 2H, J = 8.8 Hz)
ppm. 13C NMR (75 MHz, CD3OD): d 32.63, 50.38, 55.44, 55.71,
70.45, 103.26, 105.28, 115.33, 130.00, 130.77, 136.95, 144.91,
159.97, 160.30, 173.87 ppm. UV (MeOH) kmax (e): 277 nm (2850),
224 (14,500), 207 (21,900). MS m/z (rel): 304 (100, M+H base).
RP-HPLC: Eclipse XDB-C18, 150 ꢃ 4.6 mm; 10–50%/5 min ACN/
0.1%TFA-water, 1.0 mL/min, 275 nm, Rt = 5.11 min (95.38%),
2.16 min (2.79%, fumarate). Chiral-HPLC: 100 ꢃ 4.0 mm; 95/5
50 mM NH4OAc (pH 7); 0.70 mLmin; 275 nm; Rt = 13.2 (96.1% S),
16.2 min (3.9% R). [
a
]
D
+13.8 (1.0% free base in MeOH). HRMS:
m/z calcd for [C17H21NO4]H+ 304.1543, found 304.1539.
Acknowledgment
This research was supported by the National Institutes of
Health National Institute on Aging [Contract N01-AG31009] by
the Intramural Research Program of the NIA/NIH; and by the
14. Conrow, R. E.; Dean, W. D. Org. Process Res. Dev. 2008, 12, 1285–1286.
15. Koppenhoefer, B.; Schurig, V. Org Synth. 1993, Coll. 8, 119.
16. Birk, C.; Jurgen, V. C. Tetrahedron 1996, 52, 12745–12760.